WO2025029980A1 - Inhibition of hypoxia signaling pathway by plk1 inhibitor - Google Patents

Abstract

Disclosed herein include methods, compositions, and kits suitable for use in treating cancer in a subject. The method can comprise administrating to the subject a PLK1 inhibitor (for example, onvansertib) to inhibit hypoxia signaling pathway in the subject to reduce or inhibit progression of the cancer.

Description

INHIBITION OF HYPOXIA SIGNALING PATHWAY BY PLK1 INHIBITOR

RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Application No. 63/517,111, filed August 1, 2023; U.S. Provisional Application No. 63/561,218, filed March 4, 2024; and U.S. Provisional Application No. 63/575,221, filed April 5, 2024. The entire contents of these applications are hereby expressly incorporated by reference in their entireties.

BACKGROUND

Field

[0002] The present disclosure relates generally to the field of treatment of cancer. More specifically, combination therapies for treating cancer using HIF-la inhibitors in combination with polo-like kinase 1 (PLK1) inhibitors are provided. Description of the Related Art

[0003] PLK1 is a well characterized member of the 5 members of the family of serine/threonine protein kinases and strongly promotes the progression of cells through mitosis. PLK1 performs several important functions throughout mitotic (M) phase of the cell cycle, including the regulation of centrosome maturation and spindle assembly, the removal of cohesins from chromosome arms, the inactivation of anaphase-promoting complex/cyclosome (APC/C) inhibitors, and the regulation of mitotic exit and cytokinesis. PLK1 plays a key role in centrosome functions and the assembly of bipolar spindles. PLK1 also acts as a negative regulator of p53 family members leading to ubiquitination and subsequent degradation of p53/TP53, inhibition of the p73/TP73 mediated pro-apoptotic functions and phosphorylation/degradation of bora, a cofactor of Aurora kinase A. During the various stages of mitosis PLK1 localizes to the centrosomes, kinetochores and central spindle. PLK1 is a master regulator of mitosis and aberrantly overexpressed in a variety of human cancers and is correlated with cellular proliferation and poor prognosis.

[0004] Oxygen concentrations are typically reduced in many cancers compared to non- cancerous tissue. Hypoxic conditions induce hypoxia inducible factor 1 alpha (HIF-la) expression, which regulates the transcription of hundreds of genes, many involved in cancer. There is a need for novel therapies targeting the HIF-la pathway for treating cancer.

SUMMARY

[0005] Disclosed herein include methods of treating cancer. In some embodiments, the method comprises: administering a Polo-like kinase 1 (PLK1) inhibitor and a hypoxia inducible factor 1 a (HIFla) inhibitor to a subject with cancer, wherein the cancer comprises at least one tumor with a high level of hypoxia prior to the administering, wherein the administrating results in a decrease in the expression and/or activity of at least one HIFla pathway component in the at least one tumor of the subject, thereby reducing or inhibiting progression of the cancer.

[0006] In some embodiments, the at least one HIFla pathway component is HIFla, BNIP3, PFKFB4, P4HA1, ANKZF1, INSIG2, ENO2, LDHA, ALDOC, PGK1, HK1, PFKP, P4HA2, KLF6, EFNA3, NFIL3, AK4, ERRFI1, SAP30, CCNG2, ANKRD37, KDM3A, ADM, DDIT4, HK2, SLC2A3, STC2, VEGFA, WSB1, PDK1, GLUT1, GLUT3, or HK2. In some embodiments, the expression of HIFla is reduced in the at least one tumor by at least 25% following administration of the PLK1 inhibitor, the HIFla inhibitor, or both, e.g., as compared to the subject prior to the administration. In some embodiments, administering the PLK1 inhibitor and the HIFla inhibitor synergistically reduces HIFla expression in the at least one tumor, e.g., as compared to the subject prior to the administration, a subject treated with the PLK1 inhibitor alone, or a subject treated with the HIFla inhibitor alone. In some embodiments, administering the PLK1 inhibitor and the HIFla inhibitor synergistically reduces HIFla expression in the at least one tumor by at least 50%, e.g., as compared to the subject prior to the administration.

[0007] The high level of hypoxia can comprise a partial pressure of oxygen (pCh) level of about 2 mmHg to about 25 mmHg in or near the at least one tumor. In some embodiments, the high level of hypoxia comprises a pCh level of about 10 mmHg in or near the at least one tumor. In some embodiments, the high level of hypoxia comprises at least 10% of the cells of the at least one tumor being positive for one or more markers for hypoxia. In some embodiments, the cells comprise a biopsy sample. The one or more markers of hypoxia can be, e.g., HIFla, glucose transporter 1 (GLUT1), and/or carbonic anhydrase 9 (CA IX). In some embodiments, the at least one tumor comprises cells over-expressing one or more HIFla target genes relative to non- cancerous and/or non-hypoxic cells prior to the administration. In some embodiments, the one or more HIFla target genes is: BNIP3L, PFKFB4, P4HA1, ANKZF1, INSIG2, EN02, LDHA, ALDOC, PGK1, HK1, PFKP, P4HA2, KLF6, EFNA3, NFIL3, AK4, ERRFI1, SAP30, CCNG2, ANKRD37, KDM3A, ADM, DDIT4, HK2, SLC2A3, STC2, VEGFA, WSB1, and/or PDK1.

[0008] In some embodiments, the at least one tumor comprises cells comprising a mutation in a gene involved in mitochondrial metabolism. In some embodiments, the gene is SDH, FH, or ND2. In some embodiments, the at least one tumor comprises cells comprising elevated nitric oxide synthase levels, e.g., as compared to non-cancerous or non-hypoxic cells. In some embodiments, the at least one tumor comprises cells comprising elevated NAD+ levels, e.g., as compared to non-cancerous or non-hypoxic cells. In some embodiments, the at least one tumor comprises cells comprising a mutation in a gene causing dysregulation of the mTOR pathway. In some embodiments, the gene comprises LKB1, PML, PTEN, or TSC1/2. [0009] The method can comprise determining a hypoxia status of the cancer prior to the administration, after the administration, or both. In some embodiments, determining the hypoxia status of the cancer comprises electron paramagnetic resonance (EPR) oximetry, eppendorf electrode technique, optical methods, NMR relaxation techniques, or any combination thereof. In some embodiments, determining the hypoxia status of the cancer comprises an indirect method comprising: i) PET-based measurement of hypoxia-localizing drugs; ii) measurement of anaerobic and/or aerobic glycolysis; iii) detecting the presence, absence, or amount of one or more markers of hypoxia, (the one or more markers can comprise HIFla, GLUT1, and/or CA IX); and/or iv) measuring one or more physiological parameters in the subject, optionally selected from MRI perfusion, laser doppler flow, and lactate levels.

[0010] In some embodiments, administering the PLK1 inhibitor and the HIFla inhibitor synergistically reduces or inhibits progression of the cancer relative to the PLK1 inhibitor treatment alone, the HIFla inhibitor treatment alone, and/or the additive effect of the PLK1 inhibitor treatment alone and the HIFla inhibitor treatment alone. In some embodiments, administering the PLK1 inhibitor and the HIFla inhibitor improves one or more therapeutic effects in the subject relative to a control or a baseline. In some embodiments, the one or more therapeutic effects comprise size of a tumor derived from the cancer, objective response rate (ORR), duration of response (DOR), time to response, progression free survival (PFS), overall survival (OS), disease control rate (DCR), oncogenic allelic burden, and/or expression level of CD31. In some embodiments, administering the PLK1 inhibitor and the HIFla inhibitor improves the ORR in the subject, improves the DOR in the subject, improves PFS in the subject, improves OS in the subject, improves DCR in the subject, reduces oncogenic allelic burden in the subject, reduces expression level of CD31, hypoxia-inducible factor la (HIFla) and/or a factor under the regulation of HIFla, or a combination thereof, relative to subjects who have received prior treatment comprising a HIFla inhibitor. In some embodiments, administering the PLK1 inhibitor and the HIFla inhibitor improves the ORR in the subject by at least 50% relative to subjects who have received prior treatment comprising a HIFla inhibitor. In some embodiments, administering the PLK1 inhibitor and the HIFla inhibitor improves the PFS in the subject by at least 50% relative to subjects who have received prior treatment comprising a HIFla inhibitor.

[0011] The cancer can be, e.g., colorectal cancer, bladder cancer, breast cancer, kidney cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, stomach cancer, thyroid cancer, uterine cancer, renal cancer, cervical cancer, recurrent glioblastoma, head and neck cancer, soft tissue carcinoma, vulvar cancer, melanoma, brain cancer, liver cancer, bile duct cancer, or a combination thereof. In some embodiments, the cancer is KRAS-mutant cancer or ER+ cancer, e.g., KRAS-mutant colorectal cancer (mCRC) or ER+ breast cancer. The cancer can be a metastatic cancer, e.g., metastatic colorectal cancer, metastatic bladder cancer, metastatic breast cancer, metastatic kidney cancer, metastatic lung cancer, metastatic ovarian cancer, metastatic pancreatic cancer, metastatic prostate cancer, metastatic stomach cancer, metastatic thyroid cancer, metastatic uterine cancer, metastatic renal cancer, metastatic cervical cancer, metastatic recurrent glioblastoma, metastatic head and neck cancer, metastatic soft tissue carcinoma, metastatic vulvar cancer, metastatic malignant melanoma, metastatic brain cancer, metastatic liver cancer, metastatic bladder cancer, metastatic bile duct cancer, or a combination thereof. The metastatic cancer can be e.g., KRAS-mutant cancer or ER+ cancer. In some embodiments, the metastatic cancer is KRAS-mutant metastatic colorectal cancer (mCRC) or ER+ breast cancer.

[0012] In some embodiments, the PLK1 inhibitor is selective and/or specific for PLK1. In some embodiments, the PLK1 inhibitor is a dihydropteridinone, a pyridopyrimidine, an aminopyrimidine, a substituted thiazolidinone, a pteridine derivative, a dihydroimidazo[l,5- f]pteridine, a metasubstituted thiazolidinone, a benzyl styryl sulfone analogue, a stilbene derivative, or any combination thereof. In some embodiments, the PLK1 inhibitor is onvansertib, BI2536, Volasertib (BI 6727), GSK461364, AZD1775, CYC140, HMN-176, HMN-214, rigosertib (ON-01910), MLN0905, TKM-080301, TAK-960, or Ro3280.

[0013] In some embodiments, the HIFla inhibitor comprises a microRNA (miRNA), a precursor microRNA (pre-miRNA), a small interfering RNA (siRNA), a short-hairpin RNA (shRNA), an antibody, or a small molecule. In some embodiments, the HIFla inhibitor inhibits HIFla mRNA expression, inhibits HIFla protein translation, modulates HIFla protein degradation, inhibits HIFla heterodimerization, inhibits HIFla DNA binding, inhibits HIFla transcriptional activity, or any combination thereof. In some embodiments, the HIFla inhibitor is selected from the group consisting of: EZN-2968; aminoflavone; topotecan; EZN-2208; a cardiac glycoside, e.g., digoxin or PX-478; an mTOR inhibitor, e.g., temsirolimus or everolimus; an HSP90 inhibitor, e.g., galdanamycin; a histone deactylase inhibitor; an antibiotic, e.g., a quinoxaline or anthracycline; chetomin; bevacizumab; paclitaxel; and bortezomib.

[0014] In some embodiments, the HIFla inhibitor is an angiogenesis inhibitor. In some embodiments, the angiogenesis inhibitor is capable of inhibiting VEGF-A, VEGFR-1, VEGFR-2, VEGFR-3, EGFR, HER2, PDGFR family proteins, RAF, Kit (or c-Kit), FLT3, CSF- 1R, RET, Abl, Itk, LcK, c-FMS, FGFR family proteins, c-Met, P1GF, TNF-a, IFNs, ILs, bFGF, mTOR, or any combination thereof. In some embodiments, the angiogenesis inhibitor is Afatinib (Gilotrif®), Axitinib (Inlyta®), Bevacizumab (Avastin®), Cabozantinib (Cometriq®), Cetuximab (Erbitux®), Erlotinib (Tarceva®), Everolimus (Afinitor®), Gefitinib (Iressa®), Imatinib (Gleevec®), Lapatinib (Tykerb®), Lenalidomide (Revlimid®), Lenvatinib mesylate (Lenvima®), Necitumumab (Portrazza™), Neratinib (Nerlynx®), Panitumumab (Vectibix®), Pazopanib (Votrient®), Pertuzumab (Perjeta®), Ramucirumab (Cyramza®), Regorafenib (Stivarga®), Sorafenib (Nexavar®), Sunitinib (Sutent®), Thalidomide (Synovir, Thalomid®), Trastuzumab (Ontruzant®), Vandetanib (Caprelsa®), or Ziv-aflibercept (Zaltrap®).

[0015] In some embodiments, the HIFla inhibitor is a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent comprises an alkylating agent, an antimetabolite, a topoisomerase inhibitor, a mitotic inhibitor, or an antitumor antibiotic. The mitotic inhibitor can be a microtubule polymerization or depolymerization inhibitor. In some embodiments, the chemotherapeutic agent is selected from the group consisting of: Altretamine, Bendamustine, Busulfan, Carboplatin, Chlorambucil, Cisplatin, Cyclophosphamide, Dacarbazine, Ifosfamide, Mechlorethamine, Melphalan, Oxaliplatin, Procarbazine, Temozolomide, Thiotepa, Trabectedin, Carmustine, Lomustine, Streptozocin, 5-fluorouracil, 6-mercaptopurine, Azacitidine, Capecitabine, Cladribine, Clofarabine, Cytarabine, Decitabine, Floxuridine, Fludarabine, Gemcitabine, Hydroxyurea, Methotrexate, Nelarabine, Pemetrexed, Pentostatin, Pralatrexate, Thioguanine, Trifluridine/tipiracil combination, Etoposide, Irinotecan, Irinotecan liposomal, Mitoxantrone Teniposide, Topotecan, Cabazitaxel, Docetaxel, Nab-paclitaxel, Paclitaxel, Vinblastine, Vincristine, Vincristine liposomal, Vinorelbine, Daunorubicin, Doxorubicin, Doxorubicin liposomal, Epirubicin, Idarubicin, Mitoxantrone, Valrubicin, Bleomycin, Dactinomycin, Mitomycin-C, Arsenic trioxide, Asparaginase, Eribulin, Ixabepilone, Mitotane, Omacetaxine, Pegaspargase, Procarbazine, Romidepsin, Vorinostat, and a combination therapy comprising folinic acid, 5-fluorouracil, and irinotecan (FOLFIRI).

[0016] The HIFla inhibitor can be paclitaxel or bevacizumab. In some embodiments, the PLK1 inhibitor and the HIFla inhibitor are co-administered simultaneously. In some embodiments, the PLK1 inhibitor and the HIFla inhibitor are administered sequentially. In some embodiments, the PLK1 inhibitor is administered prior to the administration of the HIFla inhibitor. In some embodiments, the PLK1 inhibitor is administered prior to the administration of the HIFla inhibitor every day on which the subject is administered with the PLK1 inhibitor and the HIFla inhibitor. In some embodiments, the PLK1 inhibitor is administered about 30 minutes to about 5 hours prior to the administration of the HIFla inhibitor on a given day. In some embodiments, the administration of the PLK1 inhibitor comprises oral administration, and the administration of the HIFla inhibitor comprises intravenous administration or oral administration. In some embodiments, the HIFla inhibitor and the PLK1 inhibitor are each administered to the subject in a cycle of at least twice or at least five times within a week. In some embodiments, the HIFla inhibitor, the PLK1 inhibitor, or both are administered in a cycle of at least 7 days. In some embodiments, each cycle of treatment is at least about 21 days. In some embodiments, each cycle of treatment is from about 21 days to about 28 days. In some embodiments, the PLK1 inhibitor is administered on at least four days in the cycle. In some embodiments, the PLK1 inhibitor is not administered on at least one day in the cycle. In some embodiments, the HIFla inhibitor is administered daily, weekly, bi-weekly, every three weeks, every four weeks, or every month. In some embodiments, the subject undergoes at least two cycles of the administration of the HIFla inhibitor and the PLK1 inhibitor.

[0017] In some embodiments, the PLK1 inhibitor is onvansertib and/or the HIFla inhibitor is bevacizumab or paclitaxel. Onvansertib can be, e.g., administered at 12 mg/m² - 90 mg/m². In some embodiments, a maximum concentration (Cmax) of onvansertib in a blood of the subject is from about 100 nmol/L to about 1500 nmol/L. In some embodiments, an area under curve (AUC) of a plot of a concentration of onvansertib in a blood of the subject over time is from about 1000 nmol/L. hour to about 400000 nmol/L. hour. In some embodiments, a time (Tmax) to reach a maximum concentration of onvansertib in a blood of the subject is from about 1 hour to about 5 hours. In some embodiments, an elimination half-life (T1/2) of onvansertib in a blood of the subject is from about 10 hours to about 60 hours. Bevacizumab can be, e.g., administered at about 1 mg/kg - 20 mg/kg, e.g., about 5 mg/kg, about 7.5 mg/kg, about 10 mg/kg, or about 15 mg/kg. Paclitaxel can be, e.g., administered at about 50 mg/m² to about 175 mg/m².

[0018] In some embodiments, the subject has received at least one prior cancer treatment. In some embodiments, the prior treatment does not comprise the use of a HIFla inhibitor, aPLKl inhibitor, or both. In some embodiments, the subject was in remission for cancer. In some embodiments, the subject in remission for cancer was in complete remission (CR) or in partial remission (PR). The method can comprise one or more of (1) determining cancer status of the subject, (2) determining responsiveness of the subject to a PLK1 inhibitor treatment, and (3) administering one or more cancer therapeutics or therapies for the cancer. In some embodiments, the subject is human. In some embodiments, the one or more cancer therapeutics or therapies comprise FOLFIRI, abiraterone, FOLFOX, an anti-EGFR agent, a KRAS directed inhibitor, gemcitabine, abraxane, nanoliposomal irinotecan, 5-FU, or a combination thereof. In some embodiments, anti-EGFR agents is optionally cetuximab. In some embodiments, the KRAS directed inhibitor is optionally a G12C inhibitor, a G12D inhibitor or a combination thereof. In some embodiments, determining the responsiveness of the subject comprises determining if the subject is a responder of the treatment, if the subject is or is going to be in complete recovery (CR), or if the subject is or is going to be in partial remission (PR). In some embodiments, determining the responsiveness of the subject comprises determining objective response rate (ORR), duration of response (DOR), time to response, progression free survival (PFS), overall survival (OS), disease control rate (DCR), oncogenic allelic burden, or a combination thereof of the subject. In some embodiments, determining the responsiveness of the subject comprises determining if the subject has a partial response to the treatment, if the subject has a complete response to the treatment, if the subject has a stable disease (SD) status, or if the subject has a progressive disease (PD) status.

[0019] Disclosed herein include kits. In some embodiments, the kit comprises: a PLK1 inhibitor; and a manual providing instructions for co-administrating the PLK1 inhibitor with a HIF la inhibitor to a subject for treating a cancer, wherein the cancer comprises at least one tumor with a high level of hypoxia prior to the co-administration.

[0020] In some embodiments, the cancer is colorectal cancer, bladder cancer, breast cancer, kidney cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, stomach cancer, thyroid cancer, uterine cancer, renal cancer, cervical cancer, recurrent glioblastoma, head and neck cancer, soft tissue carcinoma, vulvar cancer, melanoma, brain cancer, liver cancer, bile duct cancer, or a combination thereof. In some embodiments, the cancer is KRAS-mutant cancer or ER+ cancer. In some embodiments, the cancer is KRAS-mutant colorectal cancer (mCRC) or ER+ breast cancer. In some embodiments, the cancer is a metastatic cancer. In some embodiments, the metastatic cancer is metastatic colorectal cancer, metastatic bladder cancer, metastatic breast cancer, metastatic kidney cancer, metastatic lung cancer, metastatic ovarian cancer, metastatic pancreatic cancer, metastatic prostate cancer, metastatic stomach cancer, metastatic thyroid cancer, metastatic uterine cancer, metastatic renal cancer, metastatic cervical cancer, metastatic recurrent glioblastoma, metastatic head and neck cancer, metastatic soft tissue carcinoma, metastatic vulvar cancer, metastatic malignant melanoma, metastatic brain cancer, metastatic liver cancer, metastatic bladder cancer, metastatic bile duct cancer, or a combination thereof. In some embodiments, the metastatic cancer is KRAS-mutant cancer or ER+ cancer. In some embodiments, the metastatic cancer is mCRC or ER+ breast cancer.

[0021] The instructions can comprise instructions for co-administrating the PLK1 inhibitor and the HIF la inhibitor simultaneously. In some embodiments, the instructions comprise instructions for co-administrating the PLK1 inhibitor and the HIFla inhibitor sequentially. In some embodiments, the instructions comprise (1) instructions for administering of the PLK1 inhibitor orally, (2) instructions for administrating the HIFla inhibitor orally, (3) instructions for administrating the HIFla inhibitor intravenously, or any combination thereof.

[0022] In some embodiments, the instructions comprise instructions wherein the subject has not received any prior treatment comprising administration of a HIFla inhibitor. In some embodiments, the instructions comprise instructions for administering each of the HIFla inhibitor and the PLK1 inhibitor to the subject in a cycle of at least twice or at least five times within a week. In some embodiments, the instructions comprise instructions for administering the HIFla inhibitor, the PLK1 inhibitor, or both in a cycle of at least 7 days. In some embodiments, each cycle of treatment is at least about 21 days. In some embodiments, each cycle of treatment is from about 21 days to about 28 days. In some embodiments, the instructions comprise instructions for administering the PLK1 inhibitor on at least four days in the cycle. In some embodiments, the instructions comprise instructions for not administering the PLK1 inhibitor on at least one day in the cycle. In some embodiments, the instructions comprise instructions for administrating the HIFla inhibitor daily, weekly, bi-weekly, every three weeks, every four weeks, or monthly. In some embodiments, the instructions comprise instructions for administrating the HIFla inhibitor and the PLK1 inhibitor for at least two cycles.

[0023] The HIFla inhibitor can comprise a microRNA (miRNA), a precursor microRNA (pre-miRNA), a small interfering RNA (siRNA), a short-hairpin RNA (shRNA), an antibody, or a small molecule. In some embodiments, the HIFla inhibitor inhibits HIFla mRNA expression, inhibits HIFla protein translation, modulates HIFla protein degradation, inhibits HIFla heterodimerization, inhibits HIFla DNA binding, inhibits HIFla transcriptional activity, or any combination thereof. In some embodiments, the HIFla inhibitor is selected from the group consisting of: EZN-2968; aminoflavone; topotecan; EZN-2208; a cardiac glycoside, e.g., digoxin or PX-478; an mTOR inhibitor, e.g., temsirolimus or everolimus; an HSP90 inhibitor, e.g., galdanamycin; a histone deactylase inhibitor; an antibiotic, e.g., a quinoxaline or anthracycline; chetomin; bevacizumab; paclitaxel; and bortezomib. In some embodiments, the HIFla inhibitor is an angiogenesis inhibitor. In some embodiments, the angiogenesis inhibitor is capable of inhibiting VEGF-A, VEGFR-1, VEGFR-2, VEGFR-3, EGFR, HER2, PDGFR family proteins, RAF, Kit (or c-Kit), FLT3, CSF-1R, RET, Abl, Itk, LcK, c-FMS, FGFR family proteins, c-Met, P1GF, TNF-a, IFNs, ILs, bFGF, mTOR, or any combination thereof. In some embodiments, the angiogenesis inhibitor is Afatinib (Gilotrif®), Axitinib (Inlyta®), Bevacizumab (Avastin®), Cabozantinib (Cometriq®), Cetuximab (Erbitux®), Erlotinib (Tarceva®), Everolimus (Afinitor®), Gefitinib (Iressa®), Imatinib (Gleevec®), Lapatinib (Tykerb®), Lenalidomide (Revlimid®), Lenvatinib mesylate (Lenvima®), Necitumumab (Portrazza™), Neratinib (Nerlynx®), Panitumumab (Vectibix®), Pazopanib (Votrient®), Pertuzumab (Perjeta®), Ramucirumab (Cyramza®), Regorafenib (Stivarga®), Sorafenib (Nexavar®), Sunitinib (Sutent®), Thalidomide (Synovir, Thalomid®), Trastuzumab (Ontruzant®), Vandetanib (Caprelsa®), or Ziv-aflibercept (Zaltrap®). In some embodiments, the HIFla inhibitor is a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent comprises an alkylating agent, an antimetabolite, a topoisomerase inhibitor, a mitotic inhibitor, or an antitumor antibiotic. The mitotic inhibitor can be a microtubule polymerization or depolymerization inhibitor. In some embodiments, the chemotherapeutic agent is selected from the group consisting of: Altretamine, Bendamustine, Busulfan, Carboplatin, Chlorambucil, Cisplatin, Cyclophosphamide, Dacarbazine, Ifosfamide, Mechlorethamine, Melphalan, Oxaliplatin, Procarbazine, Temozolomide, Thiotepa, Trabectedin, Carmustine, Lomustine, Streptozocin, 5-fluorouracil, 6-mercaptopurine, Azacitidine, Capecitabine, Cladribine, Clofarabine, Cytarabine, Decitabine, Floxuridine, Fludarabine, Gemcitabine, Hydroxyurea, Methotrexate, Nelarabine, Pemetrexed, Pentostatin, Pralatrexate, Thioguanine, Trifluridine/tipiracil combination, Etoposide, Irinotecan, Irinotecan liposomal, Mitoxantrone Teniposide, Topotecan, Cabazitaxel, Docetaxel, Nab-paclitaxel, Paclitaxel, Vinblastine, Vincristine, Vincristine liposomal, Vinorelbine, Daunorubicin, Doxorubicin, Doxorubicin liposomal, Epirubicin, Idarubicin, Mitoxantrone, Valrubicin, Bleomycin, Dactinomycin, Mitomycin-C, Arsenic trioxide, Asparaginase, Eribulin, Ixabepilone, Mitotane, Omacetaxine, Pegaspargase, Procarbazine, Romidepsin, Vorinostat, and a combination therapy comprising folinic acid, 5-fluorouracil, and irinotecan (FOLFIRI).

[0024] In some embodiments, the PLK1 inhibitor is selective and/or specific for PLK1. In some embodiments, the PLK1 inhibitor is a dihydropteridinone, a pyridopyrimidine, a aminopyrimidine, a substituted thiazolidinone, a pteridine derivative, a dihydroimidazo[l,5- f]pteridine, a metasubstituted thiazolidinone, a benzyl styryl sulfone analogue, a stilbene derivative, or any combination thereof. In some embodiments, the PLK1 inhibitor is onvansertib, BI2536, Volasertib (BI 6727), GSK461364, AZD1775, CYC140, HMN-176, HMN-214, rigosertib (ON-01910), MLN0905, TKM-080301, TAK-960 or Ro3280. In some embodiments, the PLK1 inhibitor is onvansertib.

[0025] The instructions can comprise instructions for administering onvansertib at 12 mg/m² - 90 mg/m². In some embodiments, the HIFla inhibitor is bevacizumab or paclitaxel and the PLK1 inhibitor is onvansertib. In some embodiments, the instructions comprise instructions the subject has received at least one prior treatment for the cancer. In some embodiments, the prior treatment does not comprise the use of a PLK1 inhibitor. In some embodiments, the instructions comprise instructions the subject was in remission for cancer. In some embodiments, the subject in remission for cancer was in complete remission (CR) or in partial remission (PR). The kit can comprise the HIFla inhibitor.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG. 1 depicts an exemplary Kaplan-Meier survival curve of cancer patients treated using the methods and compositions provided herein, with or without prior anti- angiogenics treatments.

[0027] FIG. 2 depicts number of events and censoring (non-reporting) patients for the study described herein.

[0028] FIG. 3 depicts objective response rates for a cohort of patients treated using the methods and compositions provided herein, broken down by patients who had (columns in darker color) or had not (columns in lighter color) received previous anti-angiogenics treatment. Numbers in boxes are the total number of patients in the indicated group.

[0029] FIG. 4 shows that onvansertib inhibits the hypoxia signaling pathway by downregulating HIFla expression in four KRAS-mutant CRC cell lines.

[0030] FIG. 5 shows that downregulation of HIFla by onvansertib in a dosedependent manner.

[0031] FIG. 6 shows subgroup analysis of baseline characteristics (n=66) of cancer patients (as of January 29, 2024) treated using the methods and compositions provided herein.

[0032] FIG. 7 shows waterfall plot of best radiographic response (n=66) of cancer patients (as of January 29, 2024) treated using the methods and compositions provided herein. * indicates patients treated with onvansertib atRP2D. CR=complete response, PR=partial response, SD= stable disease, and PD=progressive disease.

[0033] FIG. 8 shows an exemplary Kaplan-Meier curves of PFS of cancer patients (as of January 29, 2024) treated using the methods and compositions provided herein, with or without prior anti-angiogenics treatments.

[0034] FIG. 9A-FIG. 9C depict the effect of the combination of onvansertib and bevacizumab in inhibiting tumor growth and angiogenesis in KRAS-mutant colorectal cancer xenograft models. Xenografts were treated with onvansertib (Onv, oral, QD, 45 mg/kg), bevacizumab (Bev, IP, BIW, Img/kg for LoVo, 5 mg/kg for SW620 and HCT116) for 30-33 days. FIG. 9A depicts mean tumor volumes ± SEM (n=8-9/group). Tumor volumes were compared at end-of-study (EOS) by One-way ANOVA. FIG. 9B depicts photographs of tumors at EOS. FIG. 9C depicts microscopic images of the tumors. Tumors were stained with anti-CD31 antibody. Representative images of the SW620 tumors with no treatment or treated with onvansertib or bevacizumab or both are shown. The graph in FIG. 9C depicts CD31 scores of images in FIG. 9C, from left to right for each tumor type: vehicle only, Bev only, Onv only, Onv+Bev. CD31+ vessels were counted manually for each tumor (5 fields/tumor). Means ± SEM are shown and were compared by One-way ANOVA. **p<0.01, ***p<0.001, and ****p<0.0001.

[0035] FIG. 10A-FIG. 10F depict PLK1 was inhibited by onvansertib (FIG. 10A-FIG. 10D) or siPLKl (FIG. 10E-FIG. 10F) for 20 h and then cells were exposed to hypoxia for 4 h. FIG. 10A and FIG. 10E depict immunoblot showing inhibiting of HIFla by onvansertib (FIG. 10A) or siPLKl (FIG. 10E). HIFla protein levels were normalized to P-actin and 20% O2 control sample. FIG. 10B depicts study design of the RNA-seq experiment. FIG. 10C depicts enrichment scores of LoVo and SW620 receiving different treatment. Expression data were transformed into gene set enrichment scores using GSVA. FIG. 10D depicts heatmap of genes upregulated upon hypoxia and downregulated by onvansertib. In FIG. 10D, D=DMSO, Ol/Onvl=Onvansertib dose 1, and O2/Onv2=Onvansertib dose 2. FIG. 10F depicts gene expression analysis by RT-qPCR in SW620 cells, with data normalized to RPLPO gene and 20% Ch-siCtrl sample.

[0036] FIG. 11 depicts the workflow of the transcriptomic analysis.

[0037] FIG. 12 depicts the enrichment scores of the hallmarks and oncogenic signatures enriched in genes differentially expressed.

[0038] FIG. 13A-FIG. 13C depict data related to the effect of onvansertib, paclitaxel, and a combination of onvansertib and paclitaxel, on HIF-la expression in cancer cells.

DETAILED DESCRIPTION

[0039] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols typically identify similar components, unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein, and illustrated in the Figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly contemplated herein and made part of the disclosure herein.

[0040] All patents, published patent applications, other publications, and sequences from GenBank, and other databases referred to herein are incorporated by reference in their entirety with respect to the related technology.

[0041] Disclosed herein include methods of treating cancer. In some embodiments, the method comprises: administering a PLK1 inhibitor and a hypoxia inducible factor 1 a (HIFla) inhibitor to a subject with cancer, wherein the cancer comprises at least one tumor with a high level of hypoxia prior to the administering, wherein the administrating results in a decrease in the expression and/or activity of at least one HIFla pathway component in the at least one tumor of the subject, thereby reducing or inhibiting progression of the cancer.

[0042] Disclosed herein include kits. In some embodiments, the kit comprises: a PLK1 inhibitor; and a manual providing instructions for co-administrating the PLK1 inhibitor with a HIFla inhibitor to a subject for treating a cancer, wherein the cancer comprises at least one tumor with a high level of hypoxia prior to the co-administration.

Definitions

[0043] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. See e.g., Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, NY 1989). For purposes of the present disclosure, the following terms are defined below.

[0044] As used herein, the term “about” can mean plus or minus 5% of the provided value.

[0045] As used herein, a “subject” refers to an animal that is the object of treatment, observation or experiment. “Animals” include cold- and warm-blooded vertebrates and invertebrates such as fish, shellfish, reptiles and, in particular, mammals. “Mammal” includes, without limitation, mice; rats; rabbits; guinea pigs; dogs; cats; sheep; goats; cows; horses; primates, such as monkeys, chimpanzees, and apes, and, in particular, humans.

[0046] As used herein, a “patient” refers to a subject that is being treated by a medical professional, such as a Medical Doctor or a Doctor of Veterinary Medicine, to attempt to cure, or at least ameliorate the effects of, a particular disease or disorder or to prevent the disease or disorder from occurring in the first place. In some embodiments, the patient is an animal. In some embodiments, the patient is a mammal. In some embodiments, the patient is a human.

[0047] As used herein, “administration” or “administering” refers to a method of giving a dosage of a pharmaceutically active ingredient to a subject.

[0048] As used herein, a “dosage” refers to the amount of the active ingredients (e.g., PLK1 inhibitor (e.g., onvansertib) and/or HIF la inhibitor (e.g., bevacizumab or paclitaxel)).

[0049] As used herein, a “unit dosage” refers to an amount of therapeutic agent administered to a patient in a single dose.

[0050] As used herein, the term “daily dose” or “daily dosage” refers to a total amount of a pharmaceutical composition or a therapeutic agent that is to be taken within 24 hours.

[0051] As used herein, the term “delivery” refers to approaches, formulations, technologies, and systems for transporting a pharmaceutical composition or a therapeutic agent into the body of a patient as needed to safely achieve its desired therapeutic effect. In some embodiments, an effective amount of the composition or agent is formulated for delivery into the blood stream of a patient. In some embodiments, an effective amount of the composition or agent is formulated for intravenous administration. In some embodiments, an effective amount of the composition or agent is formulated for oral administration.

[0052] As used herein, the term “formulated” or “formulation” refers to the process in which different chemical substances, including one or more pharmaceutically active ingredients, are combined to produce a dosage form. In some embodiments, two or more pharmaceutically active ingredients can be co-formulated into a single dosage form or combined dosage unit, or formulated separately and subsequently combined into a combined dosage unit. A sustained release formulation is a formulation which is designed to slowly release a therapeutic agent in the body over an extended period of time, whereas an immediate release formulation is a formulation which is designed to quickly release a therapeutic agent in the body over a shortened period of time.

[0053] As used herein, the term “pharmaceutically acceptable” indicates that the indicated material does not have properties that would cause a reasonably prudent medical practitioner to avoid administration of the material to a patient, taking into consideration the disease or conditions to be treated and the respective route of administration. For example, it is commonly required that such a material be essentially sterile.

[0054] As used herein, the term “pharmaceutically acceptable carrier” refers to pharmaceutically acceptable materials, compositions or vehicles, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting any supplement or composition, or component thereof, from one organ, or portion of the body, to another organ, or portion of the body, or to deliver an agent to a diseased tissue or a tissue adjacent to the diseased tissue. Carriers or excipients can be used to produce compositions. The carriers or excipients can be chosen to facilitate administration of a drug or pro-drug. Examples of carriers include calcium carbonate, calcium phosphate, various sugars such as lactose, glucose, or sucrose, or types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols and physiologically compatible solvents. Examples of physiologically compatible solvents include sterile solutions of water for injection (WFI), saline solution, and dextrose.

[0055] As used herein, the term “pharmaceutically acceptable salt” refers to any acid or base addition salt whose counter-ions are non-toxic to the patient in pharmaceutical doses of the salts. A host of pharmaceutically acceptable salts are well known in the pharmaceutical field. If pharmaceutically acceptable salts of the compounds of this disclosure are utilized in these compositions, those salts are preferably derived from inorganic or organic acids and bases. Included among such acid salts are the following: acetate, adipate, alginate, aspartate, benzoate, benzene sulfonate, bisulfate, butyrate, citrate, camphorate, camphor sulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, lucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3 -phenyl -propionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, undecanoate, hydrohalides (e.g., hydrochlorides and hydrobromides), sulphates, phosphates, nitrates, sulphamates, malonates, salicylates, methylene-bis-b-hydroxynaphthoates, gentisates, isethionates, di-p- toluoyltartrates, ethanesulphonates, cyclohexylsulphamates, quinates, and the like. Pharmaceutically acceptable base addition salts include, without limitation, those derived from alkali or alkaline earth metal bases or conventional organic bases, such as triethylamine, pyridine, piperidine, morpholine, N-methylmorpholine, ammonium salts, alkali metal salts, such as sodium and potassium salts, alkaline earth metal salts, such as calcium and magnesium salts, salts with organic bases, such as dicyclohexylamine salts, N-methyl-D-glucamine, and salts with amino acids such as arginine, lysine, and so forth.

[0056] As used herein, the term “hydrate” refers to a complex formed by combination of water molecules with molecules or ions of the solute. As used herein, the term “solvate” refers to a complex formed by combination of solvent molecules with molecules or ions of the solute. The solvent can be an organic compound, an inorganic compound, or a mixture of both. Solvate is meant to include hydrate, hemi-hydrate, channel hydrate etc. Some examples of solvents include, but are not limited to, methanol, N,N-dimethylformamide, tetrahydrofuran, dimethylsulfoxide, and water.

[0057] As used herein, “therapeutically effective amount” or “pharmaceutically effective amount” refers to an amount of therapeutic agent, which has a therapeutic effect. The dosages of a pharmaceutically active ingredient which are useful in treatment when administered alone or in combination with one or more additional therapeutic agents are therapeutically effective amounts. Thus, as used herein, a therapeutically effective amount refers to an amount of therapeutic agent which produces the desired therapeutic effect as judged by clinical trial results and/or model animal studies. The therapeutically effective amount will vary depending on the compound, the disease, disorder or condition and its severity and the age, weight, etc., of the subject to be treated. The dosage can be conveniently administered, e.g., in divided doses up to four times a day or in sustained-release form.

[0058] As used herein, the term “treat,” “treatment,” or “treating,” refers to administering a therapeutic agent or pharmaceutical composition to a subject for prophylactic and/or therapeutic purposes. The term “prophylactic treatment” refers to treating a subject who does not yet exhibit symptoms of a disease or condition, but who is susceptible to, or otherwise at risk of, a particular disease or condition, whereby the treatment reduces the likelihood that the patient will develop the disease or condition. The term “therapeutic treatment” refers to administering treatment to a subject already suffering from a disease or condition. As used herein, a “therapeutic effect” relieves, to some extent, one or more of the symptoms of a disease or disorder. For example, a therapeutic effect may be observed by a reduction of the subjective discomfort that is communicated by a subject (e.g., reduced discomfort noted in self-administered patient questionnaire).

[0059] As used herein, the term “prophylaxis,” “prevent,” “preventing,” “prevention,” and grammatical variations thereof as used herein refers the preventive treatment of a subclinical disease-state in a subject, e.g., a mammal (including a human), for reducing the probability of the occurrence of a clinical disease-state. The method can partially or completely delay or preclude the onset or recurrence of a disorder or condition and/or one or more of its attendant symptoms or barring a subject from acquiring or reacquiring a disorder or condition or reducing a subject’s risk of acquiring or requiring a disorder or condition or one or more of its attendant symptoms. The subject is selected for preventative therapy based on factors that are known to increase risk of suffering a clinical disease state compared to the general population. “Prophylaxis” therapies can be divided into (a) primary prevention and (b) secondary prevention. Primary prevention is defined as treatment in a subject that has not yet presented with a clinical disease state, whereas secondary prevention is defined as preventing a second occurrence of the same or similar clinical disease state.

[0060] As used herein, each of the terms “partial response” and “partial remission” can refer to the amelioration of a cancerous state, as measured by, for example, tumor size and/or cancer marker levels, in response to a treatment. In some embodiments, a “partial response” means that a tumor or tumor-indicating blood marker has decreased in size or level by about 50% in response to a treatment. The treatment can be any treatment directed against cancer, including but not limited to, chemotherapy, radiation therapy, hormone therapy, surgery, cell or bone Marrow transplantation, and immunotherapy. The size of a tumor can be detected by clinical or by radiological means. Tumor-indicating markers can be detected by means well known to those of skill, e.g., ELISA or other antibody-based tests.

[0061] As used herein, each of the terms “complete response” or “complete remission” means that a cancerous state, as measured by, for example, tumor size and/or cancer marker levels, has disappeared following a treatment, including but are not limited to, chemotherapy, radiation therapy, hormone therapy, surgery, cell or bone marrow transplantation, and immunotherapy. The presence and/or size of a tumor can be detected by clinical or by radiological means. Tumorindicating markers can be detected by means well known to those of skill, e.g., ELISA or other antibody -based tests. A “complete response” does not necessarily indicate that the cancer has been cured, however, as a complete response can be followed by a relapse.

Hypoxia and cancer

[0062] Disclosed herein includes methods and kits for treating cancer. In some embodiments, the method comprises: administering a PLK1 inhibitor and a HIFla inhibitor to a subject with cancer, wherein the cancer comprises at least one tumor with a high level of hypoxia prior to the administering, wherein the administrating results in a decrease in the expression and/or activity of at least one HIFla pathway component in the at least one tumor of the subject, thereby reducing or inhibiting progression of the cancer. In some embodiments, the kit comprises: a PLK1 inhibitor; and a manual providing instructions for co-administrating the PLK1 inhibitor with a HIF la inhibitor to a subject for treating a cancer, wherein the cancer comprises at least one tumor with a high level of hypoxia prior to the co-administration.

[0063] Hypoxia is a non-physiological level of oxygen tension and a hallmark of a wide variety of solid tumors. Tumor-hypoxia leads to advanced but dysfunctional vascularization and acquisition of epithelial-to-mesenchymal transition phenotype resulting in cell mobility and metastasis. Hypoxia alters cancer cell metabolism and contributes to therapy resistance by inducing cell quiescence.

[0064] Solid tumors depend on neovascularization for maintained growth. In tumors, hypoxia arises due to a mismatch between oxygen consumption and delivery, often as a result of inadequate vascularization. To adapt and respond to hypoxic stress, tumor cells secrete a number of growth and angiogenic factors that stimulate endothelial cell proliferation and, ultimately, the formation of new tumor-associated blood vessels. Hypoxia Inducible transcription Factor (HIF) is a central regulator of the cellular response to hypoxia and genes encoding secreted growth and angiogenic factors, including vascular endothelial growth factor (VEGF), platelet derived growth factor (PDFG), transforming growth factor (TGFa) and angiopoetins are some of the many genes targeted by HIF.

[0065] HIF is composed of two subunits belonging to the bHLH-PAS family: HIF-la or HIF-2a and aryl hydrocarbon receptor nuclear translocator (ARNT also known as HIF-ip). To induce transactivation of target genes, HIF1 a-subunits dimerize with HIF-ip and bind to consensus sequences on DNA (hypoxia responsive element, HRE) in the promoter or enhancer regions of these genes. In contrast, HIF dimers containing HIF3a subunits are not transcriptionally active and HIF3a isoforms may act as dominant negative regulators. HIF modulates the expression of many genes whose products are critical to many aspects of tumor progression, including metabolic adaptation, apoptosis resistance, angiogenesis and metastasis. These include, but are not limited to, vascular endothelial growth factor (VEGF), erythopoietin, gluocose transportes, glycolytic enzymes and tyrosine hydroxylase. Additional HIF regulated or related genes are Hk2, Pfkfb3, Slc2al, Slc2a3, Cxcr4, Plin2, Adm, Bnip3, Lep, Rora, Ndrgl, Egln3, Mt3, Plod2, Hilpda, Angptl4. In some embodiments, the methods, compositions or kits disclosed herein inhibit expression level of HIF (e.g., HIF-la) and/or a HIF regulated or related gene (e.g., Hk2, Slc2a3, Adm or VEGF gene). In some embodiments, the combination of PLK1 inhibitor (e.g., onvansertib) and the HIFla inhibitor reduces expression level of HIF (e.g., HIF-la) and/or a HIF regulated or related gene (e.g., Hk2, Slc2a3, Adm or VEGF gene) by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or a number or range between any two of these values.

[0066] In normoxia, HIF is hydroxylated and interacts with the von Hippel Lindau protein (pVHL), an E3 ubiquitin subunit that targets HIF for degradation. In the absence of oxygen, HIF hydroxylation is inhibited, preventing binding of pVHL and leading to its intracellular accumulation. HIF-2a upregulation is found predominately in cancers with VHL gene mutations.

[0067] Tumor hypoxia can increase cancer progression (e.g., malignant progression and metastasis) by promoting angiogenesis through the induction of proangiogenic proteins such as VEGF. The inhibition of HIF-mediated gene regulation can reduce tumor angiogenesis and prevent the adaptative metabolic response to hypoxia, thus suppressing tumor growth. As used herein, the term “HIF inhibitor” refers to a compound, pharmaceutically acceptable salt, prodrug, or derivative thereof that inhibits the biological activity of HIF 1, HIF2 and HIF3; interferes with HIF1, HIF2, and HIF3, signal transduction pathway; or down regulates expression or availability of HIF 1, HIF2, and HIF3 in a cell or organism. A HIF inhibitor can act to inhibit the cellular production or activity of HIF by cells. In some embodiments, the HIF inhibitors inhibit HIF la expression or activity by decreasing HIF la protein levels, and/or inhibiting HIF la mRNA translation.

[0068] Provided herein are methods for treating cancer. In some embodiments, the method comprises: administering a PLK1 inhibitor and a HIF la inhibitor to a subject with cancer, wherein the cancer comprises at least one tumor with a high level of hypoxia prior to the administering, wherein the administrating results in a decrease in the expression and/or activity of at least one HIF la pathway component in the at least one tumor of the subject, thereby reducing or inhibiting progression of the cancer.

[0069] The at least one HIF la pathway component can be HIF la, BNIP3, PFKFB4, P4HA1, ANKZF1, INSIG2, ENO2, LDHA, ALDOC, PGK1, HK1, PFKP, P4HA2, KLF6, EFNA3, NFIL3, AK4, ERRFI1, SAP30, CCNG2, ANKRD37, KDM3A, ADM, DDIT4, HK2, SLC2A3, STC2, VEGF A, WSB1, PDK1, GLUT1, GLUT3, or HK2. The expression of HIFla, or any HIFla pathway component, can be reduced in the at least one tumor by at least 25% (e.g., 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%,

42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,

59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,

76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,

93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values) following administration of the PLK1 inhibitor, the HIFla inhibitor, or both, e.g., as compared to the subject prior to the administration. [0070] In some embodiments, administering the PLK1 inhibitor and the HIFla inhibitor synergistically reduces HIFla expression in the at least one tumor, e.g., as compared to the subject prior to the administration, a subject treated with the PLK1 inhibitor alone, or a subject treated with the HIFla inhibitor alone. In some embodiments, administering the PLK1 inhibitor and the HIFla inhibitor synergistically reduces HIFla expression in the at least one tumor by at least 50% (50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values), e.g., as compared to the subject prior to the administration.

[0071] In some embodiments, the cancer comprises at least one tumor that is hypoxic. In some embodiments, a hypoxic tumor comprises cells having a higher level of hypoxia, e.g., as compared to non-hypoxic cells (e.g., normoxic cells) or noncancerous cells. In some embodiments, the oxygenation status of cells or tissue (or a cancer) can be defined by a partial pressure of oxygen measurement in the cells or tissue. In some embodiments, “hypoxic” cells or tissue will have a lower partial pressure of oxygen level than “non-hypoxic” cells or tissue. A “normoxic” or “non-hypoxic” partial pressure can be different for different tissue types. Partial pressures can also vary, e.g., depending on the time of measurement, and other factors. Thus, in some embodiments, the partial pressure is a median partial pressure value. In some embodiments, the at least one tumor is hypoxic relative to, e.g., non-cancerous cells of the same tissue type. In some embodiments, normoxic cells or tissues comprise a partial pressure (e.g., a median partial pressure) of at least 20 mmHg. In some embodiments, normoxic cells or tissues comprise a partial pressure (e.g., a median partial pressure) of about 65 mmHg. In some embodiments, normoxic cells or tissues comprise a partial pressure (e.g., a median partial pressure) of at least 20 mmHg to at least 70 mmHg (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 or a number or a range between any two of these values). The high level of hypoxia can comprise a partial pressure of oxygen (pCh) level (e.g., a median partial pressure) of about 2 mmHg to about 25 mmHg in or near the at least one tumor (e.g., in tumor cells and/or the tumor microenvironment). The high level of hypoxia can comprise a pCh level of about 10 mmHg in or near the at least one tumor. The high level of hypoxia can comprise a pCh level of less than 10 mmHg in or near the at least one tumor.

[0072] In some embodiments, hypoxia status is determined by measuring the expression of one or more markers of hypoxia. The high level of hypoxia can comprise at least 10% of the cells (e.g., 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values) of the at least one tumor being positive for one or more markers for hypoxia. The cells can comprise a biopsy sample. The one or more markers of hypoxia can be HIFla, glucose transporter 1 (GLUT1), and/or carbonic anhydrase 9 (CA IX). The at least one tumor can comprise cells over-expressing one or more HIFla target genes relative to non-cancerous and/or non-hypoxic cells prior to the administration. In some embodiments, the one or more HIF 1 a target genes is: BNIP3L, PFKFB4, P4HA1, ANKZF1, INSIG2, EN02, I.DHA, ALDOC, PGK1, HK1, PFKP, P4HA2, KLF6, EFNA3, NFIL3, AK4, ERRFI1, SAP 30, CCNG2, ANKRD37, KDM3A, ADM, DDIT4, HK2, SLC2A3, STC2, VEGFA, WSB1, and/or PDKL The expression of one or more markers of hypoxia can be measured by a method described herein (e.g., immunohistochemistry), or by any method known in the art.

[0073] Several cellular pathways have also been shown to promote hypoxia when, e.g., disrupted. The at least one tumor can comprise cells comprising a mutation in a gene involved in mitochondrial metabolism. The gene can be SDH, FH, or ND2. The at least one tumor can comprise cells comprising elevated nitric oxide synthase levels, e.g., as compared to non- cancerous or non-hypoxic cells. The at least one tumor can comprise cells comprising elevated NAD+ levels, e.g., as compared to non-cancerous or non-hypoxic cells. The at least one tumor can comprise cells comprising a mutation in a gene causing dysregulation of the mTOR pathway. The gene can comprise LKB1, PML, PTEN, or TSC1/2.

[0074] The method can comprise determining a hypoxia status of the cancer prior to the administration, after the administration, or both. In some embodiments, determining the hypoxia status of the cancer comprises electron paramagnetic resonance (EPR) oximetry, Eppendorf electrode technique, optical methods, NMR relaxation techniques, or any combination thereof. In some embodiments, determining the hypoxia status of the cancer comprises an indirect method comprising: i) PET-based measurement of hypoxia-localizing drugs; ii) measurement of anaerobic and/or aerobic glycolysis; iii) detecting the presence, absence, or amount of one or more markers of hypoxia, (the one or more markers can comprise HIFla, GLUT1, and/or CA IX); and/or iv) measuring one or more physiological parameters in the subject, optionally selected from MRI perfusion, laser doppler flow, and lactate levels. Methods for determining hypoxia status and/or effect of treatment

[0075] In some embodiments, the administrating (e.g., a PLK1 inhibitor and a HIFla inhibitor) results in a decrease in the expression and/or activity of at least one HIFla pathway component in the at least one tumor of the subject, thereby reducing or inhibiting progression of the cancer.

[0076] The at least one HIFla pathway component can be HIFla, BNIP3, PFKFB4, P4HA1, ANKZF1, INSIG2, ENO2, LDHA, ALDOC, PGK1, HK1, PFKP, P4HA2, KLF6, EFNA3, NFIL3, AK4, ERRFI1, SAP30, CCNG2, ANKRD37, KDM3A, ADM, DDIT4, HK2, SLC2A3, STC2, VEGFA, WSB1, PDK1, GLUT1, GLUT3, or HK2. The expression of HIFla, or any HIFla pathway component, can be reduced in the at least one tumor by at least 25% (e.g., 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values) following administration of the PLK1 inhibitor, the HIFla inhibitor, or both, e.g., as compared to the subject prior to the administration.

[0077] In some embodiments, administering the PLK1 inhibitor and the HIFla inhibitor synergistically reduces HIFla expression in the at least one tumor, e.g., as compared to the subject prior to the administration, a subject treated with the PLK1 inhibitor alone, or a subject treated with the HIFla inhibitor alone. In some embodiments, administering the PLK1 inhibitor and the HIFla inhibitor synergistically reduces HIFla expression in the at least one tumor by at least 50% (50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values), e.g., as compared to the subject prior to the administration.

[0078] The method can comprise determining a hypoxia status of the cancer prior to the administration, after the administration, or both. In some embodiments, determining the hypoxia status of the cancer comprises electron paramagnetic resonance (EPR) oximetry, eppendorf electrode technique, optical methods, NMR relaxation techniques, or any combination thereof. In some embodiments, determining the hypoxia status of the cancer comprises an indirect method comprising: i) PET-based measurement of hypoxia-localizing drugs; ii) measurement of anaerobic and/or aerobic glycolysis; iii) detecting the presence, absence, or amount of one or more markers of hypoxia, (the one or more markers can comprise HIFla, GLUT1, and/or CA IX); and/or iv) measuring one or more physiological parameters in the subject, optionally selected from MRI perfusion, laser doppler flow, and lactate levels.

[0079] Methods for determining hypoxia status of a cancer are known in the art. Exemplary methods for determining hypoxia status are described in more detail below.

Hypoxia markers and other indirect methods

[0080] The activity or level of a marker of hypoxia can be detected and/or quantified by detecting or quantifying the expressed polypeptide and/or transcript, e.g., from a biopsy or other sample obtained from a subject. The polypeptide can be detected and quantified by any of a number of means well known to those of skill in the art. Any method known in the art for detecting polypeptides can be used. Such methods include, but are not limited to, immunodiffusion, immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, Western blotting, binder-ligand assays, immunohistochemical techniques, agglutination, complement assays, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, and the like.

[0081] Described below are non-limiting examples of techniques that may be used to detect a marker protein according to a practitioner's preference based upon the present disclosure. One such technique is Western blotting, wherein a suitably treated sample is run on an SDS-PAGE gel before being transferred to a solid support, such as a nitrocellulose filter. Anti-marker protein antibodies (unlabeled) are then brought into contact with the support and assayed by a secondary immunological reagent, such as labeled protein A or anti-immunoglobulin (suitable labels including¹²⁵I, horseradish peroxidase, alkaline phosphatase, fluorophore). Chromatographic detection may also be used.

[0082] Immunohistochemistry may be used to detect expression of marker protein. A suitable antibody is brought into contact with, for example, a thin layer of cells, washed, and then contacted with a second, labeled antibody. Labeling may be by fluorescent markers, enzymes, such as peroxidase, avidin, or radiolabelling. The assay can be scored visually, using microscopy. In some embodiments, immunohistochemistry is performed, e.g., on a biopsy sample obtained from a subject and contacted with an antibody capable of binding to HIFla.

[0083] Anti-marker protein antibodies, such as intrabodies, may also be used for imaging purposes, for example, to detect the presence of marker protein in cells or, e.g., an EP structure. Suitable labels include radioisotopes, iodine (¹²⁵I,¹²¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (¹¹²In), and technetium ("mTc), fluorescent labels, such as fluorescein and rhodamine, and biotin.

[0084] Antibodies that may be used to detect marker protein include any antibody, whether natural or synthetic, full length or a fragment thereof, monoclonal or polyclonal, that binds sufficiently strongly and specifically to the marker protein to be detected. An antibody may have a Ka of at most about 10⁶M, 10⁷M, 10⁸M, 10⁹M, 10^l()M, 10 "M, 10^{l 2}M. The phrase “specifically binds” refers to binding of, for example, an antibody to an epitope or antigen or antigenic determinant in such a manner that binding can be displaced or competed with a second preparation of identical or similar epitope, antigen or antigenic determinant. An antibody may bind preferentially to the marker protein relative to other proteins, such as related proteins. Antibodies are commercially available or may be prepared according to methods known in the art.

[0085] Antibodies and derivatives thereof that may be used encompass polyclonal or monoclonal antibodies, chimeric, human, humanized, primatized (CDR-grafted), veneered or single-chain antibodies as well as functional fragments, i.e., marker protein binding fragments, of antibodies. For example, antibody fragments capable of binding to a marker protein or portions thereof, including, but not limited to, Fv, Fab, Fab' and F(ab') 2 fragments can be used. Such fragments can be produced by enzymatic cleavage or by recombinant techniques. For example, papain or pepsin cleavage can generate Fab or F(ab') 2 fragments, respectively. Other proteases with the requisite substrate specificity can also be used to generate Fab or F(ab') 2 fragments. Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons have been introduced upstream of the natural stop site. For example, a chimeric gene encoding a F(ab') 2 heavy chain portion can be designed to include DNA sequences encoding the CH, domain and hinge region of the heavy chain. In some embodiments, agents that specifically bind to a marker protein other than antibodies are used, such as peptides. Peptides that specifically bind to a marker protein can be identified by any means known in the art. For example, specific peptide binders of a marker protein can be screened for using peptide phage display libraries.

[0086] In some embodiments, the expression of a gene (e.g., RNA transcript levels) of one or more HIFla target genes (or other HIFla pathway component) is measured. Any method known in the art can be used to measure the expression of genes regulated by HIFla. In general, methods of gene expression profiling can be divided into two large groups: methods based on hybridization analysis of polynucleotides, and methods based on sequencing of polynucleotides. Methods known in the art for the quantification of mRNA expression in a sample include northern blotting and in situ hybridization, RNAse protection assays, and reverse transcription polymerase chain reaction (RT-PCR). Alternatively, antibodies are employed that recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Representative methods for sequencing-based gene expression analysis include Serial Analysis of Gene Expression (SAGE), and gene expression analysis by massively parallel signature sequencing (MPSS). For example, RT-PCR is used to compare mRNA levels in different sample populations, in normal and tumor tissues, with or without drug treatment, to characterize patterns of gene expression, to discriminate between closely related mRNAs, and/or to analyze RNA structure.

[0087] In some cases, a first step in gene expression profiling by RT-PCR is the reverse transcription of the RNA template into cDNA, followed by amplification in a PCR reaction. For example, extracted RNA is reverse-transcribed using a GeneAmp RNA PCR kit (Perkin Elmer, Calif., USA), following the manufacturer's instructions. The cDNA is then used as template in a subsequent PCR amplification and quantitative analysis using, for example, a TaqMan assay (Life Technologies, Inc., Grand Island, N.Y.) or other probe-based assay.

[0088] In certain embodiments, sequencing may be performed by any method known in the art, for example, methods of high-throughput sequencing, also known as next generation sequencing. In some embodiments, a nucleic acid target molecule is labeled with a barcode (for example, an origin-specific barcode) and can be sequenced with the barcode to produce a single read and/or contig containing the sequence, or portions thereof, of both the target molecule and the barcode. Exemplary next generation sequencing technologies include, for example, Illumina sequencing, Ion Torrent sequencing, 454 sequencing, SOLiD sequencing, and nanopore sequencing amongst others. Methods for constructing sequencing libraries and performing and analyzing next-generation sequencing runs are known in the art.

[0089] As discussed above, endogenous hypoxia markers (e.g., HIF-la, GLUT-1, CA IX) are often used to judge the oxygenation status of malignant tumors. Responses to low oxygen levels in normal and cancer cells are mainly initiated by HIF-la, a key regulator for genes responsible for mammalian oxygen homeostasis. These responses (among others) include an increased erythropoiesis, angiogenesis, and glycolysis. The latter is mainly initiated by an enhanced cellular glucose uptake through GLUT-1 transporters.

[0090] There is also a group of physiological measurements that are sometimes indirectly linked to O2. Measurements of blood flow, for example, MRI perfusion and laser Doppler flow are often used clinically to obtain a parameter that can be linked to the supply of 02. Because of the many factors that affect how much O2 is delivered and the impact of utilization on the amount of O2 is available, these techniques may, in some embodiments, not by themselves provide reliable insights into the oxygenation status of the tissues. Measurements of metabolites that may be affected by O2 levels, for example, lactate and redox intermediates, are frequently used. These physiological measurements, especially if repeated and related to appropriate other parameters, may in some instances provide useful clinical information. In vivo oxygen measurement

[0091] In some embodiments, obtaining or measuring hypoxia in, e.g., the at least one tumor comprises obtaining a median partial pressure of oxygen (pCh) level. In some embodiments, a non-hypoxic cell or tissue can have a pCh (or median pCh) greater than 10 mmHg. In some embodiments, a non-hypoxic cell or tissue can have a pCh (or median pCh) of about 20 mmHg to about 70 mmHg. Exemplary methods for in vivo pCh measurement are described further below.

[0092] Using appropriate particulate paramagnetic materials, EPR oximetry can provide direct measurements of O2, that is, the EPR signal is directly proportional to the amount of O2 Because each multisite sensor senses a volume that is much larger than capillary networks, these techniques provide a volume averaged sampling of all compartments within the tissues. The time resolution of the techniques can be milliseconds or shorter.

[0093] The measured parameter of an EPR spectrum that indicates the amount of O2 present is the line width of the observed resonance peak. There usually is a fixed relationship between the line width and the amount of O2, with the relationship being specific for each type of paramagnetic material, for example, carbon, charcoal, or phthalocyanine particulates. Using particulate oximetric materials, measurements can be continuous over any span of time and can be repeated indefinitely. The method requires that the sensing material be injected or implanted in one or more regions of interest, but thereafter all measurements can be made entirely noninvasively. Importantly the measurements can be carried out in a clinical setting and can fit into the workflow needed for patient care.

[0094] The initial clinical EPR measurements of oxygen in tissues have used India Ink as the oxygen sensor. The carbon particles are the components that respond to oxygen. After injection of 30-50 pl of the suspension through a small needle, the carbon particles disperse nonuniformly through the local region as small extracellular aggregates. They are often engulfed by macrophages. The resulting EPR spectra in the region probed by the resonator (i.e., the surface coil used for signal detection) are a composite of the oxygen-dependent line widths from each of the particles. Another method of clinical EPR oximetry is based on the use of micro-crystalline probes (e.g., LiPc, LiNc-BuO), encapsulated in biocompatible polymers. Clinical measurements currently are being performed using the “OxyChip” which consists of oxygen sensitive microcrystals of lithium octa-n-butoxynaphthalocyanine (LiNc-BuO) embedded in polydimethylsiloxane.

[0095] The Eppendorf electrode has been used clinically to provide direct tissue pO2 measurements along electrode tracks. To create a track, a series of points is obtained by progressing the 200 pm microelectrode through the tissue in a sequence of “Pilgrim steps,” that is, the probe is advanced a prescribed distance and then withdrawn a fraction of that distance to minimize pressure effects. The volume sampled by each point is estimated to be 100-500 cells around the tip of the probe. Therefore, the measurement usually reflects the average of a range of pO2 values, especially in the presence of pathophysiology. The histogram of values of a tumor was thought to be a representative sample of three to seven tracks through the volume, although this still was only a sampling of the true heterogeneity of the tissue O2. Nevertheless, in tumors, very useful clinical correlations have been found with the number of points below a threshold value, for example, median pCh or hypoxic fractions. Note that this does not require individual, true absolute pCh values. Instead, the separation between severely hypoxic and less hypoxic values relies on averaged oxygen tensions. When used with multiple point measurements (>70), this is a good but not infallible technique to determine the presence of hypoxic regions. In some embodiments, optical methods can be used. For example, the OxyLite technology is based on quenching by O2 of fluorescence with a sensor whose diameter is 230-750 pm at the end of a fiber-optic fiber. This cross-section corresponds to the diameter of a tissue subvolume of 10-75 mammalian cells. And, therefore, the sensor will provide an average of the distribution of pCh values throughout this volume. The temporal resolution can be quite rapid. It may be used in a manner similar to the Eppendorf electrode to obtain a series of similarly averaged measurements at different locations.

[0096] Direct injection of phosphorescent agents can provide one way to directly sample tissue O2, where the signal comes from the phosphorescence lifetime changes that result from excited triplet state quenching of O2. High resolution mapping with very fast time resolution can be provided, although care must be taken in choosing the probe that localizes in the compartments of interest, as some provide intracellular information, some provide purely extracellular information, and some are simply perivascular in nature. A key part of advancing these molecular probes has been to ensure that they sample the oxygen in the environment with a buffer around them such that the oxygen diffuses into the sensor and any measurement of oxygen by the triplet state quenching does not alter the local oxygen level, making the signal potentially nonlinear. Many unprotected or bare nanoparticle probes can have a signal dependence which is not ideal for linearity with oxygen or repeated measurements, or have uncertain localization. Dendrimer particles of the Oxyphor complex have been used as biocompatible large particles with pegylation to the exterior to ensure a known biodistribution. Their localization is largely extracellular as well, but of course the localization can be tailored by specifically altering the surface chemistry, and they have been used to create probes for many unique biological environments such as the gut, bone marrow, or brain.

[0097] Another method for oxygen measurement in vivo is through delayed phosphorescence from protoporphyrin IX, which is present in all tissues to some extent, produced in the mitochondria, and can be proactively enhanced by administration of aminolevulinic acid. This molecule has triplet state quenching by oxygen as well, and the reverse intersystem crossing that can occur in the absence of oxygen allows for a delayed fluorescence signal which is uniquely sensitive to the local oxygen environment.

[0098] Photoacoustic tomography (PAT) is another optical technique that is becoming increasingly used to define perfusion and O2 saturation in preclinical cancer and investigational studies of breast tumors, with ongoing commercial development for broader clinical use in a number of ventures. The method relies on optical absorption of a pulsed laser source by chromophores in the tissue, including hemoglobin, to produce ultrasound waves which are detected at the surface. The major benefits of PAT in lesion imaging are high resolution for neovasculature imaging through ~l-2 cm of tissue, and the ability to image features of blood oxygen saturation with spectroscopic PAT. Quantitation of tissue oxygenation using PAT remains difficult because an indirect indicator of oxygenation is measured (i.e., blood oxygen saturation) and unknown tissue properties skew the light penetration in tissue, making accurate spectroscopic measurements challenging at nonsuperficial depths.

[0099] Another method comprises NMR relaxation methods. Based on the principle that molecular O2 impacts the relaxation time of nuclei, several different approaches have been developed to try to use the power of NMR to measure O2 in tissues. The measurement of O2 by NMR using isotopes with spin (e.g., O¹⁷ for NMR) which is measured when the O2 is incorporated into water and O¹⁵ for PET is possible, but these can be extraordinarily expensive and may not be suitable for routine clinical use. The most developed method has been the use of the relaxation time of fluorine nuclei in fluorinated hydrocarbons injected directly into tissues. The measurements are based on the relaxation time of the F atoms in the emulsion, but the volume of the fluorine containing hydrocarbon is much larger than several capillary networks. Consequently, the data obtained are an average of a range of partial pressures.

[0100] Indirect methods of assessing oxygen in tissues can also be employed. There are a number of methods used clinically that can provide indications of O2 levels in tissues, which are based on plausible but indirect relationships to the actual O2 levels. While these therefore intrinsically do not directly measure O2 in tissues, because of their widespread availability, they are sometimes considered to provide direct measurements of O2.

[0101] The use of molecules such as the nitroimidazoles and Cu-ATSM that selectively localize in hypoxic tissues (O2 content <1%) has been widely employed, most often using PET labels to indicate their location. The principle is that with critically low levels of O2 these can be reduced to reactive intermediates which, if not reoxidized by O2, can then bind to cellular components. These methods can provide a qualitative indication of regions with moderate to good perfusion that were hypoxic at the time that the tracer was delivered. Another widely used PET clinical imaging technique is based on the observation that many tumors have a high rate of anaerobic and aerobic glycolysis and therefore a high uptake of glucose, which in turn can be followed by using the imaging agent 18F-FDG (18F -fluorodeoxy glucose). The amount of uptake is based on a number of different factors including perfusion rate, extent of hypoxia, expression of the Warburg effect (i.e., aerobic glycolysis), number of cells, uptake of glucose analogs and rate of glycolysis. Clinical use of this technique is widespread and considered clinically useful for a number of contexts, especially in identifying regions where metabolically active tumor cells are located. Some clinicians even consider areas of high uptake as potentially indicating tumor hypoxia although the Warburg effect is mainly responsible for the high glucose uptake rates. A major complication is the high rate of false positives, due to many other causes of uptake of the agent. There can be very significant discrepancies between the pattern of localization of FDG and the histological evidence of hypoxia.

Cancers

[0102] The methods, compositions and kits disclosed herein can be used to treat various types of cancer, including but not limited to, melanoma (e.g., metastatic malignant melanoma), renal cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone refractory prostate adenocarcinoma), pancreatic adenocarcinoma, breast cancer, colon cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC) and small-cell lung cancer (SCLC)), esophageal cancer, squamous cell carcinoma of the head and neck, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma, and other neoplastic malignancies. Additionally, the disease or condition provided herein includes refractory or recurrent malignancies whose growth may be inhibited using the methods and compositions disclosed herein. In some embodiments, the cancer is carcinoma, squamous carcinoma, adenocarcinoma, sarcomata, endometrial cancer, breast cancer, ovarian cancer, cervical cancer, fallopian tube cancer, primary peritoneal cancer, colon cancer, colorectal cancer, squamous cell carcinoma of the anogenital region, melanoma, renal cell carcinoma, lung cancer, non-small cell lung cancer, squamous cell carcinoma of the lung, stomach cancer, bladder cancer, gall bladder cancer, liver cancer, thyroid cancer, laryngeal cancer, salivary gland cancer, esophageal cancer, head and neck cancer, glioblastoma, glioma, squamous cell carcinoma of the head and neck, prostate cancer, pancreatic cancer, mesothelioma, sarcoma, hematological cancer, leukemia, lymphoma, neuroma, or a combination thereof. In some embodiments, the cancer is carcinoma, squamous carcinoma (e.g., cervical canal, eyelid, tunica conjunctiva, vagina, lung, oral cavity, skin, urinary bladder, tongue, larynx, and gullet), and adenocarcinoma (e.g., prostate, small intestine, endometrium, cervical canal, large intestine, lung, pancreas, gullet, rectum, uterus, stomach, mammary gland, and ovary). In some embodiments, the cancer is sarcomata (e.g., myogenic sarcoma), leukosis, neuroma, melanoma, and lymphoma.

[0103] The cancer can be a solid tumor, a liquid tumor, or a combination thereof. In some embodiments, the cancer is a solid tumor, including but not limited to, melanoma, renal cell carcinoma, lung cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, gall bladder cancer, laryngeal cancer, liver cancer, thyroid cancer, stomach cancer, salivary gland cancer, prostate cancer, pancreatic cancer, Merkel cell carcinoma, brain and central nervous system cancers, and any combination thereof. In some embodiments, the cancer is a liquid tumor. In some embodiments, the cancer is a hematological cancer. Non-limiting examples of hematological cancer include Diffuse large B cell lymphoma (“DLBCL”), Hodgkin's lymphoma (“HL”), NonHodgkin's lymphoma (“NHL”), Follicular lymphoma (“FL”), acute myeloid leukemia (“AML”), and Multiple myeloma (“MM”).

[0104] The cancer can be colorectal cancer, bladder cancer, breast cancer, kidney cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, stomach cancer, thyroid cancer, uterine cancer, renal cancer, cervical cancer, recurrent glioblastoma, head and neck cancer, soft tissue carcinoma, vulvar cancer, melanoma, brain cancer, liver cancer, bile duct cancer, or a combination thereof. The cancer can be KRAS-mutant cancer or ER+ cancer. The cancer can be KRAS-mutant colorectal cancer (mCRC) or ER+ breast cancer. In some embodiments, the cancer is a metastatic cancer. As used herein, “metastatic cancer” can refer to when a cancer spreads (metastasizes) from its original site to another area of the body. Virtually all cancers have the potential to spread this way. Whether metastases develop depends on the complex interaction of many tumor cell factors, including the type of cancer, the degree of maturity (differentiation) of the tumor cells, the location and how long the cancer has been present, as well as other incompletely understood factors. As used herein, the term “metastasis” can refer to formation of progressively growing secondary tumor foci at sites discontinuous from the primary lesion. The metastatic process is a multi-step mechanism in which a metastatic cancer cell escapes from the primary tumor, enters the circulation, invades a distant tissue site and grows into a macroscopic tumor at the target site. The metastatic cancer can be metastatic colorectal cancer, metastatic bladder cancer, metastatic breast cancer, metastatic kidney cancer, metastatic lung cancer, metastatic ovarian cancer, metastatic pancreatic cancer, metastatic prostate cancer, metastatic stomach cancer, metastatic thyroid cancer, metastatic uterine cancer, metastatic renal cancer, metastatic cervical cancer, metastatic recurrent glioblastoma, or a combination thereof.

[0105] The metastatic cancer can be metastatic colorectal cancer, metastatic bladder cancer, metastatic breast cancer, metastatic kidney cancer, metastatic lung cancer, metastatic ovarian cancer, metastatic pancreatic cancer, metastatic prostate cancer, metastatic stomach cancer, metastatic thyroid cancer, metastatic uterine cancer, metastatic renal cancer, metastatic cervical cancer, metastatic recurrent glioblastoma, metastatic head and neck cancer, metastatic soft tissue carcinoma, metastatic vulvar cancer, metastatic malignant melanoma, metastatic brain cancer, metastatic liver cancer, metastatic bladder cancer, metastatic bile duct cancer, or a combination thereof. The metastatic cancer can be KRAS-mutant cancer or ER+ cancer. The metastatic cancer can be KRAS-mutant metastatic colorectal cancer (mCRC) or ER+ breast cancer.

[0106] In some embodiments, the cancer (or metastatic cancer) is KRAS-mutant cancer. The KRAS gene (also known as Kirsten rat sarcoma viral oncogene homolog, KRAS Proto-Oncogene, GTPase, K-Ras, KRAS2) is a proto-oncogene that encodes a GTPase that is part of signal transduction pathways that regulate mitosis. Several mutations in KRAS activate the protein and are implicated in cancer such as acute myelogenous leukemia (AML), juvenile myelomonocytic leukemia (JMML), gastric cancer, colorectal cancer, pancreatic cancer and lung cancer. Tumors having KRAS mutations and resistant to treatment can emerge during treatment. This resistance is particularly common with anti-EGFR treatment of metastatic colorectal cancer (mCRC). Without being bound to any particular theory, PLK1 gene is among the genes identified to be necessary for KRAS-mutated tumor cells to drive tumor growth. It is believed that KRAS mutants are experiencing mitotic stress and exacerbating this stress in particular ways such that interference with PLK1 leads to stress overload and tumor cell death. KRAS mutants together with PLK1 inhibition can block the anaphase promoting complex (APC/C), which is crucial for mitosis to occur.

[0107] In some embodiments, the cancer is KRAS-mutant metastatic colorectal cancer (mCRC). KRAS-mutant mCRC represents -50% of mCRC patients and have poorer prognosis than RAS wild-type patients. First- and second-line treatments for mCRC include chemotherapy (e.g., FOLFIRI/FOLFOX) ± bevacizumab (Bev). Second-line regimens are available but have limited efficacy. For example, ORR of previous second-line regimens is 5%-10%, while the median PFS and median OS are -6 months and -12 months, respectively.

[0108] The cancer can be a breast cancer, e.g., an Estrogen Receptor (ER) positive (ER+) breast cancer. In some embodiments, the ER+ breast cancer is progesterone receptor negative (PR-). The ER+ breast cancer can be, e.g., HER2 negative. In some embodiments, the ER+ breast cancer has a histological or cytological profile with ER > 1%, 10%, 20%, or higher. In some embodiments, the subject with the ER+ breast cancer is resistant to or does not respond effectively to a hormone therapy, optionally the hormone therapy comprises a selective estrogen receptor degrader (SERD), a selective estrogen receptor modulator (SERM), an aromatase inhibitor, or a combination thereof. In some embodiments, the subject with the ER+ breast cancer is resistant to a kinase inhibitor, optionally the kinase inhibitor is a CDK inhibitor, further optionally the CDK inhibitor is a CDK 4/6 inhibitor. In some embodiments, the subject with the ER+ breast cancer develops stable disease, progressive disease or resistance to a CDK4/6 inhibitor and/or a SERD. In some embodiments, the subject with the ER+ breast cancer is resistant to palbociclib, fulvestrant, or both or develops stable or progressive disease following treatment with palbociclib, fulvestrant, or both.

[0109] In some embodiments, the at least one tumor of a subject comprises, for example, cells comprising a mutation in a gene or other genotype or phenotype that results in increased hypoxia signaling. The at least one tumor can comprise cells comprising a mutation in a gene involved in mitochondrial metabolism. The gene can be SDH, FH, or ND2. The at least one tumor can comprise cells comprising elevated nitric oxide synthase levels, e.g., as compared to non-cancerous or non-hypoxic cells. The at least one tumor can comprise cells comprising elevated NAD+ levels, e.g., as compared to non-cancerous or non-hypoxic cells. The at least one tumor can comprise cells comprising a mutation in a gene causing dysregulation of the mTOR pathway. The gene can comprise LKB1, PML, PTEN, or TSC1/2.

PLK1 Inhibitors

[0110] Polo-like kinases (PLK) are a family of five highly conserved serine/threonine protein kinases. PLK1 is a master regulator of mitosis and is involved in several steps of the cell cycle, including mitosis entry, centrosome maturation, bipolar spindle formation, chromosome separation, and cytokinesis. PLK1 has been shown to be overexpressed in solid tumors and hematologic malignancies, including AML. PLK1 inhibition induces G2-M-phase arrest with subsequent apoptosis in cancer cells, and has emerged as a promising targeted therapy. Several PLK inhibitors have been studied in clinical trials. In a randomized phase II study of patients with AML who were treatment naive yet unsuitable for induction therapy, the pan-PLK inhibitor, volasertib (BI6727), administered intravenously in combination with LDAC showed a significant increase in OS when compared with LDAC alone. A subsequent randomized phase III study identified no benefit of the combination and described an increased risk of severe infections. PLK1 facilitates HR during Double Strand DNA Break (DSB) Repair. PLK1 phosphorylates Rad51 and BRCA1, facilitating their recruitment to DSB sites and thereby HR-mediated DNA repair. The PLK1 inhibitor can be selective and/or specific for PLK1.

[OHl] The PLK1 inhibitor can be a dihydropteridinone, a pyridopyrimidine, a aminopyrimidine, a substituted thiazolidinone, a pteridine derivative, a dihydroimidazo[l,5- f]pteridine, a metasubstituted thiazolidinone, a benzyl styryl sulfone analogue, a stilbene derivative, or any combination thereof. The PLK1 inhibitor can be onvansertib, BI2536, Volasertib (BI 6727), GSK461364, AZD1775, CYC140, HMN-176, HMN-214, rigosertib (ON- 01910), MLN0905, TKM-080301, TAK-960, or Ro3280.

[0112] Onvansertib (also known as PCM-075, NMS-1286937, NMS-937, “compound of formula (I)” in US8,927,530, IUPAC name l-(2-hydroxyethyl)-8-{[5-(4-methylpiperazin-l- yl)-2-(trifluoromethoxy) phenyl] amino}-4,5-dihydro-lH-pyrazolo[4,3-h] quinazoline-3- carboxamide) is a highly selective ATP-competitive PLK1 inhibitor. Biochemical assays demonstrated high specificity of onvansertib for PLK1 among a panel of 296 kinases, including other PLK members. Onvansertib has potent in vitro and in vivo antitumor activity in models of both solid and hematologic malignancies. Onvansertib is the first PLK1 specific ATP competitive inhibitor administered by oral route to enter clinical trials with proven antitumor activity in different preclinical models. Onvansertib inhibited cell proliferation at nanomolar concentrations in AML cell lines and tumor growth in xenograft models of AML. Onvansertib also significantly increased cytarabine antitumor activity in disseminated models of AML.

Onvansertib

[0113] Onvansertib shows high potency in proliferation assays having low nanomolar activity on a large number of cell lines, both from solid as well as hematologic tumors. Onvansertib potently causes a mitotic cell-cycle arrest followed by apoptosis in cancer cell lines and inhibits xenograft tumor growth with a clear PLKl-related mechanism of action at well tolerated doses in mice after oral administration. In addition, onvansertib shows activity in combination therapy with approved cytotoxic drugs, such as irinotecan, in which there is enhanced tumor regression in HT29 human colon adenocarcinoma xenografts compared to each agent alone, and shows prolonged survival of animals in a disseminated model of AML in combination therapy with cytarabine. Onvansertib has favorable pharmacologic parameters and good oral bioavailability in rodent and nonrodent species, as well as proven antitumor activity in different nonclinical models using a variety of dosing regimens, which may potentially provide a high degree of flexibility in dosing schedules, warranting investigation in clinical settings. Onvansertib has several advantages over volasertib (BI6727, another PLK1 inhibitor), including a higher degree of potency and specificity for the PLK1 isozyme, and oral bioavailability. Therefore, onvansertib can be a promising therapeutic option for KRAS-mutant mCRC.

[0114] A phase I, first-in-human, dose-escalation study of onvansertib in patients with advanced/metastatic solid tumors identified neutropenia and thrombocytopenia as the primary dose-limiting toxicities. These hematologic toxicities were anticipated on the basis of the mechanism of action of the drug and were reversible, with recovery occurring within 3 weeks. The half-life of onvansertib was established between 20 and 30 hours. The oral bioavailability of onvansertib plus its short half-life provide the opportunity for convenient, controlled, and flexible dosing schedules with the potential to minimize toxicities and improve the therapeutic window. Pharmacodynamics and biomarker studies, including baseline genomic profiling, serial monitoring of mutant allele fractions in plasma, and the extent of PLK1 inhibition in circulating blasts, have been performed to identify biomarkers associated with clinical response and are described in WO2021/146322, the content of which is incorporated herein by reference in its entirety.

[0115] It was surprisingly found by the inventors that PLK1 inhibition can result in decrease the expression and/or activity of at least one HIFla pathway component, for example, decreased expression of HIFla in a cell.

HIFla inhibitors

[0116] As described herein, HIFla inhibitors can act one or more points in the HIF1 pathway. In some embodiments, the HIFla inhibitor inhibits HIFla mRNA expression, inhibits HIFla protein translation, modulates HIFla protein degradation, inhibits HIFla heterodimerization, inhibits HIFla DNA binding, inhibits HIFla transcriptional activity, or any combination thereof. The HIFla inhibitor can comprise a microRNA (miRNA), a precursor microRNA (pre-miRNA), a small interfering RNA (siRNA), a short-hairpin RNA (shRNA), an antibody, or a small molecule. In some embodiments, the HIFla inhibitor is selected from the group consisting of: EZN-2968; aminoflavone; topotecan; EZN-2208; a cardiac glycoside, e.g., digoxin or PX-478; an mTOR inhibitor, e.g., temsirolimus or everolimus; an HSP90 inhibitor, e.g., galdanamycin; a histone deactylase inhibitor; an antibiotic, e.g., a quinoxaline or anthracycline; chetomin; bevacizumab; paclitaxel; and bortezomib. In some embodiments, the HIFla inhibitor is an angiogenesis inhibitor or a chemotherapeutic agent, as described further below.

Angiogenesis Inhibitors

[0117] The HIFla inhibitor can be an angiogenesis inhibitor. [0118] Angiogenesis as used herein is the inappropriate formation of new blood vessels, and is typically required for cancer metastasis. “Angiogenesis” often occurs in tumors when endothelial cells secrete a group of growth factors that are mitogenic for endothelium causing the elongation and proliferation of endothelial cells which results in a generation of new blood vessels.

[0119] The inhibition of angiogenesis can cause tumor regression in animal models and has also been found to be effective in humans for treatment of metastatic cancer. The terms “anti-angiogenics” and “angiogenesis inhibitor” shall have their ordinary meaning, and can also be used interchangeably herein to refer to any agent that can inhibit angiogenesis. Several angiogenesis inhibitors have been approved for use in humans for the treatment of cancer. In some embodiments of the methods disclosed herein, the subject has not received any prior treatment comprising administration of an angiogenesis inhibitor.

[0120] The angiogenesis inhibitor can be capable of inhibiting VEGF-A, VEGFR-1, VEGFR-2, VEGFR-3, EGFR, HER2, PDGFR family proteins, RAF, Kit (or c-Kit), FLT3, CSF- 1R, RET, Abl, Itk, LcK, c-FMS, FGFR family proteins, c-Met, P1GF, TNF-a, IFNs, ILs, bFGF, mTOR, or any combination thereof.

[0121] The angiogenesis inhibitor can be Afatinib (Gilotrif®), Axitinib (Inlyta®), Bevacizumab (Avastin®), Cabozantinib (Cometriq®), Cetuximab (Erbitux®), Erlotinib (Tarceva®), Everolimus (Afinitor®), Gefitinib (Iressa®), Imatinib (Gleevec®), Lapatinib (Tykerb®), Lenalidomide (Revlimid®), Lenvatinib mesylate (Lenvima®), Necitumumab (Portrazza™), Neratinib (Nerlynx®), Panitumumab (Vectibix®), Pazopanib (Votrient®), Pertuzumab (Perjeta®), Ramucirumab (Cyramza®), Regorafenib (Stivarga®), Sorafenib (Nexavar®), Sunitinib (Sutent®), Thalidomide (Synovir, Thalomid®), Trastuzumab (Ontruzant®), Vandetanib (Caprelsa®), or Ziv- aflibercept (Zaltrap®).

[0122] Bevacizumab (also referred to herein as “bev”) can be used in combination with chemotherapy in both first and second lines of therapy for treating cancer. Other angiogenesis inhibitors, e.g., ramucirumab and aflibercept, can be used in second line setting. Prior to the presently disclosed method, overall survival (OS) and median progression free survival (mPFS) benefit in second line have been shown to be independent of whether bevacizumab was given in first line (e.g., the subject received prior treatment comprising inhibiting angiogenesis). For example, in studies examining OS and PFS in patients with and without prior bevacizumab treatment, it was found that OS was 13.9 months without prior bevacizumab vs. 12.5 months with prior bevacizumab. For PFS, mPFS was 6.9 months without prior bevacizumab vs. 6.7 months with prior bevacizumab. Anti-angiogenic therapies incrementally improve response rates in patients with prior bevacizumab vs without prior bevacizumab. The objective response rate of patients who had received prior bevacizumab is 5% to 13% as compared to -25% with no prior bevacizumab.

Chemotherapeutic agents

[0123] The HIFla inhibitor can be a chemotherapeutic agent. Chemotherapy involves treatments with one or more chemotherapeutic agents. These agents typically act to inhibit cell division (e.g., mitosis) and/or induce DNA damage to cancer cells. Chemotherapeutic agents can be, but are not limited to: alkylating agents, antimetabolites, topoisomerase inhibitors, mitotic inhibitors, and antitumor antibiotics. The mitotic inhibitor can be a microtubule polymerization or depolymerization inhibitor. Examples of specific chemotherapeutic agents are provided below. As provided herein, a chemotherapeutic agent (e.g., paclitaxel) in combination with a PLK1 inhibitor (e.g., onvansertib) can synergistically inhibit hypoxia signaling in cancer cells as evidence by, e.g., reduced HIFla protein expression.

[0124] Exemplary chemotherapeutic agents are described below. The chemotherapeutic agent can comprise an alkylating agent, an antimetabolite, a topoisomerase inhibitor, a mitotic inhibitor, or an antitumor antibiotic. The chemotherapeutic agent can be an alkylating agent (including nitrosoureas). Alkylating agents damage cell DNA to prevent cancer cells from dividing. Nitrosoureas are a particular type of alkylating agent. Unlike other alkylating agents, nitrosoureas can travel into the brain and kill cancer cells there. Nitrosoureas are used to treat some brain tumors. Exemplary alkylating agents include: Altretamine, Bendamustine, Busulfan, Carboplatin, Chlorambucil, Cisplatin, Cyclophosphamide, Dacarbazine, Ifosfamide, Meehl orethamine, Melphalan, Oxaliplatin, Procarbazine, Temozolomide, Thiotepa, and Trabectedin. Exemplary nitrosoureas include: Carmustine, Lomustine, and Streptozocin.

[0125] The chemotherapeutic agent can be an antimetabolite. Antimetabolites prevent cancer cells from making the genetic material they need to create new cells. Exemplary antimetabolites include: 5-fluorouracil, 6-mercaptopurine, Azacitidine, Capecitabine, Cladribine, Clofarabine, Cytarabine, Decitabine, Floxuridine, Fludarabine, Gemcitabine, Hydroxyurea, Methotrexate, Nelarabine, Pemetrexed, Pentostatin, Pralatrexate, Thioguanine, and Trifluridine/tipiracil combination.

[0126] The chemotherapeutic agent can be a topoisomerase inhibitor. Topoisomerase inhibitors inhibit DNA replication. Stopping this enzyme prevents cancer cells from multiplying and can also damage the cell DNA. Exemplary topoisomerase inhibitors include: Etoposide, Irinotecan, Irinotecan liposomal, Mitoxantrone (also classified as an antitumor antibiotic, see below), Teniposide, and Topotecan.

[0127] The chemotherapeutic agent can be a mitotic inhibitor (e.g., plant alkaloids). The mitotic inhibitor can be a microtubule polymerization or depolymerization inhibitor. Mitotic inhibitors are also called plant alkaloids because they are made of the same material plants use to protect against predators. Exemplary mitotic inhibitors include: Cabazitaxel, Docetaxel, Nab- paclitaxel, Paclitaxel, Vinblastine, Vincristine, Vincristine liposomal, and Vinorelbine.

[0128] The chemotherapeutic agent can be an antitumor antibiotic (including anthracy clines). Antitumor antibiotics prevent the cancer-cell DNA from replication. Sometimes, they induce DNA damage. Anthracyclines are a specific type of antitumor antibiotic. Exemplary anthracyclines include: Daunorubicin, Doxorubicin, Doxorubicin liposomal, Epirubicin, Idarubicin, Mitoxantrone, and Valrubicin. Other antitumor antibiotics include: Bleomycin, Dactinomycin, and Mitomycin-C.

[0129] Additional exemplary chemotherapeutic agents include, but are not limited to: All-trans-retinoic acid, Arsenic trioxide, Asparaginase, Eribulin, Ixabepilone, Mitotane, Omacetaxine, Pegaspargase, Procarbazine, Romidepsin, and Vorinostat. In some embodiments, the chemotherapeutic agent is a combination therapy comprising folinic acid, 5-fluorouracil, and irinotecan (e g., FOLFIRI).

Combination therapy

[0130] As disclosed herein, a combination therapy of a HIFla inhibitor and a PLK1 inhibitor (including onvansertib) can surprisingly result in significantly enhanced efficacy against cancer in a subject causing tumor regression and cancer survival. In some embodiments, the subject has not received prior treatment comprising inhibiting hypoxia signaling. The resulting tumor regression and cancer survival rate/duration by the combination can be surprisingly synergistic (i.e., more than additive, superior to the cumulated anti-tumor efficacy caused by the HIFla inhibitor and the PLK1 inhibitor separately). The PLK1 inhibitor can be onvansertib. Provided herein include methods, compositions and kits for treating cancer in a subject (e.g., a human patient suffering from cancer). The method comprises administrating a HIFla inhibitor and a PLK1 inhibitor to the patient in a manner sufficient to inhibit progression of the cancer. For example, the HIFla inhibitor and the PLK1 inhibitor can be administrated to a subject with cancer simultaneously, separately, or sequentially.

[0131] In some embodiments, administering the PLK1 inhibitor and the HIFla inhibitor synergistically reduces or inhibits progression of the cancer relative to the PLK1 inhibitor treatment alone, the HIFla inhibitor treatment alone, and/or the additive effect of the PLK1 inhibitor treatment alone and the HIFla inhibitor treatment alone. In some embodiments, the inhibition or reduction of cancer progression is not merely additive, but is enhanced or synergistic (that is, the inhibition is greater than the combined inhibition of progression caused by the HIFla inhibitor alone plus the PLK1 inhibitor alone). The enhanced or synergistic efficacy or inhibition of any combination of a HIFla inhibitor and a PLK1 inhibitor of the present disclosure can be different in different embodiments. In some embodiments, the enhanced or synergistic efficacy or inhibition of any combination of a HIFla inhibitor and a PLK1 inhibitor of the present disclosure is, is about, is at least, is at least about, is at most, or is at most about, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, or a number or a range between any two of these values, higher than the combined inhibition of progression caused by the HIFla inhibitor alone plus the PLK1 inhibitor alone.

[0132] In some embodiments, administering the PLK1 inhibitor and the HIFla inhibitor improves one or more therapeutic effects in the subject relative to a control or a baseline. The one or more therapeutic effects can comprise size of a tumor derived from the cancer, objective response rate (ORR), duration of response (DOR), time to response, progression free survival (PFS), overall survival (OS), disease control rate (DCR), oncogenic allelic burden, and/or expression level of CD31. In some embodiments, administering the PLK1 inhibitor and the HIFla inhibitor improves the ORR in the subject, improves the DOR in the subject, improves PFS in the subject, improves OS in the subject, improves DCR in the subject, reduces oncogenic allelic burden in the subject, reduces expression level of CD31, hypoxia-inducible factor la (HIFla) and/or a factor under the regulation of HIFla, or a combination thereof, relative to untreated subjects or subjects who have received prior treatment comprising a HIFla inhibitor.

[0133] In some embodiments, administering the PLK1 inhibitor and the HIFla inhibitor improves the ORR in the subject by at least 50% (e.g., 50%, 51%, 52%, 53%, 54%, 55%,

56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,

73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,

90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values) relative to untreated subjects or subjects who have received prior treatment comprising a HIFla inhibitor. In some embodiments, administering the PLK1 inhibitor and the HIFla inhibitor improves the PFS in the subject by at least 50% (e.g., 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values) relative to untreated subjects or subjects who have received prior treatment comprising a HIFla inhibitor.

[0134] The molar ratio of the PLK1 inhibitor (e.g., onvansertib) to the HIFla inhibitor

(e.g., bevacizumab or paclitaxel) can be, for example, about 1 :200, 1 : 100, 1:90, 1 :80, 1 :70, 1 :60, 1 :50, 1 :40, 1 :30, 1 :20, 1 : 10, 1 : 1, 10: 1, 20: 1, 30: 1, 40: 1, 50: 1, 100: 1, 1000: 1, 2000:1, or 5000: 1, or a number or a range between any two of these values. In some embodiments, the enhanced or synergistic efficacy or inhibition of cancer progression caused by a combination of the HIFla inhibitor and the PLK1 inhibitor (e.g., onvansertib) is, is about, is at least, is at least about, is at most, or is at most about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, or a number or a range between any two of these values, higher than the combined inhibition of progression caused by the HIFla inhibitor alone plus the PLK1 inhibitor (e.g., onvansertib) alone. For example, a combination of the HIFla inhibitor and the PLK1 inhibitor can cause a 50%, 60%, 70%, 80%, 90%, or more, inhibition of cancer progression (cancer cell viability of 50%, 40%, 30%, 20%, 10%, or less), whereas under the same conditions the combined inhibition of the HIFla inhibitor alone plus the PLK1 inhibitor alone can be 10%, 20%, 25%, 30%, or less) inhibition of cancer progression (cancer cell viability of 90%, 80%, 75%, 70%, or more). Thus, the enhanced or synergistic efficacy or inhibition of cancer progression caused by the combination of the HIFla inhibitor and the PLK1 inhibitor for example, 50%, 60%, 70%, 80%, 90%, 100%, or more than the combined inhibition of progression caused by the HIFla inhibitor alone plus the PLK1 inhibitor alone. In some embodiments, the HIFla inhibitor is bevacizumab or paclitaxel and the PLK1 inhibitor is onvansertib.

[0135] The HIFla inhibitor and the PLK1 inhibitor can be administered to the patient in any manner deemed effective to treat the cancer. The HIFla inhibitor can be administered together with, or separately from, the PLK1 inhibitor. When administered separately, HIFla inhibitor can be administered before or after the PLK1 inhibitor, or in different administration cycles. The administration of the PLK1 inhibitor can be oral administration. The administration of the HIFla inhibitor can be intravenous administration or oral administration.

[0136] The PLK1 inhibitor and the HIFla inhibitor can be co-administered (i.e., simultaneously) or sequentially. In some embodiments, it can be advantageous to administer the PLK1 inhibitor (e.g., onvansertib) to the subject before the HIFla inhibitor, e.g., on one or more days, or each day, of the days on which the PLK1 inhibitor and the HIFla inhibitor are administered to the subject. The time interval between the administration of the PLK1 inhibitor and the administration of the HIFla inhibitor can be, for example, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, a range between any two of these values, or any value between 30 minutes and 12 hours. In some embodiments, the PLK1 inhibitor (e.g., onvansertib) and the HIFla inhibitor are both administered to the subject on, or on at least about, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the days in a cycle (e.g., in each cycle during the combination treatment), and optionally the PLK1 inhibitor is administered to the subject prior to the HIFla inhibitor on each of the days both are administered, for example the PLK1 inhibitor is administered 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, a range between any two of these values, or any value between 30 minutes and 12 hours, prior to the administration of the HIFla inhibitor.

[0137] The HIFla inhibitor and the PLK1 inhibitor can each be administered in any schedule, e.g., once or multiple times per day or week; once, twice, three times, four times, five times, six times or seven times (daily) per week; for one or multiple weeks; etc. In some embodiments, the HIFla inhibitor and the PLK1 inhibitor are each administered to the patient in a cycle of at least twice within a week. In other embodiments, the HIFla inhibitor and the PLK1 inhibitor are each administered to the patient in a cycle of at least five times within a week. In some embodiments, the PLK1 inhibitor is administered daily, and the HIFla inhibitor are administered daily, weekly, bi-weekly, every four weeks, every five weeks, or monthly. In further embodiments, the patient undergoes at least two cycles of administration. The patient can undergo one cycle or more than one cycle of administrations, for example, two cycles, three cycles, three cycles, four cycles, five cycles, or more. Two adjacent cycles of administration can be continuous, i.e., no break between the last day of the first cycle and the first day of the second cycle. In some embodiments, two adjacent cycles of administration have a break between them, i.e., an interval between the last day of the first cycle and the first day of the second cycle. The break (i.e., the interval) can be or be at least, one day, two days, three days, five days, seven days, ten days, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, or a number or a range between any two of these values. In some embodiments, the patient undergoes three or four cycles of administration in which each cycle comprises at least five times within a week (e.g., 5 days per week). Each of the cycle in a multi-cycle administration can have the same dosing schedule, or different. For example, one of the cycles in the multi-cycle administration can be five continuous days of daily administration of the PLK1 inhibitor and HIFla inhibitor and two days of break in one week for four weeks, and one or more other cycles in the same multi-cycle administration be 28 continuous days of daily administration of the PLK1 inhibitor and the HIFla inhibitor in a four-week period. The PLK1 inhibitor can be administered on at least four days in the cycle. In some embodiments, PLK1 inhibitor is not administered on at least one day in the cycle.

[0138] The HIFla inhibitor can be administered to the patient at any appropriate dosage, e.g., a dosage of about, at least or at most 0.1 mg/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 200 mg/kg, 300 mg/kg, 400 mg/kg, 500 mg/kg, 600 mg/kg, 700 mg/kg, 800 mg/kg, 900 mg/kg, 1000 mg/kg, 1500 mg/kg, 2000 mg/kg, or a number between any two of these values. The dosage unit based on the body weight (mg/kg) can be converted to another unit (e.g., mg/m²) using a conversion chart such as the body surface area (BSA) conversion chart as will be understood by a person skilled in the art. The HIFla inhibitor (e.g., bevacizumab) can be administered at about 1 mg/kg - 20 mg/kg. In some embodiments, the HIFla inhibitor is bevacizumab, which is administered at a dosage of about, at least or at most 1 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 200 mg/kg, 300 mg/kg, 400 mg/kg, 500 mg/kg, 600 mg/kg, 700 mg/kg, 800 mg/kg, 900 mg/kg, 1000 mg/kg, or a number between any two of these values. The bevacizumab can be administered at about 5 mg/kg, about 7.5 mg/kg, about 10 mg/kg, or about 15 mg/kg. A chemotherapeutic agent, e.g., paclitaxel, can be administered at about 50 mg/m² to about 175 mg/m². In some embodiments, the chemotherapeutic agent (e.g., paclitaxel) is administered at a dosage of about, at least or at most 50 mg/m², 50 mg/m², 51 mg/m², 52 mg/m², 53 mg/m², 54 mg/m², 55 mg/m², 56 mg/m², 57 mg/m²,

58 mg/m², 59 mg/m², 60 mg/m², 61 mg/m², 62 mg/m², 63 mg/m², 64 mg/m², 65 mg/m², 66 mg/m²,

67 mg/m², 68 mg/m², 69 mg/m², 70 mg/m², 71 mg/m², 72 mg/m², 73 mg/m², 74 mg/m², 75 mg/m²,

76 mg/m², 77 mg/m², 78 mg/m², 79 mg/m², 80 mg/m², 81 mg/m², 82 mg/m², 83 mg/m², 84 mg/m²,

85 mg/m², 86 mg/m², 87 mg/m², 88 mg/m², 89 mg/m², 90 mg/m², 91 mg/m², 92 mg/m², 93 mg/m²,

94 mg/m², 95 mg/m², 96 mg/m², 97 mg/m², 98 mg/m², 99 mg/m², 100 mg/m², 150 mg/m², 175 mg/m², 200 mg/m², or a number or a range between any two of these values.

[0139] The HIFla inhibitor can be administrated to the patient once daily, twice daily, or three times daily. The HIFla inhibitor can be administered daily, weekly, bi-weekly, every three weeks, every four weeks, or every month. In some embodiments, the HIFla inhibitor is administered in a cycle of 7-56 days of daily, weekly, bi-weekly, tri-weekly, every four weeks, or monthly. In some embodiments, the HIFla inhibitor is administered in a cycle of 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 32 days, 35 days, 42 days, 49 days, or 56 days. In some embodiments, the HIFla inhibitor is administered in 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 32 days, 35 days, 42 days, 49 days, or 56 days, in a cycle. In some embodiments, the HIFla inhibitor is administered in day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, day 14, day 15, day 16, day 17, day 18, day 19, day 20, day 21, day 22, day 23, day 24, day 25, day 26, day 27, day 28, day 29, day 30, day 31, day 32, day 33, day 34, day 35, day 36, day 37, day 38, day 39, day 40, day 41, day 42, day 43, day 44, day 45, day 46, day 47, day 48, day 49, day 50, day 51, day 52, day 52, day 53, day 54, day 55, and/or day 56. In some embodiments, the HIFla inhibitor is not administered in day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, day 14, day 15, day 16, day 17, day 18, day 19, day 20, day 21, day 22, day 23, day 24, day 25, day 26, day 27, day 28, day 29, day 31, day 32, day 33, day 34, day 35, day 36, day 37, day 38, day 39, day 40, day 41, day 42, day 43, day 44, day 45, day 46, day 47, day 48, day 49, day 50, day 51, day 52, day 52, day 53, day 54, day 55, and/or day 56.

[0140] Any PLK1 inhibitor, now known or later discovered, can be used in these methods, including PLK1 inhibitors that are selective for PLK1, and PLK1 inhibitors that also inhibit the activity of other proteins. In some embodiments, the PLK1 inhibitor is a dihydropteridinone, a pyridopyrimidine, a aminopyrimidine, a substituted thiazolidinone, a pteridine derivative, a dihydroimidazo[l,5-f]pteridine, a metasubstituted thiazolidinone, a benzyl styryl sulfone analogue, a stilbene derivative, or a combination thereof. In some of these embodiments, the PLK1 inhibitor is onvansertib, BI2536, Volasertib (BI 6727), GSK461364, AZD1775, CYC140, HMN-176, HMN-214, rigosertib (ON-01910), MLN0905, TKM-080301, TAK-960 or Ro3280.

[0141] In some embodiments, the PLK1 inhibitor is onvansertib. In these embodiments, the onvansertib is administered to the patient at any appropriate dosage, e.g., a dosage of less than 12 mg/m², less than or equal to 24 mg/m², or greater than 24 mg/m². In some embodiments, the onvansertib is administered to the patient at about 12 mg/m², at about 15 mg/m², or at about 18 mg/m². In some embodiments, the onvansertib is administered to the patient daily. In additional embodiments, the onvansertib is administered in a cycle of 3-10 days of daily onvansertib administration with 2-16 days with no onvansertib administration. In some embodiments, the onvansertib is administered to the patient in a cycle of at least five times within a week. The patient can undergo two, three, or four cycles of administration. In some embodiments, the patient undergoes four cycles of administration in a cycle of at least five days of daily onvansertib administration with 1-2 days with no onvansertib administration.

[0142] In some embodiments, a PLK1 inhibitor alone or in combination with a HIFla inhibitor is administrated to a patient who has taken a drug holiday after undergoing one or more cycles of administration. A drug holiday as used herein refers to a period of time when a patient stops taking a PLK1 inhibitor and/or a HIFla inhibitor. A drug holiday can be a few days to several months. In some embodiments, the drug holiday can be 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, or any value or a range between any two of these values.

[0143] As can be appreciated by one of skill in the art, the amount of co-administration of the HIFla inhibitor and the PLK1 inhibitor, and the timing of co-administration, can depend on the type (species, gender, age, weight, etc.) and condition of the subject being treated and the severity of the disease or condition being treated. The HIF la inhibitor and the PLK1 inhibitor can formulated into a single pharmaceutical composition, or two separate pharmaceutical compositions. The active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interracial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.

[0144] Methods, compositions, kits and systems disclosed herein can be applied to different types of subjects. For example, the subject can be a subject receiving a cancer treatment, a subject at cancer remission, a subject has received one or more cancer treatment, or a subject suspected of having cancer. The subject can have a stage I cancer, a stage II cancer, a stage III cancer, and/or a stage IV cancer. In some embodiments, the subject has stage IV cancer. In some embodiments, the subject has metastatic cancer. In some embodiments, the subject has not received any prior treatment comprising a HIF la inhibitor.

[0145] The treatment of the present disclosure can comprise administration of a PLK1 inhibitor (onvansertib) for a desired duration in a cycle. The administration of the PLK1 inhibitor (and/or the HIF la inhibitor) can be daily or with break(s) between days of administrations. The break can be, for example, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, or more. The administration can be once, twice, three times, four times, or more on a day when the PLK1 inhibitor (and/or the HIF la inhibitor) is administered to the patient. The administration can be, for example, once every two days, every three days, every four days, every five days, every six days, or every seven days. The length of the desired duration can vary, for example, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, or more days. Each cycle of treatment can have various lengths, for example, at least 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, or more. For example, a single cycle of the treatment can comprise administration of the PLK1 inhibitor (e.g., onvansertib) and/or the HIFla inhibitor for four days, five days, six days, seven days, eight days, nine days, ten days, eleven days, twelve days, thirteen days, fourteen days, fifteen days, sixteen days, seventeen days, eighteen days, nineteen days, twenty days, twenty-one days, twenty -two days, twenty -three days, twenty-four days, twenty-five days, twenty- six days, twenty-seven days, twenty-eight days, or more in a cycle (e.g., in a cycle of at least 21 days (e.g., 21 to 28 days)). In some embodiments, the treatment can comprise administration of the PLK1 inhibitor (e.g., onvansertib) and/or the HIFla inhibitor for, or for at least, four days, five days, six days, seven days, eight days, nine days, ten days, eleven days, twelve days, thirteen days, fourteen days, fifteen days, sixteen days, seventeen days, eighteen days, nineteen days, twenty days, or a range between any two of these values, in a cycle (e.g., a cycle of at least 21 days (e.g., 21 to 28 days)). The administration of the PLK1 inhibitor (e.g., onvansertib) and/or the HIFla inhibitor in a single cycle of the treatment can be continuous or with one or more intervals (e.g., one day or two days of break). In some embodiments, the treatment comprises administration of the PLK1 inhibitor (e.g., onvansertib) for five days in a cycle of 21 to 28 days.

[0146] In some embodiments, the PLK1 inhibitor (e.g., onvansertib) is administered to the subject in need thereof on twenty days (e.g., Days 1-10 and 15-24) during a 28-day cycle. The twenty days can be, for example, a continuous daily administration for ten days (e.g., Days 1-10) and another continuous daily administration (e.g., Days 15-24) for ten days, or a continuous daily administration for four sets of five days (e.g., Days 1-5, 8-12, 15-19, and 22-26), In some embodiments, for example when the patient is identified to have low tolerance to the PLK1 inhibitor (e.g., onvansertib), the PLK1 inhibitor is administered to the subject in need thereof on ten days (e.g., Days 1-5 and 15-19) during a 28-day cycle. The ten days can be, for example, a continuous daily administration for ten days (e.g., Days 1-10) or two continuous daily admiration for five days each (e.g., Days 1-5 and Days 15-19). In some embodiments, the PLK1 inhibitor (e.g., onvansertib) is administered to the subject in need thereof daily throughout the whole cycle (e.g., daily for 28 days in a cycle of 28 days). Depending on the needs of inhibition/reversion of cancer progression in the subject, the subject can receive one, two, three, four, five, six, or more cycles of treatment. For combination treatment, the administration cycles, dosing schedules, and/or dosage amounts of the HIFla inhibitor and the PLK1 inhibitor can be the same or different. For combination treatment, the administration cycle, dosing schedule, and/or dosage amount of the HIFla inhibitor can be adjusted according to the administration cycle, dosing schedule, and/or dosage amount of the PLK1 inhibitor. For example, the HIFla inhibitor can be administered in four 7-day cycles (e.g., daily dose on Days 1-5 and no dose on Days 6-7, repeated for 4 weeks), which corresponds to a 28-day cycle for administration of the PLK1 inhibitor (e.g., onvansertib).

[0147] The treatment can comprise administration of the PLK1 inhibitor (e.g., onvansertib) at, or at about, 6 mg/m² - 90 mg/m², for example, as a daily dose. For example, the treatment can comprise daily administration of the PLK1 inhibitor (e.g., onvansertib) at, or at about, 6 mg/m², 8 mg/m², 10 mg/m², 12 mg/m², 14 mg/m², 16 mg/m², 18 mg/m², 20 mg/m², 23 mg/m², 27 mg/m², 30 mg/m², 35 mg/m², 40 mg/m², 45 mg/m², 50 mg/m², 55 mg/m², 60 mg/m², 65 mg/m², 70 mg/m², 80 mg/m², 85 mg/m², 90 mg/m², a number or a range between any two of these values, or any value between 8 mg/m² - 90 mg/m². In some embodiments, the daily dose of the PLK1 inhibitor (e.g., onvansertib) can be adjusted (e.g., increased or decreased with the range) during the treatment, or during a single cycle (e.g., the first cycle, the second cycle, the third cycle, and a subsequent cycle) of the treatment, for the subject. In some embodiments, the PLK1 inhibitor (e.g., onvansertib) is administered at 12 mg/m² on twenty days (e.g., Days 1-10 and 15-24) during a 28-day cycle. In some embodiments, the PLK1 inhibitor (e.g., onvansertib) is administered at 15 mg/m² on ten days (e.g., Days 1-5 and 15-19) during a 28-day cycle. In some embodiments, the PLK1 inhibitor (e.g., onvansertib) is administered at 8 mg/m² or 10 mg/m² everyday (e.g., Days 11-28) during a 28-day cycle. In some embodiments, the daily dose of the PLK1 inhibitor (e.g., onvansertib) can be adjusted (e.g., increased or decreased with the range) during the treatment, or during a single cycle (e.g., the first cycle, the second cycle, the third cycle, and a subsequent cycle) of the treatment, for the subject. In some embodiments, the PLK1 inhibitor is administered at or at about 12 mg/m². In some embodiments, the PLK1 inhibitor is administered at or at about 15 mg/m². In some embodiments, the PLK1 inhibitor is administered at or at about 18 mg/m².

[0148] A maximum concentration (Cmax) of the PLK1 inhibitor (e.g., onvansertib) in a blood of the subject (during the treatment or after the treatment) when the PLK1 inhibitor is administered alone or in combination with the HIFla inhibitor can be from about 100 nmol/L to about 1500 nmol/L. For example, the Cmax of the PLK1 inhibitor (e.g., onvansertib) in a blood of the subject when the PLK1 inhibitor is administered alone or in combination with the HIFla inhibitor can be, or be about, 100 nmol/L, 200 nmol/L, 300 nmol/L, 400 nmol/L, 500 nmol/L, 600 nmol/L, 700 nmol/L, 800 nmol/L, 900 nmol/L, 1000 nmol/L, 1100 nmol/L, 1200 nmol/L, 1300 nmol/L, 1400 nmol/L, 1500 nmol/L, a range between any two of these values, or any value between 200 nmol/L to 1500 nmol/L.

[0149] An area under curve (AUC) of a plot of a concentration of the PLK1 inhibitor (e.g., onvansertib) in a blood of the subject over time (e.g., AUC0-24 for the first 24 hours after administration) when the PLK1 inhibitor is administered alone or in combination with the HIFla inhibitor can be from about 1000 nmol/L. hour to about 400000 nmol/L. hour. For example, the AUC of a plot of a concentration of the PLK1 inhibitor (e.g., onvansertib) in a blood of the subject over time (e.g., AUC0-24 for the first 24 hours after administration) when the PLK1 inhibitor is administered alone or in combination with the HIFla inhibitor can be, or be about, 1000 nmol/L. hour, 5000 nmol/L. hour, 10000 nmol/L. hour, 15000 nmol/L. hour, 20000 nmol/L. hour, 25000 nmol/L. hour, 30000 nmol/L. hour, 35000 nmol/L. hour, 40000 nmol/L. hour, a range between any two of these values, or any value between 1000 nmol/L. hour and 400000 nmol/L. hour.

[0150] A time (T max ) to reach a maximum concentration of the PLK1 inhibitor (e.g., onvansertib) in a blood of the subject when the PLK1 inhibitor is administered alone or in combination with the HIFla inhibitor can be from about 1 hour to about 5 hours. For example, the time (Tmax) to reach a maximum concentration of the PLK1 inhibitor (e.g., onvansertib) in a blood of the subject when the PLK1 inhibitor is administered alone or in combination with the HIFla inhibitor can be, or be about, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, a range between any two of these values, or any value between 1 hour and 5 hours.

[0151] An elimination half-life (T1/2) of the PLK1 inhibitor (e.g., onvansertib) in a blood of the subject when the PLK1 inhibitor is administered alone or in combination with the HIFla inhibitor can be from about 10 hours to about 60 hours. For example, the elimination halflife (T1/2) of the PLK1 inhibitor (e.g., onvansertib) in a blood of the subject when the PLK1 inhibitor is administered alone or in combination with the HIFla inhibitor can be, or be about, 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 hours, 50 hours, 55 hours, 60 hours, a range between any two of these values, or any value between 10 hours and 60 hours.

Additional Cancer Therapeutics or Therapy

[0152] Methods, compositions and kits disclosed herein can be used for treating cancer. In some embodiments, a method for treating cancer comprises administrating a HIFla inhibitor and a PLK1 inhibitor (e.g., onvansertib) to a subject (e.g., a patient) in need thereof. The method can comprise administering a therapeutically effective amount of the HIFla inhibitor and a therapeutically effective amount of the PLK1 inhibitor. The treatment can comprise administration of at least one additional cancer therapeutics or cancer therapy. In some embodiments, the subject has not received any prior treatment comprising inhibiting HIFla.

[0153] The one or more cancer therapeutics or therapies can comprise FOLFIRI, abiraterone, FOLFOX, an anti-EGFR agent, a KRAS directed inhibitor, gemcitabine, abraxane, nanoliposomal irinotecan, 5-FU, or a combination thereof. In some embodiments, anti-EGFR agents is optionally cetuximab. In some embodiments, the KRAS directed inhibitor is optionally a G12C inhibitor, a G12D inhibitor or a combination thereof. In some embodiments, the additional cancer therapy is FOLFOX (leucovorin, fluorouracil, and oxaliplatin) and/or FOLFIRI (leucovorin, fluorouracil, and irinotecan).

Methods for Predicting/Determining Treatment Efficacy and Status of Cancer

[0154] Disclosed herein include methods of treating cancer. In some embodiments, the method comprises: administering a PLK1 inhibitor and a HIFla inhibitor to a subject with a cancer, thereby reducing or inhibiting progression of cancer. The method described herein using the combination of the HIFla inhibitor and the PLK1 inhibitor is expected to be effective with various cancers, for example colorectal cancer, bladder cancer, breast cancer, kidney cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, stomach cancer, thyroid cancer, uterine cancer, renal cancer, cervical cancer, recurrent glioblastoma, head and neck cancer, soft tissue carcinoma, vulvar cancer, melanoma, brain cancer, liver cancer, bile duct cancer, or a combination thereof. The cancer can be metastatic cancer.

[0155] The method can comprise one or more of (1) determining cancer status of the subject, (2) determining responsiveness of the subject to a PLK1 inhibitor treatment, and (3) administering one or more cancer therapeutics or therapies for the cancer.

[0156] In some embodiments, administering the PLK1 inhibitor and the HIFla inhibitor improves one or more therapeutic effects in the subject relative to a control or a baseline. The one or more therapeutic effects can comprise size of a tumor derived from the cancer, objective response rate (ORR), duration of response (DOR), time to response, progression free survival (PFS), overall survival (OS), disease control rate (DCR), oncogenic allelic burden, and/or expression level of CD31. In some embodiments, administering the PLK1 inhibitor and the HIFla inhibitor synergistically improves the ORR and/or the PFS of the subject relative to the PLK1 inhibitor treatment alone, the HIFla inhibitor treatment alone, and/or the additive effect of the PLK1 inhibitor treatment alone and the HIFla inhibitor treatment alone.

[0157] Tumor evaluations and assessment of tumor burden and therapeutic efficacy can be made based on RECIST criteria (Therasse et al., 2000), New Guidelines to Evaluate the Response to Treatment in Solid Tumors, Journal of National Melanoma Institute, Vol. 92; 205-16 and is made within the presently disclosed methods, in some embodiments, according to the revised RECIST guidelines (version 1.1) (Eisenhauer et al 2009, New response evaluation criteria in solid tumors: revised RECIST guideline (version 1.1). Eur J Melanoma, 45(2):228-47.), which is hereby incorporated by reference in its entirety.

[0158] In some embodiments, the treatment results in a sustained response in the subject after cessation of the treatment. “Sustained response” can refer to the sustained effect on reducing tumor growth after cessation of a treatment. For example, the tumor size may remain the same or smaller as compared to the size at the beginning of the administration phase. In some embodiments, the sustained response has a duration at least the same as the treatment duration, at least 1.5x, 2. Ox, 2.5x, or 3. Oxlength of the treatment duration.

[0159] The treatment methods disclosed herein may result in a partial or complete response. As used herein, “complete response” or “CR” can refer to disappearance of all target lesions; “partial response” or “PR” can refer to at least a 30% decrease in the sum of the longest diameters (SLD) of target lesions, taking as reference the baseline SLD; and “stable disease” or “SD” can refer to neither sufficient shrinkage of target lesions to qualify for PR, nor sufficient increase to qualify for PD, taking as reference the smallest SLD since the treatment started. As used herein, “objective response rate” (ORR) can refer to the sum of complete response (CR) rate and partial response (PR) rate.

[0160] The treatment methods disclosed herein can lead to an increase in progression free survival (PFS) and overall survival (OS) of the subject administered the combination therapy. As used herein, “progression free survival” (PFS) refers to the length of time during and after treatment during which the disease being treated (e.g., cancer) does not get worse. Progression-free survival may include the amount of time patients have experienced a complete response or a partial response, as well as the amount of time patients have experienced stable disease. As used herein, “overall survival” refers to the percentage of subjects in a group who are likely to be alive after a particular duration of time. As used herein, “disease control rate” refers to complete response (CR) plus partial response (PR) plus stable disease (SD).

[0161] In some embodiments, administering the PLK1 inhibitor and the HIFla inhibitor improves the ORR in the subject by, by about, by at least, or by at least about 50% (e.g., 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values) relative to untreated subjects or subjects who have received prior treatment comprising a HIFla inhibitor. In some embodiments, administering the PLK1 inhibitor and the HIFla inhibitor improves the PFS in the subject by, by about, by at least, or by at least about 50% (e.g., 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values) relative to untreated subjects or subjects who have received prior treatment comprising a HIFla inhibitor.

[0162] In some embodiments, administering a PLK1 inhibitor and a HIFla inhibitor to a subject who has not received any prior treatment comprising a HIFla inhibitor can result in an ORR of, of at least, of about, or of at least about 69% (e.g., 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values). In some embodiments, administering a PLK1 inhibitor and a HIFla inhibitor to a subject who has received any prior treatment comprising a HIFla inhibitor can result in an ORR of about 23% or less (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, or a number or a range between any two of these values). In some embodiments, administering a PLK1 inhibitor and a HIFla inhibitor to a subject who has not received any prior treatment comprising a HIFla inhibitor can result in an median PFS of, of at least, of about, or of at least about 13.5 months (e.g., 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 21 years, 22 years, 23 years, 24 years, 25 years, 26 years, 27 years, 28 years, 29 years, 30 years, 31 years, 32 years, 33 years, 34 years, 35 years, 36 years, 37 years, 38 years, 39 years, 40 years, 41 years, 42 years, 43 years, 44 years, 45 years, 46 years, 47 years, 48 years, 49 years, 50 years, 55 years, 60 years, 65 years, or more than 65 years). In some embodiments, administering a PLK1 inhibitor and a HIFla inhibitor to a subject who has received any prior treatment comprising inhibiting HIFla can result in a median PFS of about 7.8 months or less (e.g., 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, or a number or a range between any two of these values).

[0163] The term “inhibition of tumor growth” or “reduced tumor growth” can refer to causing a reduction in or complete cessation of tumor growth and/or causing a regression in tumor size (e.g., diameter and/or volume). The term “tumor volume” or “tumor size” can refer to the total size of the tumor, which can include the tumor itself plus affected lymph nodes if applicable. The presence or absence of a tumor, and tumor size can be determined by a variety of methods known in the art, such as, by measuring the dimensions of the tumor using calipers, computed tomography (CT) or magnetic resonance imaging (MRI) scans, mammography, and X- ray. The volume can be calculated using equations based on, for example, the z-axis diameter, or on standard shapes such as the sphere, ellipsoid, or cube. Tumor size may be assessed at any time before, during or following at least one cycle of treatment with onvansertib and HIFla inhibitor. Tumor size can be assessed at a first timepoint, and at one or more additional timepoints. In some embodiments, tumor size can be assessed in the subject and, e.g., an untreated subject at equivalent timepoints (e.g., at a first timepoint, and at one or more additional timepoints). Tumor growth can be determined by, e.g., measuring tumor size at a first timepoint and measuring tumor size at one or more additional timepoints. In some embodiments, increased inhibition of tumor growth in the subject (e.g., improved ORR) indicates the subject as responsive to the cancer treatment.

[0164] The inhibition of growth of at least one of one or more tumors in the subject can be, can be about, can be at least, or can be at least about 1.1 times greater, 1.2 times greater, 1.3 times greater, 1.4 times greater, 1.5 times greater, 1.6 times greater, 1.7 times greater, 1.8 times greater, 1.9 times greater, 2 times greater, or a number or a range between any two of these values, or more, than the inhibition of growth caused by onvansertib and the HIFla inhibitor in a subject (e.g., a subject who has received a previous treatment with a HIFla inhibitor) who has received previous treatment for inhibiting HIFla, following one or more cycles of treatment. The inhibition of growth of at least one of the one or more tumors in the subject can be increased by, by about, by at least, or by at least about 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or a number or a range between any two of these values, relative to a subject prior to the administration and/or who has received previous treatment with a HIFla inhibitor, following one or more cycles of treatment. The growth of at least one of the one or more tumors in the subject can be inhibited by, by about, by at least, or by at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or a number or a range between any two of these values relative to an untreated subject or a subject who has received prior HIFla inhibitor treatment, following one or more cycles of treatment with the combination. The growth of at least one of the one or more tumors in the subject can be inhibited by, by about, by at least, or by at least about 70%, 75%, 80%, 85%, 90%, 95%, 100% or a number or a range between any two of these values relative to an untreated subject, following one or more cycles of treatment. The subject can be tumor-free following one or more cycles of treatment.

[0165] The inhibition of growth of at least one of the one or more tumors in the subject can be, can be about, can be at least, or can be at least about 1.1 times greater, 1.2 times greater, 1.3 times greater, 1.4 times greater, 1.5 times greater, 1.6 times greater, 1.7 times greater, 1.8 times greater, 1.9 times greater, 2 times greater, or a number or a range between any two of these values, or more, than the inhibition of growth caused by onvansertib alone or the HIFla inhibitor alone, following one or more cycles of treatment. The inhibition of growth of at least one of the one or more tumors in the subject can be increased by, by about, by at least, or by at least about 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or a number or a range between any two of these values, relative to a subject treated with onvansertib alone or the HIFla inhibitor alone, following one or more cycles of treatment. The growth of at least one of the one or more tumors in the subject can be inhibited by, by about, by at least, or by at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or a number or a range between any two of these values relative to an untreated subject, following one or more cycles of treatment. The growth of at least one of the one or more tumors in the subject can be inhibited by, by about, by at least, or by at least about 70%, 75%, 80%, 85%, 90%, 95%, 100% or a number or a range between any two of these values relative to an untreated subject, following one or more cycles of treatment. The subject can be tumor-free following one or more cycles of treatment.

[0166] In some embodiments, the first time point is prior or immediately prior to the combination treatment, and at least one of the one or more additional time points are at the end of or after at least one cycle of the combination treatment. In some embodiments, the cycle of the combination treatment is the first cycle of the combination treatment. In some embodiments, the first time point is prior or immediately prior to a first cycle of the combination treatment, and the one or more additional time points are at the end of or after a second cycle of the combination treatment.

[0167] In some embodiments, the first cycle of the combination treatment is immediately prior to the second cycle of the combination treatment. In some embodiments, the method comprises continuing the combination treatment to the subject if the subject is indicated as responsive to the combination treatment. In some embodiments, the method comprises discontinuing the combination treatment to the subject and/or starting a different combination treatment to the subject if the subject is not indicated as responsive to the combination treatment.

[0168] In some embodiments, the first time point is prior or immediately prior to the combination treatment, and the one or more additional time points are at the end of or after at least a cycle of the combination treatment, optionally the cycle of the combination treatment is the first cycle of the combination treatment. In some embodiments, the first time point is prior or immediately prior to a first cycle of the combination treatment, and the one or more additional time points are at the end of or after a second cycle of the combination treatment, optionally the first cycle of the combination treatment is immediately prior to the second cycle of the combination treatment.

[0169] Determining the responsiveness of the subject can comprise determining if the subject is a responder of the treatment, if the subject is or is going to be in complete recovery (CR), or if the subject is or is going to be in partial remission (PR). Determining the responsiveness of the subject can comprise determining objective response rate (ORR), duration of response (DOR), time to response, progression free survival (PFS), overall survival (OS), disease control rate (DCR), oncogenic allelic burden, or a combination thereof of the subject. Determining the responsiveness of the subject can comprise determining if the subject has a partial response to the treatment, if the subject has a complete response to the treatment, if the subject has a stable disease (SD) status, or if the subject has a progressive disease (PD) status.

[0170] In some embodiments, the method comprises starting an additional treatment to the subject if the subject is indicated as in cancer relapse. The additional treatment can be the same or different from the current or prior combination treatment.

[0171] Additional methods for assessing cancer status of the subject include determining ECOG status. As used herein, ECOG status refers to Eastern Cooperative Oncology Group (ECOG) Performance Status (Oken M, etal., Toxicity and response criteria of the Eastern Cooperative Oncology Group. Am J Clin Oncol 1982; 5(6):649-655), as shown below: 0, Fully active, able to carry on all pre-disease performance without restriction; 1, Restricted in physically strenuous activity but ambulatory and able to carry out work of a light or sedentary nature, e.g., light house work, office work; 2, Ambulatory and capable of all selfcare but unable to carry out any work activities; up and about more than 50% of waking hours; 3, Capable of only limited selfcare; confined to bed or chair more than 50% of waking hours; 4, Completely disabled; cannot carry on any selfcare; totally confined to bed or chair; and 5, Dead.

[0172] As described herein, the patient can achieve complete response or partial response after treatment with the HIFla inhibitor and the PLK1 inhibitor. In some embodiments, the patient achieves a complete response. In some embodiments, the patient achieves a partial response. In some embodiments, the patient did not receive any prior HIFla inhibitor treatment.

[0173] Disclosed herein include methods, compositions, kits, and systems for predicting/determining clinical outcome for a combination treatment of cancer of the present disclosure, monitoring of the combination treatment, predicting/determining responsiveness of a subject to the combination treatment, determining the status of the cancer in a subject, and improving combination treatment outcome. The methods, compositions, kits and systems can be used to guide the combination treatment, provide combination treatment recommendations, reduce or avoid unnecessary ineffective combination treatment for patients. In some embodiments, the combination treatment disclosed herein can improve oncogenic allelic burden in a subject. As used herein, “allelic burden” can refer to the ratio between mutant (e.g., oncogenic) and wild-type alleles in clinical samples employed for genotyping. In some embodiments, the sample can comprise circulating tumor DNA (ctDNA). ctDNA can be analyzed to predict/determine clinical outcome for cancer treatment, monitor cancer treatment, predict/determine responsiveness of a subject to a cancer treatment, determine cancer status in a subject, improve cancer treatment outcome, guide cancer treatment, provide treatment recommendations, and/or to reduce or avoid ineffective cancer treatment. Such analysis of ctDNA has been described in PCT Application published as WO2021146322, the content of which is incorporated herein by reference in its entirety.

[0174] A method of determining responsiveness of a subject to a combination treatment comprising a HIFla inhibitor and a PLK1 inhibitor of the disclosure can comprise, for example, analyzing circulating tumor DNA (ctDNA) of a subject with cancer, wherein the subject is undergoing a treatment and/or has received the combination treatment, thereby determining the responsiveness of the subject to the combination treatment. In some embodiments, determining the responsiveness of the subject comprises determining if the subject is a responder of the treatment, if the subject is or is going to be in CR, or if the subject is or is going to be in partial remission (PR). For example, analyzing ctDNA can comprise detecting variant allele frequency in the ctDNA in a first sample obtained from the subject at a first time point, detecting variant allele frequency in the ctDNA obtained from the subject at one or more additional time points in one or more additional samples, and determining the difference of the variant allele frequency in ctDNA between the first and at least one of the one or more additional samples, a decrease in the variant allele frequency in at least one of the additional samples relative to the first sample indicates the subject as responsive to the cancer treatment.

[0175] In some embodiments, the first time point is prior or immediately prior to the combination treatment, and at least one of the one or more additional time points are at the end of or after at least a cycle of the combination treatment. In some embodiments, the cycle of the combination treatment is the first cycle of the combination treatment. In some embodiments, the first time point is prior or immediately prior to a first cycle of the combination treatment, and the one or more additional time points are at the end of or after a second cycle of the combination treatment.

[0176] In some embodiments, the first cycle of the combination treatment is immediately prior to the second cycle of the combination treatment. In some embodiments, the method comprises continuing the combination treatment to the subject if the subject is indicated as responsive to the combination treatment. In some embodiments, the method comprises discontinuing the combination treatment to the subject and/or starting a different combination treatment to the subject if the subject is not indicated as responsive to the combination treatment.

[0177] Disclosed herein include methods of determining cancer status of a subject, comprising analyzing circulating tumor DNA (ctDNA) of a subject, thereby determining cancer status of the subject. The subject can be a subject undergoing a current combination treatment comprising a HIFla inhibitor and a PLK1 inhibitor of the present disclosure, a subject that has received a prior combination treatment of the present disclosure, and/or a subject that is in remission for the cancer. The subject in remission for cancer can be in complete remission (CR), or in partial remission (PR).

[0178] In some embodiments, analyzing the ctDNA comprises detecting variant allele frequency in the ctDNA. In some embodiments, analyzing the ctDNA comprises detecting variant allele frequency in the ctDNA obtained from the subject at a first time point in a first sample, detecting variant allele frequency in the ctDNA obtained from the subject at one or more additional time points in one or more additional samples, and determining the difference of the variant allele frequency in ctDNA between the first and at least one of the one or more additional samples, an increase in the variant allele frequency at the additional sample(s) relative to the first sample indicates that the subject is at risk of cancer relapse or is in cancer relapse.

[0179] In some embodiments, the first time point is prior or immediately prior to the combination treatment, and the one or more additional time points are at the end of or after at least a cycle of the combination treatment, optionally the cycle of the combination treatment is the first cycle of the combination treatment. In some embodiments, the first time point is prior or immediately prior to a first cycle of the combination treatment, and the one or more additional time points are at the end of or after a second cycle of the combination treatment, optionally the first cycle of the combination treatment is immediately prior to the second cycle of the combination treatment. In some embodiments, the method comprises starting an additional treatment to the subject if the subject is indicated as in cancer relapse. The additional treatment can be the same or different from the current or prior combination treatment.

[0180] The variant allele frequency in ctDNA can be determined, for example, by total mutation count in the ctDNA in each of the first sample and one or more additional samples, or by the mean variant allele frequency in each of the first sample and one or more additional samples. In some embodiments, the variant allele frequency is mutant allelic frequency (MAF) for a driver mutation of the cancer. In some embodiments, the variant allele frequency is MAF for one or more driver mutations of the cancer. In some embodiments, Log2(Ci/Co) < a MAF threshold indicates a decrease in ctDNA MAF Co is ctDNA MAF in the first sample and Ci is ctDNA MAF in one of the additional samples. In some embodiments, the MAF threshold is, or is about, 0.01 to -0.10. In some embodiments, the MAF threshold is, or is about, 0.06. In some embodiments, the MAF threshold is, or is about, 0.05.

[0181] In some embodiments, the first sample comprises ctDNA from the subject before treatment, and the one of additional samples comprises ctDNA from the subject after treatment. In some embodiments, the driver mutation is a mutation in one of the below 75 genes ABL1, ANKRD26, ASXL1, ATRX, BCOR, BCORL1, BRAF, BTK, CALR, CBL, CBLB, CBLC, CCND2, CDC25C, CDKN2A, CEBPA, CSF3R, CUX1, CXCR4, DCK, DDX41, DHX15, DNMT3A, ETNK1, ETV6, EZH2, FBXW7, FLT3, GATA1, GATA2, GNAS, HRAS, IDH1, IDH2, IKZF1, JAK2, JAK3, KDM6A, KIT, KMT2A, KRAS, LUC7L2, MAP2K1, MPL, MYC, MYD88, NF1, NOTCH1, NPM1, NRAS, PDGFRA, PHF6, PPM1D, PTEN, PTPN11, RAD21, RBBP6, RPS14, RUNX1, SETBP1, SF3B1, SH2B3, SLC29A1, SMC1A, SMC3, SRSF2, STAG2, STAT3, TET2, TP53, U2AF1, U2AF2, WT1, XPO1, and ZRSR2. In some embodiments, at least one of the one or more the driver mutations is a mutation in in the 75 genes. In some embodiments, one or more the driver mutations are mutations in the 75 genes.

[0182] The driver mutation or at least one of the one or more driver mutations can be in a gene selected from the group consisting of TP53, ASXL1, DNMT3A, NRAS, SRSF2, TET2, SF3B1, FLT3, FLT3 ITD, IDH2, NPM1, RUNX1, CDKN2A, KRAS, STAG2, CALR, CBL, CSF3R, DDX41, GATA2, JAK2, PHF6, and SETBP1. In some embodiments, the driver mutation or at least one of the one or more driver mutations is in a gene selected from the group consisting of DNMT3A, TET2, NPM1, SRSF2, NRAS, CDKN2A, SF3B1, FLT3, ASXL1, SRSF2, IDH2, NRAS, and SF3B1. In some embodiments, the method further comprises determining variant allele frequency in one or more of the ctDNA, PBMCs and BMMCs of the subject.

[0183] The ctDNA can be analyzed using, for example, polymerase chain reaction (PCR), next generation sequencing (NGS), and/or droplet digital PCR (ddPCR). The sample disclosed herein can be derived from, for example, whole blood of the subject, plasma of the subject, serum of the subject, or a combination thereof. In some embodiments, the ctDNA is from whole blood of the subject, plasma of the subject, serum of the subject, or a combination thereof.

[0184] In some embodiments, the method comprises analyzing ctDNA of the subject before the treatment. In some embodiments, the treatment comprises one or more cycles, and the ctDNA is analyzed before, during and after each cycle of the treatment. Each cycle of treatment can be at least 21 days. In some embodiments, each cycle of treatment is from about 21 days to about 28 days. In some embodiments, the subject is human.

[0185] Disclosed herein include methods of improving treatment outcome for the cancer. The method can comprise: detecting variant allele frequency in circulating tumor DNA (ctDNA) obtained from a subject at a first time point in a first sample before the subject undergoes a combination treatment comprising a HIFla inhibitor and a PLK1 inhibitor of the present disclosure; detecting variant allele frequency in ctDNA obtained from the subject at one or more additional time points in one or more additional samples after the subject undergoes the combination treatment; determining the difference of the variant allele frequency in ctDNA between the first and at least one of the one or more additional samples, a decrease in the oncogenic allelic burden and/or variant allele frequency in at least one of the additional samples relative to the first sample indicates the subject as responsive to the combination treatment; and continuing the combination treatment to the subject if the subject is indicated as responsive to the combination treatment, or discontinuing the combination treatment to the subject and/or starting a different cancer treatment to the subject if the subject is not indicated as responsive to the combination treatment.

[0186] Also disclosed herein include methods of treating cancer. The method can comprise: administering a combination treatment comprising a HIFla inhibitor and a PLK1 inhibitor of the present disclosure to a subject in need thereof; determining a decrease, relative to an oncogenic allelic burden and/or variant allele frequency in a first sample of the subject obtained at a first time point before the subject receives the combination treatment, in a variant allele frequency in a second sample of the subject obtained at a second time point after the subject receives the combination treatment; and continuing with the combination treatment. In some embodiments, the subject is a subject newly diagnosed with cancer, for example a subject that has not received any prior cancer treatment before the combination treatment. In some embodiments, the subject has received prior cancer treatment and was in remission for the cancer, for example a subject in complete remission (CR), or in partial remission (PR) after receiving the prior combination treatment. In some embodiments, the prior treatment does not comprise the use of a HIF la inhibitor, a PLKl inhibitor, or both.

[0187] The first time point can be, for example, prior or immediately prior to the combination treatment. The at least one of the one or more additional time points can be, for example, at the end of or after at least a cycle of the combination treatment. In some embodiments, the cycle of the combination treatment is the first cycle of the combination treatment. In some embodiments, the first time point is prior or immediately prior to a first cycle of the combination treatment, and the one or more additional time points are at the end of or after a second cycle of the combination treatment. In some embodiments, the first cycle of the combination treatment is immediately prior to the second cycle of the combination treatment.

[0188] The variant allele frequency in ctDNA can be determined, for example, by total mutation count in the ctDNA in each of the first sample and one or more additional samples, and/or by the mean variant allele frequency in each of the first sample and one or more additional samples. In some embodiments, the variant allele frequency is mutant allelic frequency (MAF) for a driver mutation of the cancer. In some embodiments, the variant allele frequency is mutant allelic frequency (MAF) for one or more driver mutations of the cancer. In some embodiments, Log2(Ci/Co) < a MAF threshold indicates a decrease in ctDNA MAF Co is ctDNA MAF in the first sample and Ci is ctDNA MAF in one of the additional samples. In some embodiments, the MAF threshold is -0.05.

[0189] The driver mutation can be, for example, a mutation in one of the 75 genes set forth below: ABL1, ANKRD26, ASXL1, ATRX, BCOR, BCORL1, BRAF, BTK, CALR, CBL, CBLB, CBLC, CCND2, CDC25C, CDKN2A, CEBPA, CSF3R, CUX1, CXCR4, DCK, DDX41, DHX15, DNMT3A, ETNK1, ETV6, EZH2, FBXW7, FLT3, GATA1, GATA2, GNAS, HRAS, IDH1, IDH2, IKZF1, JAK2, JAK3, KDM6A, KIT, KMT2A, KRAS, LUC7L2, MAP2K1, MPL, MYC, MYD88, NF1, NOTCH1, NPM1, NRAS, PDGFRA, PHF6, PPM1D, PTEN, PTPN11, RAD21, RBBP6, RPS14, RUNX1, SETBP1, SF3B1, SH2B3, SLC29A1, SMC1A, SMC3, SRSF2, STAG2, STAT3, TET2, TP53, U2AF1, U2AF2, WT1, XPO1, and ZRSR2, and/or one or more the driver mutations are mutations in the 75 genes. In some embodiments, the driver mutation or at least one of the one or more driver mutations is in a gene selected from the group consisting of TP53, ASXL1, DNMT3A, NRAS, SRSF2, TET2, SF3B1, FLT3, FLT3 ITD, IDH2, NPM1, RUNX1, CDKN2A, KRAS, STAG2, CALR, CBL, CSF3R, DDX41, GATA2, JAK2, PHF6, and SETBP1. In some embodiments, the driver mutation or at least one of the one or more driver mutations is in a gene selected from the group consisting of DNMT3 A, TET2, NPM1, SRSF2, NRAS, CDKN2A, SF3B1, FLT3, ASXL1, SRSF2, IDH2, NRAS, and SF3B1.

[0190] In some embodiments, the method further comprises determining variant allele frequency in one or more of the ctDNA, PBMCs and BMMCs of the subject. The variant allele frequency in ctDNA can be detected, for example, using polymerase chain reaction (PCR) or next generation sequencing (NGS). In some embodiments, the variant allele frequency in ctDNA is detected using droplet digital PCR (ddPCR).

[0191] At least one of the first sample, the one or more additional samples, and the second sample can be derived from whole blood of the subject, plasma of the subject, serum of the subject, or a combination thereof. In some embodiments, the ctDNA is from whole blood of the subject, plasma of the subject, serum of the subject, or a combination thereof.

[0192] In some embodiments, the subject whose ctDNA is analyzed is undergoing or will be undergoing treatment for the cancer. The method can comprise analyzing ctDNA of the subject before the treatment. The treatment can comprise one or more cycles, and the ctDNA is analyzed before, during and after one or more cycles of the treatment. For example, the ctDNA can be analyzed before, during and after two or more cycle of the treatment, three or more cycle of the treatment, or each cycle of the treatment. Each cycle of treatment can be at least 21 days, for example, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, or more, or a range between any two of these values. In some embodiments, each cycle of treatment is from about 21 days to about 28 days. In some embodiments, each cycle of treatment is from 21 days to 28 days. In some embodiments, the subject is human.

Compositions and Kits

[0193] Disclosed herein include kits, e.g., kit comprising: a PLK1 inhibitor; and a manual providing instructions for co-administrating the PLK1 inhibitor with a HIFla inhibitor to a subject for treating a cancer, wherein the cancer comprises at least one tumor with a high level of hypoxia prior to the co-administration.

[0194] In some embodiments, the cancer is colorectal cancer, bladder cancer, breast cancer, kidney cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, stomach cancer, thyroid cancer, uterine cancer, renal cancer, cervical cancer, recurrent glioblastoma, head and neck cancer, soft tissue carcinoma, vulvar cancer, melanoma, brain cancer, liver cancer, bile duct cancer, or a combination thereof. The cancer can be KRAS-mutant cancer or ER+ cancer. The cancer can be KRAS-mutant colorectal cancer (mCRC) or ER+ breast cancer. The cancer can be a metastatic cancer. The metastatic cancer can be metastatic colorectal cancer, metastatic bladder cancer, metastatic breast cancer, metastatic kidney cancer, metastatic lung cancer, metastatic ovarian cancer, metastatic pancreatic cancer, metastatic prostate cancer, metastatic stomach cancer, metastatic thyroid cancer, metastatic uterine cancer, metastatic renal cancer, metastatic cervical cancer, metastatic recurrent glioblastoma, metastatic head and neck cancer, metastatic soft tissue carcinoma, metastatic vulvar cancer, metastatic malignant melanoma, metastatic brain cancer, metastatic liver cancer, metastatic bladder cancer, metastatic bile duct cancer, or a combination thereof. The metastatic cancer can be KRAS-mutant cancer or ER+ cancer. The metastatic cancer can be KRAS-mutant metastatic colorectal cancer (mCRC) or ER+ breast cancer.

[0195] The instructions can comprise instructions for co-administrating the PLK1 inhibitor and the HIFla inhibitor simultaneously. The instructions can comprise instructions for co-administrating the PLK1 inhibitor and the HIFla inhibitor sequentially. The instructions can comprise (1) instructions for administering of the PLK1 inhibitor orally, (2) instructions for administrating the HIFla inhibitor orally, (3) instructions for administrating the HIFla inhibitor intravenously, or any combination thereof. The instructions can comprise instructions wherein the subject has not received any prior treatment comprising administration of a HIFla inhibitor. The instructions can comprise instructions for administering each of the HIFla inhibitor and the PLK1 inhibitor to the subject in a cycle of at least twice or at least five times within a week. The instructions can comprise instructions for administering the HIFla inhibitor, the PLK1 inhibitor, or both in a cycle of at least 7 days. Each cycle of treatment can be at least about 21 days. Each cycle of treatment can be from about 21 days to about 28 days. The instructions can comprise instructions for administering the PLK1 inhibitor on at least four days in the cycle. The instructions can comprise instructions for not administering the PLK1 inhibitor on at least one day in the cycle. The instructions can comprise instructions for administrating the HIFla inhibitor daily, weekly, bi-weekly, every three weeks, every four weeks, or monthly. The instructions can comprise instructions for administrating the HIFla inhibitor and the PLK1 inhibitor for at least two cycles.

[0196] The HIFla inhibitor can comprise a microRNA (miRNA), a precursor microRNA (pre-miRNA), a small interfering RNA (siRNA), a short-hairpin RNA (shRNA), an antibody, or a small molecule. In some embodiments, the HIFla inhibitor inhibits HIFla mRNA expression, inhibits HIFla protein translation, modulates HIFla protein degradation, inhibits HIFla heterodimerization, inhibits HIFla DNA binding, inhibits HIFla transcriptional activity, or any combination thereof. In some embodiments, the HIFla inhibitor is selected from the group consisting of: EZN-2968; aminoflavone; topotecan; EZN-2208; a cardiac glycoside, e.g., digoxin or PX-478; an mTOR inhibitor, e.g., temsirolimus or everolimus; an HSP90 inhibitor, e.g., galdanamycin; a histone deactylase inhibitor; an antibiotic, e.g., a quinoxaline or anthracycline; chetomin; bevacizumab; paclitaxel; and bortezomib. The HIFla inhibitor can be an angiogenesis inhibitor. The angiogenesis inhibitor can be capable of inhibiting VEGF-A, VEGFR-1, VEGFR- 2, VEGFR-3, EGFR, HER2, PDGFR family proteins, RAF, Kit (or c-Kit), FLT3, CSF-1R, RET, Abl, Itk, LcK, c-FMS, FGFR family proteins, c-Met, P1GF, TNF-a, IFNs, ILs, bFGF, mTOR, or any combination thereof. The angiogenesis inhibitor can be Afatinib (Gilotrif®), Axitinib (Inlyta®), Bevacizumab (Avastin®), Cabozantinib (Cometriq®), Cetuximab (Erbitux®), Erlotinib (Tarceva®), Everolimus (Afinitor®), Gefitinib (Iressa®), Imatinib (Gleevec®), Lapatinib (Tykerb®), Lenalidomide (Revlimid®), Lenvatinib mesylate (Lenvima®), Necitumumab (Portrazza™), Neratinib (Nerlynx®), Panitumumab (Vectibix®), Pazopanib (Votrient®), Pertuzumab (Perjeta®), Ramucirumab (Cyramza®), Regorafenib (Stivarga®), Sorafenib (Nexavar®), Sunitinib (Sutent®), Thalidomide (Synovir, Thalomid®), Trastuzumab (Ontruzant®), Vandetanib (Caprelsa®), or Ziv-aflibercept (Zaltrap®). The HIFla inhibitor can be a chemotherapeutic agent. The chemotherapeutic agent can comprise an alkylating agent, an antimetabolite, a topoisomerase inhibitor, a mitotic inhibitor, or an antitumor antibiotic. The mitotic inhibitor can be a microtubule polymerization or depolymerization inhibitor. In some embodiments, the chemotherapeutic agent is selected from the group consisting of: Altretamine, Bendamustine, Busulfan, Carboplatin, Chlorambucil, Cisplatin, Cyclophosphamide, Dacarbazine, Ifosfamide, Meehl orethamine, Melphalan, Oxaliplatin, Procarbazine, Temozolomide, Thiotepa, Trabectedin, Carmustine, Lomustine, Streptozocin, 5-fluorouracil, 6-mercaptopurine, Azacitidine, Capecitabine, Cladribine, Clofarabine, Cytarabine, Decitabine, Floxuridine, Fludarabine, Gemcitabine, Hydroxyurea, Methotrexate, Nelarabine, Pemetrexed, Pentostatin, Pralatrexate, Thioguanine, Trifluridine/tipiracil combination, Etoposide, Irinotecan, Irinotecan liposomal, Mitoxantrone Teniposide, Topotecan, Cabazitaxel, Docetaxel, Nab-paclitaxel, Paclitaxel, Vinblastine, Vincristine, Vincristine liposomal, Vinorelbine, Daunorubicin, Doxorubicin, Doxorubicin liposomal, Epirubicin, Idarubicin, Mitoxantrone, Valrubicin, Bleomycin, Dactinomycin, Mitomycin-C, Arsenic trioxide, Asparaginase, Eribulin, Ixabepilone, Mitotane, Omacetaxine, Pegaspargase, Procarbazine, Romidepsin, Vorinostat, and a combination therapy comprising folinic acid, 5-fluorouracil, and irinotecan (FOLFIRI). The PLK1 inhibitor can be selective and/or specific for PLK1. The PLK1 inhibitor can be a dihydropteridinone, a pyridopyrimidine, a aminopyrimidine, a substituted thiazolidinone, a pteridine derivative, a dihydroimidazo[l,5-f]pteridine, a metasubstituted thiazolidinone, a benzyl styryl sulfone analogue, a stilbene derivative, or any combination thereof. The PLK1 inhibitor can be onvansertib, BI2536, Volasertib (BI 6727), GSK461364, AZD1775, CYC140, HMN-176, HMN- 214, rigosertib (ON-01910), MLN0905, TKM-080301, TAK-960 or Ro3280. In some embodiments, the PLK1 inhibitor is onvansertib. [0197] The instructions can comprise instructions for administering onvansertib at 12 mg/m² - 90 mg/m². The HIF la inhibitor can be bevacizumab or paclitaxel and the PLK1 inhibitor can be onvansertib. The instructions can comprise instructions the subject has received at least one prior treatment for the cancer. In some embodiments, the prior treatment does not comprise the use of a PLK1 inhibitor. The instructions can comprise instructions the subject was in remission for cancer. In some embodiments, the subject in remission for cancer was in complete remission (CR) or in partial remission (PR). The kit can comprise the HIF la inhibitor.

EXAMPLES

[0198] Some aspects of the embodiments discussed above are disclosed in further detail in the following examples, which are not in any way intended to limit the scope of the present disclosure.

Example 1

Clinical trial of onvansertib in combination with bevacizumab and FOLFIRI

[0199] This example describes the results (as of July 25, 2022) of a Phase lb/2 clinical trial (NCT03829410) for determining the safety and efficacy of onvansertib in combination with FOLFIRI and bevacizumab (which is the current standard of care for the second line treatment of patients whose disease has relapsed or progressed following first line treatment with oxaliplatin and fluoropyrimidine). Patients received the standard doses and schedule of FOLFIRI and bevacizumab (“bev”) on Days 1 and 15 of a 28-day treatment cycle, and onvansertib orally once daily on Days 1 through 5, and Days 15 through 19 of the 28-day cycle. Patients were scanned at baseline and every eight weeks on treatment for disease response assessment. The trial was conducted to show that onvansertib safely complements and improves the efficacy of the standard or care.

[0200] The three efficacy endpoints for the Phase 2 portion of the trial are: (1) the primary efficacy endpoint: the objective response rate (or ORR); (2) secondary endpoints: other measures of disease response, including progression free survival and duration of response; and (3) for one of the exploratory endpoints, the correlation between changes in the KRAS mutation burden in circulating tumor DNA and radiographic disease response was evaluated.

[0201] Table 1 shows the enrollment and patient characteristics for the study.

TABLE 1 : PATIENT ENROLLMENT

TABLE 2: PATIENT CHARACTERISTICS

TABLE 3: STRONG, DURABLE RESPONSE ACHIEVED IN PATIENTS

[0202] Initial PRs up to eight months on treatment were observed. It was found that onvansertib in combination with FOLFIRI-Bev was well tolerated. Neutropenia was the most commonly reported TEAE, and over half of the events observed were Grade 3 or 4. Although it is part of standard FOLFIRI, the 5FU bolus is thought to add only to the toxicity of the regimen but not to the efficacy. Early in the Phase lb portion of the trial, based on feedback from our investigators, we amended the protocol to permit discontinuation of the 5FU bolus from the treatment regimen if neutropenia of grade 2 or higher was observed. Discontinuation of the 5FU bolus, with or without the use of growth factors, has subsequently been shown to ameliorate the severity of neutropenia observed.

[0203] First line use of bevacizumab in prior clinical trials had minimal impact on the efficacy of second line use. In those prior trials (e.g., Hansen et al., Cancers 2021, 13, 1031; Tabernaro et al. Eur J Cancer, 2014, 50, 320-332; Bennouna etal., Lancet Oncol. 2013, 14, 29- 37; Van Cutsem et al., J. Clin. Oncol. 2012, 30,3499-3506; Tabenaro et al., Lancet Oncol 2015, 16: 499-508; Beretta et al., Med Oncol 2013, 30:486; Moriwakij et al., Med Oncol 2012, 29:2842-2848), mPFS (mo) was 6.7 and 6.9 months and mOS (mo) was 12.5 and 13.9 months for patients that have received prior bevacizumab treatment (“prior bev”) versus patients that didn’t receive prior bevacizumab treatment (“bev naive”), respectively. The conclusion from previous trials is that the mPFS and mOS are similar in second line regardless of whether patients were bevacizumab naive or had prior bevacizumab in first line. For ORR, there is an incremental increase of 12 to 20%. Surprisingly, it was found in the present trial that the “No Prior Bev” subset had a much greater than expected ORR and mPFS (See, FIG. 1-FIG. 2). For ORR for 33 patients with prior bevacizumab treatment, there were 8/35 responders (22.9%) ORR (CI: 10.4% - 40.1%). The ORR for 15 patients without prior bevacizumab was 9/13 responders (69.2%) ORR (CI: 38.6% - 90.9%). Therefore, the ORR for patients with no prior anti-angiogenesis treatment was more than 2-fold greater than for those with prior anti-angiogenesis treatment, with an odds ratio (OR) of 0.14 (0.03-0.56, p=0.0049). PFS for patients with vs without prior bevacizumab also showed a surprising improvement for patients without prior bevacizumab treatment. In the present trial, the mPFS was 7.8 months for patients with prior bevacizumab and 13.5 months for patients without prior bevacizumab treatment (p = 0.14) (FIG. 1).

TABLE 4: EVENTS

[0204] It was found that ORR is consistently greater for bevacizumab naive patients across characteristics. No single patient characteristic explains the observed ORR difference. As shown in FIG. 3 and Table 5, the lowest ORR for the “No Prior Bev” group was 50%, with the highest 80% in RP2D. ORR ranges from 14% to 31.3% depending on the variable for patients with prior bevacizumab treatment.

TABLE 5: OBSERVED ORR

[0205] The results presented herein show a surprising improvement in multiple outcomes (e.g., ORR, PFS) for metastatic cancer patients treated with a combination of onvansertib and bevacizumab in patients without prior bevacizumab treatment. Without being bound by any particular theory, it is believed that there is a synergy between onvansertib and bevacizumab in a bevacizumab naive setting. Example 2

Onvansertib regulates the hypoxia signaling pathway

[0206] The KRAS-mutant CRC cell lines were cultured under normoxia (20% O2, Nx) or hypoxia (1% O2, Hx), in the presence (+) or absence (-) of onvansertib. As shown in FIG. 4, HIFla expression was strongly induced under hypoxia. FIG. 4 shows that onvansertib inhibited the hypoxia signaling pathway by downregulating HIFla expression in four KRAS-mutant CRC cell lines. Similarly, PLK1 inhibition using siRNA against PLK1 (siPLKl) prevented hypoxia- induced HIFla expression. FIG. 5 shows that onvansertib downregulates HIFla in a dosedependent manner. KRAS-mutant cell lines shown in FIG. 5 were cultured under hypoxic conditions (1% O2) and treated with two different doses of onvansertib. HIFla expression was assessed by ProteinSimple immunoblot. HIFla is stabilized under low levels of oxygen and promotes the gene expression of downstream targets resulting in angiogenesis, metabolic changes and survival/proliferation of tumor cells. FIG. 10A-FIG. 10F show that onvansertib inhibits the activation of the hypoxia pathway via the regulation of HIFla.

Example 3

A phase lb/2 clinical study of onvansertib in combination with FOLFIRI/bevacizumab revealed a new role of PLK1 in regulating the hypoxia pathway in KRAS-mutant colorectal cancer

[0207] Background: Onvansertib (Onv) is an oral, small molecule, selective PLK1 kinase inhibitor that demonstrated clinical activity at tolerated drug exposures in combination with FOLFIRI/Bevacizumab (Bev) in the second-line treatment of mutated KRAS (mKRAS) metastatic colorectal cancer (mCRC) (Lenz, JCO, 2022). Biomarkers of response to the combination therapy and their associated biology were explored.

[0208] Methods: In a Phase lb/2 study, mKRAS mCRC patients with prior exposure to oxaliplatin (with or without Bevacizumab) were treated with Onv (Days 1-5 and 15-19) in combination with FOLFIRI/Bev (Days 1 and 15) of each 28-day cycle (NCT03829410). Efficacy endpoints included objective response rate (ORR, RECIST vl. l), progression-free survival (PFS) and duration of response (DOR). Preclinically, the combination of Onvansertib and bevacizumab was tested in 3 mKRAS CRC xenograft models. The role of PLK1 in hypoxia was assessed through protein and RNA analyses of mKRAS CRC cell lines exposed to low oxygen levels and treated with Onvansertib.

[0209] Results: As of August 25, 2023, 66 patients were evaluable for efficacy in the Phase lb/2 and ORR of Onv + FOLFIRI/Bev was 28.8%. Patients had a median PFS of 9.8 months [95%CI: 7.6, 12.6] and median DoR of 11.7 months [95%CI: 9.0, not reached (NR)]. An analysis of baseline characteristics revealed that patients not exposed to Bevacizumab in the first-line setting (Bev-naive patients, n=15) achieved superior clinical benefit compared to Bev-exposed patients (n=51). Bev-naive patients had significantly higher ORR (73.3% versus 15.7%, p<0.0001) and longer median PFS (14.9 months [95%CI: 10.5, NR] versus 7.8 months [95%CI: 5.6, 9.8], p=0.0013) than Bev-exposed patients. These data prompted us to assess the effect of Onv+Bev in mKRAS CRC xenograft models. Onv+Bev significantly reduced tumor growth in the 3 xenograft models tested. The combination resulted in a greater decrease in tumor vascularization and tumor cell proliferation, and a corresponding increase in apoptosis, compared to either monotherapy. Further, the effect of Onvansertib on the hypoxia pathway was assessed in mKRAS CRC cell lines. Our findings indicated that under low oxygen levels, Onvansertib inhibited the hypoxia pathway by reducing the expression of the hypoxia-inducible factor 1 -alpha (HIFla) protein, and the mRNA expression of its downstream targets.

[0210] Conclusions: Onv + FOLFIRI/Bev resulted in greater clinical benefit in Bev- naive than Bev-exposed mKRAS CRC patients. Preclinical studies revealed synergistic effect of Onvansertib and bevacizumab in mKRAS CRC xenografts, and a new function of PLK1 in modulating the hypoxia pathway through the regulation of HIFla. Collectively, this data supports a crosstalk between PLK1 and angiogenesis.

Example 4

Clinical trial of onvansertib in combination with bevacizumab and FOLFIRI

[0211] This example describes the results (as of January 29, 2024) of a Phase lb/2 clinical trial (NCT03829410) for determining the safety and efficacy of onvansertib in combination with FOLFIRI and bevacizumab. Patients in this study were diagnosed with mCRC with KRAS mutation who failed or were intolerant to first-line treatment of fluoropyrimidine and oxaliplatin with or without bevacizumab. Patients received the treatment of onvansertib in combination with FOLFIRI/Bev on a 28-day cycle. During each cycle, onvansertib was given on Days 1-5 and 15-19 and FOLFIRI/Bev was given on Days 1 and 15. The Phase lb study demonstrated the safety and promising efficacy of the combined therapy. In particular, onvansertib RP2D was established at 15 mg/m². ORR was 44%, with median PFS of 12.6 months and median duration of response of 9.5 months. In this example, responses of biomarkers to onvansertib + FOLFIRI/Bev therapy in the Phase lb/2 study and their associated biology were explored.

[0212] Between July 2019 and October 2022, 68 patients were enrolled in 7 sites in the U.S., including 53 patients treated at the RP2D. As of January 29, 2024 (cut-off date), all patients have completed treatment and follow-up. Median follow-up was 7.1 months (range from 0.4-30.3 months). Reasons for discontinuation included progressive disease (n=40, 59%), pursue curative surgery (n=13, 19%), patient’s choice (n=8, 12%), adverse event (n=6, 9%), and transition to extended access program (n=l, 1%). The efficacy of onvansertib + FOLFIRI/Bev combined therapy is show in Table 6 below. TABLE 6: EFFICACY OF ONVANSERTIB + FOLFIRI/BEV ACIZUMAB,

[0213] In Table 6, “a” indicates inclusion of patients receiving all onvansertib doses, and “b” indicates inclusion of patients receiving onvansertib at RP2D. All patients receiving onvansertib at RP2D had confirmed responses with regard to ORR. Patients who received at least 1 cycle of treatment were included in the analysis. Radiographic response determined per RECIST vl. l. ORR included unconfirmed responses; and DCR included complete response, partial response and stable disease. Median duration of response (mDOR) is defined as time between first response and progression. CI indicates 95% confidence interval.

[0214] The results of this experiment show that the combination of onvansertib + FOLFIRI/Bev provided superior clinical benefits to bev-naive patients. FIG. 6 shows a subgroup analysis of baseline characteristics identified superior clinical benefit to onvansertib + FOLFIRE Bev in patients who did not receive bevacizumab in the first-line setting (Bev-naive) compared to patients who received bevacizumab in first-line treatment (Bev-exposed). ORs were based on the comparison of ORR. OR>1 indicates higher likelihood of response. Hazard ratios (HR) are based on the comparison of PFS. HR<1 indicates lower likelihood of progression. Moreover, bev-naive patients had significantly greater ORR (OR=14.8, p<0.001) than bev-exposed patient. As shown in FIG. 7, mean ORR of bev-naive patients was 73.3%, compared to 15.7% of bev-exposed patients. Bev-naive patients also had significantly longer PFS (HR=0.21, p<0.001) than bev- exposed patients, as shown in FIG. 8. In FIG. 8, the 15 bev-naive patients have mPFS of 14.9 months (95% confidence interval of [13-5 months - not reached]), and the 51 bev-exposed patients have mPFS of 7.8 months (95% confidence interval of [5.7 months - 9.8 months]). There was no evidence of differences in treatment benefit (e.g., ORR and PFS) for the other subgroups shown in FIG. 6.

[0215] In the Phase lb/2 study for second-line treatment for KRAS-mutant mCRC, the combination of onvansertib and FOLFIRI/Bev showed superior clinical benefit in patients who did not receive bevacizumab in first-line treatment (Bev-naive) compared to patients who did.

Example 5 Onvansertib + bevacizumab inhibited tumor growth and angiogenesis in KRAS-mutant colorectal cancer xenograft models

[0216] As indicated by the tumor volume shown in FIG. 9A and FIG. 9B, the combination of onvansertib and bevacizumab effectively inhibited tumor growth in 3 KRAS- mutant CRC xenograft models (e.g., SW620, LoVo and HCT116), resulting in tumor regression or stasis. Combination treatment had significantly superior antitumor activity compared to the monotherapies. Moreover, onvansertib reduced tumor vascularization. As shown in FIG. 9C, the combination of onvansertib and bevacizumab treatment resulted in greater decrease in tumor vascularization than onvansertib alone and bevacizumab alone, as indicated by reduced amount of CD31+ blood vessels. CD31, or PECAM-1 (platelet endothelial adhesion molecule), is a cellcell adhesion and signaling protein with established roles in angiogenesis.

[0217] Based on the clinical data described above, the effect of onvansertib and bevacizumab was investigated in KRAS-mutant CRC preclinical models. In vivo, onvansertib + bevacizumab resulted in potent antitumor activity in the 3 xenograft models. Onvansertib alone reduced tumor vascularization and the combination of onvansertib and bevacizumab resulted in greater decrease in tumor vascularization. In vitro, onvansertib inhibited the activation of the hypoxia pathway through the regulation of the transcription factor HIFla and its downstream targets.

[0218] The clinical activity of a combined therapy of onvansertib and chemotherapy/bevacizumab for the first-line treatment of RAS-mutated, Bev-naive mCRC patients is explored in a randomized study (NCT06106308).

Example 6

Transcriptomic changes associated with bevacizumab treatment

[0219] FIG. 12 shows the enrichment score of a number of hallmarks and oncogenic signatures related to tumors. Bev-exposed tumors were enriched for several cancer hallmarks related to PLK1 functions (e.g., hypoxia, mitotic spindle, G2M checkpoint) and oncogenic signatures related to the angiogenic factors VEGF and Pl GF. These transcriptional changes may confer resistance to onvansertib and bevacizumab.

Example 7

Combination of onvansertib and paclitaxel and HIFla in cancer

[0220] This example provides exemplary data related to the combination of onvansertib and paclitaxel according to the methods disclosed herein.

[0221] Immunoblots and quantification of HIFla expression for indicated treatments are shown in FIG. 13A-FIG. 13C. Onvansertib treatment resulted in a dose-dependent decrease in HIFla protein expression in the breast cancer cell lines, MCF7. The combination of onvansertib and paclitaxel further decreased HIFla expression. In these experiments, MCF7 cell line was treated with onvansertib and/or paclitaxel at the indicated doses for 20h and then exposed to hypoxia (1%O2) for 4h. HIFla protein expression was assessed by immunoblot and normalized to P-actin and control sample.

[0222] In at least some of the previously described embodiments, one or more elements used in an embodiment can interchangeably be used in another embodiment unless such a replacement is not technically feasible. It will be appreciated by those skilled in the art that various other omissions, additions and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter, as defined by the appended claims.

[0223] With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. The various singular/plural permutations may be expressly set forth herein for sake of clarity. As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Any reference to “or” herein is intended to encompass “and/or” unless otherwise stated.

[0224] It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (e.g., “a” and/or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of “two recitations,” without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). In those instances where a convention analogous to “at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms.

[0225] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0226] As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

[0227] While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method of treating cancer, the method comprising: administering a Polo-like kinase 1 (PLK1) inhibitor and a hypoxia inducible factor 1 a (HIFla) inhibitor to a subject with cancer, wherein the cancer comprises at least one tumor with a high level of hypoxia prior to the administering, wherein the administrating results in a decrease in the expression and/or activity of at least one HIFla pathway component in the at least one tumor of the subject, thereby reducing or inhibiting progression of the cancer.

2. The method of claim 1, wherein the at least one HIFla pathway component is HIFla, BNIP3, PFKFB4, P4HA1, ANKZF1, INSIG2, EN02, LDHA, ALDOC, PGK1, HK1, PFKP, P4HA2, KLF6, EFNA3, NFIL3, AK4, ERRFI1, SAP30, CCNG2, ANKRD37, KDM3A, ADM, DDIT4, HK2, SLC2A3, STC2, VEGFA, WSB1, PDK1, GLUT1, GLUT3, or HK2.

3. The method of any one of claims 1-2, wherein the expression of HIFla is reduced in the at least one tumor by at least 25% following administration of the PLK1 inhibitor, the HIFla inhibitor, or both, optionally as compared to the subject prior to the administration.

4. The method of any one of claims 1-3, wherein administering the PLK1 inhibitor and the HIFla inhibitor synergistically reduces HIFla expression in the at least one tumor, optionally as compared to the subject prior to the administration, a subject treated with the PLK1 inhibitor alone, or a subject treated with the HIFla inhibitor alone.

5. The method of any one of claims 1-4, wherein administering the PLK1 inhibitor and the HIFla inhibitor synergistically reduces HIFla expression or activity in the at least one tumor by at least 50%, optionally as compared to the subject prior to the administration.

6. The method of any one of claims 1-5, wherein the high level of hypoxia comprises a partial pressure of oxygen (pCh) level of about 2 mmHg to about 25 mmHg in or near the at least one tumor.

7. The method of claim 6, wherein the high level of hypoxia comprises a pCh level of about 10 mmHg in or near the at least one tumor.

8. The method of any one of claims 1-7, wherein the high level of hypoxia comprises at least 10% of the cells of the at least one tumor being positive for one or more markers for hypoxia, optionally the cells comprise a biopsy sample.

9. The method of claim 8, wherein the one or more markers of hypoxia is HIFla, glucose transporter 1 (GLUT1), and/or carbonic anhydrase 9 (CAIX).

10. The method of any one of claims 1-9, wherein the at least one tumor comprises cells over-expressing one or more HIFla target genes relative to non-cancerous and/or non- hypoxic cells prior to the administration, optionally wherein the one or more HIFla target genes is: BNIP3L, PFKFB4, P4HA1, ANKZF1, INSIG2, EN02, LDHA, A I. DOC, PGK1, HK1, PFKP, P4HA2, KLF6, EFNA3, NF1 L 3, AK4, ERRFI1, SAP 30, CCNG2, ANKRD37, KDM3A, ADM, DDIT4, HK2, SLC2A3, STC2, VEGFA, WSB1, and/or PDKE

11. The method of any one of claims 1-10, wherein the at least one tumor comprises cells comprising a mutation in a gene involved in mitochondrial metabolism, optionally the gene s SDH, FH, o ND2.

12. The method of any one of claims 1-11, wherein the at least one tumor comprises cells comprising elevated nitric oxide synthase levels, optionally as compared to non-cancerous or non-hypoxic cells.

13. The method of any one of claims 1-12, wherein the at least one tumor comprises cells comprising elevated NAD+ levels, optionally as compared to non-cancerous or non-hypoxic cells.

14. The method of any one of claims 1-13, wherein the at least one tumor comprises cells comprising a mutation in a gene causing dysregulation of the mTOR pathway, optionally the gene comprises LKB1, PML, PTEN, or TSC1/2.

15. The method of any one of claims 1-14, comprising determining a hypoxia status of the cancer prior to the administration, after the administration, or both.

16. The method of claim 15, wherein determining the hypoxia status of the cancer comprises electron paramagnetic resonance (EPR) oximetry, eppendorf electrode technique, optical methods, NMR relaxation techniques, or any combination thereof.

17. The method of any one of claims 15-16, wherein determining the hypoxia status of the cancer comprises an indirect method comprising: i) PET -based measurement of hypoxia-localizing drugs; ii) measurement of anaerobic and/or aerobic glycolysis; iii) detecting the presence, absence, or amount of one or more markers of hypoxia, optionally the one or more markers comprise HIFla, GLUT1, and/or CAIX; and/or iv) measuring one or more physiological parameters in the subject, optionally selected from MRI perfusion, laser doppler flow, and lactate levels.

18. The method of any one of claims 1-17, wherein administering the PLK1 inhibitor and the HIFla inhibitor synergistically reduces or inhibits progression of the cancer relative to the PLK1 inhibitor treatment alone, the HIFla inhibitor treatment alone, and/or the additive effect of the PLK1 inhibitor treatment alone and the HIFla inhibitor treatment alone.

19. The method of claim 18, wherein administering the PLK1 inhibitor and the HIFla inhibitor improves one or more therapeutic effects in the subject relative to a control or a baseline, and optionally wherein the one or more therapeutic effects comprise size of a tumor derived from the cancer, objective response rate (ORR), duration of response (DOR), time to response, progression free survival (PFS), overall survival (OS), disease control rate (DCR), oncogenic allelic burden, and/or expression level of CD31.

20. The method of claim 18, wherein administering the PLK1 inhibitor and the HIFla inhibitor improves the ORR in the subject, improves the DOR in the subject, improves PFS in the subject, improves OS in the subject, improves DCR in the subject, reduces oncogenic allelic burden in the subject, reduces expression level of CD31, hypoxia-inducible factor la (HIFla) and/or a factor under the regulation of HIFla, or a combination thereof, relative to subjects who have received prior treatment comprising a HIFla inhibitor.

21. The method of any one of claims 18-20, wherein administering the PLK1 inhibitor and the HIFla inhibitor improves the ORR in the subject by at least 50% relative to subjects who have received prior treatment comprising a HIFla inhibitor.

22. The method of any one of claims 18-21, wherein administering the PLK1 inhibitor and the HIFla inhibitor improves the PFS in the subject by at least 50% relative to subjects who have received prior treatment comprising a HIFla inhibitor.

23. The method of any one of claims 1-22, wherein the cancer is colorectal cancer, bladder cancer, breast cancer, kidney cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, stomach cancer, thyroid cancer, uterine cancer, renal cancer, cervical cancer, recurrent glioblastoma, head and neck cancer, soft tissue carcinoma, vulvar cancer, melanoma, brain cancer, liver cancer, bile duct cancer, or a combination thereof, optionally wherein the cancer is KRAS-mutant cancer or ER+ cancer, further optionally wherein the cancer is KRAS-mutant colorectal cancer (mCRC) or ER+ breast cancer.

24. The method of any one of claims 1-23, wherein the cancer is a metastatic cancer.

25. The method of claim 24, wherein the metastatic cancer is metastatic colorectal cancer, metastatic bladder cancer, metastatic breast cancer, metastatic kidney cancer, metastatic lung cancer, metastatic ovarian cancer, metastatic pancreatic cancer, metastatic prostate cancer, metastatic stomach cancer, metastatic thyroid cancer, metastatic uterine cancer, metastatic renal cancer, metastatic cervical cancer, metastatic recurrent glioblastoma, metastatic head and neck cancer, metastatic soft tissue carcinoma, metastatic vulvar cancer, metastatic malignant melanoma, metastatic brain cancer, metastatic liver cancer, metastatic bladder cancer, metastatic bile duct cancer, or a combination thereof, optionally wherein the metastatic cancer is KRAS- mutant cancer or ER+ cancer, further optionally wherein the metastatic cancer is KRAS-mutant metastatic colorectal cancer (mCRC) or ER+ breast cancer.

26. The method of any one of claims 1-25, wherein the PLK1 inhibitor is selective and/or specific for PLK1.

27. The method of any one of claims 1-26, wherein the PLK1 inhibitor is a dihydropteridinone, a pyridopyrimidine, an aminopyrimidine, a substituted thiazolidinone, a pteridine derivative, a dihydroimidazo[l,5-f]pteridine, a metasubstituted thiazolidinone, a benzyl styryl sulfone analogue, a stilbene derivative, or any combination thereof.

28. The method of any one of claims 1-26, wherein the PLK1 inhibitor is onvansertib, BI2536, Volasertib (BI 6727), GSK461364, AZD1775, CYC140, HMN-176, HMN-214, rigosertib (ON-01910), MLN0905, TKM-080301, TAK-960, or Ro3280.

29. The method of any one of claims 1-28, wherein the HIFla inhibitor comprises a microRNA (miRNA), a precursor microRNA (pre-miRNA), a small interfering RNA (siRNA), a short-hairpin RNA (shRNA), an antibody, or a small molecule.

30. The method of any one of claims 1-29, wherein the HIFla inhibitor inhibits HIFla mRNA expression, inhibits HIFla protein translation, modulates HIFla protein degradation, inhibits HIFla heterodimerization, inhibits HIFla DNA binding, inhibits HIFla transcriptional activity, or any combination thereof.

31. The method of any one of claims 1-30, wherein the HIFla inhibitor is selected from the group consisting of: EZN-2968; aminoflavone; topotecan; EZN-2208; a cardiac glycoside, optionally digoxin or PX-478; an mTOR inhibitor, optionally temsirolimus or everolimus; an HSP90 inhibitor, optionally galdanamycin; a histone deactylase inhibitor; an antibiotic, optionally a quinoxaline or anthracycline; chetomin; bevacizumab; paclitaxel; and bortezomib.

32. The method of any one of claims 1-31, wherein the HIFla inhibitor is an angiogenesis inhibitor.

33. The method of claim 32, wherein the angiogenesis inhibitor is capable of inhibiting VEGF-A, VEGFR-1, VEGFR-2, VEGFR-3, EGFR, HER2, PDGFR family proteins, RAF, Kit (or c-Kit), FLT3, CSF-1R, RET, Abl, Itk, LcK, c-FMS, FGFR family proteins, c-Met, P1GF, TNF-a, IFNs, ILs, bFGF, mTOR, or any combination thereof.

34. The method of any one of claims 32-33, wherein the angiogenesis inhibitor is Afatinib (Gilotrif®), Axitinib (Inlyta®), Bevacizumab (Avastin®), Cabozantinib (Cometriq®), Cetuximab (Erbitux®), Erlotinib (Tarceva®), Everolimus (Afinitor®), Gefitinib (Iressa®), Imatinib (Gleevec®), Lapatinib (Tykerb®), Lenalidomide (Revlimid®), Lenvatinib mesylate (Lenvima®), Necitumumab (Portrazza™), Neratinib (Nerlynx®), Panitumumab (Vectibix®), Pazopanib (Votrient®), Pertuzumab (Perjeta®), Ramucirumab (Cyramza®), Regorafenib (Stivarga®), Sorafenib (Nexavar®), Sunitinib (Sutent®), Thalidomide (Synovir, Thalomid®), Trastuzumab (Ontruzant®), Vandetanib (Caprelsa®), or Ziv-aflibercept (Zaltrap®).

35. The method of any one of claims 1-31, wherein the HIFla inhibitor is a chemotherapeutic agent; optionally the chemotherapeutic agent comprises an alkylating agent, an antimetabolite, a topoisomerase inhibitor, a mitotic inhibitor, or an antitumor antibiotic; further optionally, the mitotic inhibitor is a microtubule polymerization or depolymerization inhibitor.

36. The method of claim 35, wherein the chemotherapeutic agent is selected from the group consisting of: Altretamine, Bendamustine, Busulfan, Carboplatin, Chlorambucil, Cisplatin, Cyclophosphamide, Dacarbazine, Ifosfamide, Mechlorethamine, Melphalan, Oxaliplatin, Procarbazine, Temozolomide, Thiotepa, Trabectedin, Carmustine, Lomustine, Streptozocin, 5- fluorouracil, 6-mercaptopurine, Azacitidine, Capecitabine, Cladribine, Clofarabine, Cytarabine, Decitabine, Floxuridine, Fludarabine, Gemcitabine, Hydroxyurea, Methotrexate, Nelarabine, Pemetrexed, Pentostatin, Pralatrexate, Thioguanine, Trifluridine/tipiracil combination, Etoposide, Irinotecan, Irinotecan liposomal, Mitoxantrone Teniposide, Topotecan, Cabazitaxel, Docetaxel, Nab-paclitaxel, Paclitaxel, Vinblastine, Vincristine, Vincristine liposomal, Vinorelbine, Daunorubicin, Doxorubicin, Doxorubicin liposomal, Epirubicin, Idarubicin, Mitoxantrone, Valrubicin, Bleomycin, Dactinomycin, Mitomycin-C, Arsenic trioxide, Asparaginase, Eribulin, Ixabepilone, Mitotane, Omacetaxine, Pegaspargase, Procarbazine, Romidepsin, Vorinostat, and a combination therapy comprising folinic acid, 5 -fluorouracil, and irinotecan (FOLFIRI).

37. The method of any one of claims 1-31, wherein the HIFla inhibitor is paclitaxel.

38. The method of any one of claims 1-31, wherein the HIFla inhibitor is bevacizumab.

39. The method of any one of claims 1-38, wherein the PLK1 inhibitor and the HIFla inhibitor are co-administered simultaneously.

40. The method of any one of claims 1-38, wherein the PLK1 inhibitor and the HIFla inhibitor are administered sequentially.

41. The method of claim 40, wherein the PLK1 inhibitor is administered prior to the administration of the HIFla inhibitor, and optionally wherein the PLK1 inhibitor is administered prior to the administration of the HIFla inhibitor every day on which the subject is administered with the PLK1 inhibitor and the HIFla inhibitor.

42. The method of claim 41, wherein the PLK1 inhibitor is administered about 30 minutes to about 5 hours prior to the administration of the HIFla inhibitor on a given day.

43. The method of any one of claims 1-42, wherein the administration of the PLK1 inhibitor comprises oral administration, and the administration of the HIFla inhibitor comprises intravenous administration or oral administration.

44. The method of any one of claims 1-43, wherein the HIFla inhibitor and the PLK1 inhibitor are each administered to the subject in a cycle of at least twice or at least five times within a week.

45. The method of any one of claims 1-44, wherein the HIFla inhibitor, the PLK1 inhibitor, or both are administered in a cycle of at least 7 days; optionally each cycle of treatment is at least about 21 days; and further optionally each cycle of treatment is from about 21 days to about 28 days.

46. The method of any one of claims 44-45, wherein the PLK1 inhibitor is administered on at least four days in the cycle.

47. The method of any one of claims 45-46, wherein the PLK1 inhibitor is not administered on at least one day in the cycle.

48. The method of any one of claims 1-47, wherein the HIFla inhibitor is administered daily, weekly, bi-weekly, every three weeks, every four weeks, or every month.

49. The method of claims 44-48, wherein the subject undergoes at least two cycles of the administration of the HIFla inhibitor and the PLK1 inhibitor.

50. The method of any one of claims 1-49, wherein the PLK1 inhibitor is onvansertib and/or the HIF 1 a inhibitor is bevacizumab or paclitaxel.

51. The method of claim 50, wherein onvansertib is administered at 12 mg/m² - 90 mg/m².

52. The method of any one of claims 50-51 , wherein a maximum concentration (Cmax) of onvansertib in a blood of the subject is from about 100 nmol/L to about 1500 nmol/L.

53. The method of any one of claims 50-52, wherein an area under curve (AUC) of a plot of a concentration of onvansertib in a blood of the subject over time is from about 1000 nmol/L. hour to about 400000 nmol/L. hour.

54. The method of any one of claims 50-53, wherein a time (Tmax) to reach a maximum concentration of onvansertib in a blood of the subject is from about 1 hour to about 5 hours.

55. The method of any one of claims 50-54, wherein an elimination half-life (T1/2) of onvansertib in a blood of the subject is from about 10 hours to about 60 hours.

56. The method of any one of claims 50-55, wherein the bevacizumab is administered at about 1 mg/kg - 20 mg/kg.

57. The method of claim 56, wherein the bevacizumab is administered at about 5 mg/kg, about 7.5 mg/kg, about 10 mg/kg, or about 15 mg/kg.

58. The method of any one of claims 50-55, wherein the paclitaxel is administered at about 50 mg/m² to about 175 mg/m².

59. The method of any one of claims 1-58, wherein the subject has received at least one prior cancer treatment, and optionally wherein the prior treatment does not comprise the use of a HIFla inhibitor, a PLK1 inhibitor, or both.

60. The method of any one of claims 1-59, wherein the subject was in remission for cancer, optionally wherein the subject in remission for cancer was in complete remission (CR) or in partial remission (PR).

61. The method of any one of claims 1-60, further comprising one or more of (1) determining cancer status of the subject, (2) determining responsiveness of the subject to a PLK1 inhibitor treatment, and (3) administering one or more cancer therapeutics or therapies for the cancer.

62. The method of any one of claims 1-61, wherein the subject is human.

63. The method of any one of claims 61-62, wherein the one or more cancer therapeutics or therapies comprise FOLFIRI, abiraterone, FOLFOX, an anti-EGFR agent, a KRAS directed inhibitor, gemcitabine, abraxane, paclitaxel, nanoliposomal irinotecan, 5-FU, or a combination thereof; wherein the anti-EGFR agents is optionally cetuximab, and KRAS directed inhibitor is optionally a G12C inhibitor, a G12D inhibitor or a combination thereof.

64. The method of any one of claims 61-63, wherein determining the responsiveness of the subject comprises determining if the subject is a responder of the treatment, if the subject is or is going to be in complete recovery (CR), or if the subject is or is going to be in partial remission (PR).

65. The method of any one of claims 61-64, wherein determining the responsiveness of the subject comprises determining objective response rate (ORR), duration of response (DOR), time to response, progression free survival (PFS), overall survival (OS), disease control rate (DCR), oncogenic allelic burden, or a combination thereof of the subject.

66. The method of any one of claims 61-65, wherein determining the responsiveness of the subject comprises determining if the subject has a partial response to the treatment, if the subject has a complete response to the treatment, if the subject has a stable disease (SD) status, or if the subject has a progressive disease (PD) status.

67. A kit, comprising a Polo-like kinase 1 (PLK1) inhibitor; and a manual providing instructions for co-administrating the PLK1 inhibitor with a hypoxia inducible factor 1 a (HIFla) inhibitor to a subject for treating a cancer, wherein the cancer comprises at least one tumor with a high level of hypoxia prior to the co-administration.

68. The kit of claim 67, wherein the cancer is colorectal cancer, bladder cancer, breast cancer, kidney cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, stomach cancer, thyroid cancer, uterine cancer, renal cancer, cervical cancer, recurrent glioblastoma, head and neck cancer, soft tissue carcinoma, vulvar cancer, melanoma, brain cancer, liver cancer, bile duct cancer, or a combination thereof, optionally wherein the cancer is KRAS-mutant cancer or ER+ cancer, further optionally wherein the cancer is KRAS-mutant colorectal cancer (mCRC) or ER+ breast cancer.

69. The kit of any one of claims 67-68, wherein the cancer is a metastatic cancer.

70. The kit of claim 69, wherein the metastatic cancer is metastatic colorectal cancer, metastatic bladder cancer, metastatic breast cancer, metastatic kidney cancer, metastatic lung cancer, metastatic ovarian cancer, metastatic pancreatic cancer, metastatic prostate cancer, metastatic stomach cancer, metastatic thyroid cancer, metastatic uterine cancer, metastatic renal cancer, metastatic cervical cancer, metastatic recurrent glioblastoma, metastatic head and neck cancer, metastatic soft tissue carcinoma, metastatic vulvar cancer, metastatic malignant melanoma, metastatic brain cancer, metastatic liver cancer, metastatic bladder cancer, metastatic bile duct cancer, or a combination thereof, optionally wherein the metastatic cancer is KRAS- mutant cancer or ER+ cancer, further optionally wherein the metastatic cancer is KRAS-mutant metastatic colorectal cancer (mCRC) or ER+ breast cancer.

71. The kit of any one of claims 67-70, wherein the instructions comprise instructions for co-administrating the PLK1 inhibitor and the HIFla inhibitor simultaneously.

72. The kit of any one of claims 67-70, wherein the instructions comprise instructions for co-administrating the PLK1 inhibitor and the HIFla inhibitor sequentially.

73. The kit of any one of claims 67-72, wherein the instructions comprise (1) instructions for administering of the PLK1 inhibitor orally, (2) instructions for administrating the HIFla inhibitor orally, (3) instructions for administrating the HIFla inhibitor intravenously, or any combination thereof.

74. The kit of any one of claims 67-73, wherein the instructions comprise instructions wherein the subject has not received any prior treatment comprising administration of a HIFla inhibitor.

75. The kit of any one of claims 67-74, wherein the instructions comprise instructions for administering each of the HIFla inhibitor and the PLK1 inhibitor to the subject in a cycle of at least twice or at least five times within a week.

76. The kit of any one of claims 67-75, wherein the instructions comprise instructions for administering the HIFla inhibitor, the PLK1 inhibitor, or both in a cycle of at least 7 days; and optionally wherein each cycle of treatment is at least about 21 days, and further optionally each cycle of treatment is from about 21 days to about 28 days.

77. The kit of claim 76, wherein the instructions comprise instructions for administering the PLK1 inhibitor on at least four days in the cycle.

78. The kit of any one of claims 76-77, wherein the instructions comprise instructions for not administering the PLK1 inhibitor on at least one day in the cycle.

79. The kit of any one of claims 67-78, wherein the instructions comprise instructions for administrating the HIFla inhibitor daily, weekly, bi-weekly, every three weeks, every four weeks, or monthly.

80. The kit of any one of claims 75-79, wherein the instructions comprise instructions for administrating the HIFla inhibitor and the PLK1 inhibitor for at least two cycles.

81. The kit of any one of claims 67-80, wherein the HIFla inhibitor comprises a microRNA (miRNA), a precursor microRNA (pre-miRNA), a small interfering RNA (siRNA), a short-hairpin RNA (shRNA), an antibody, or a small molecule.

82. The kit of any one of claims 67-81, wherein the HIFla inhibitor inhibits HIFla mRNA expression, inhibits HIFla protein translation, modulates HIFla protein degradation, inhibits HIFla heterodimerization, inhibits HIFla DNA binding, inhibits HIFla transcriptional activity, or any combination thereof.

83. The kit of any one of claims 67-82, wherein the HIFla inhibitor is selected from the group consisting of: EZN-2968; aminoflavone; topotecan; EZN-2208; a cardiac glycoside, optionally digoxin or PX-478; an mTOR inhibitor, optionally temsirolimus or everolimus; an HSP90 inhibitor, optionally galdanamycin; a histone deactylase inhibitor; an antibiotic, optionally a quinoxaline or anthracycline; chetomin; bevacizumab; paclitaxel; and bortezomib.

84. The kit of any one of claims 67-83, wherein the HIFla inhibitor is an angiogenesis inhibitor.

85. The kit of claim 84, wherein the angiogenesis inhibitor is capable of inhibiting VEGF-A, VEGFR-1, VEGFR-2, VEGFR-3, EGFR, HER2, PDGFR family proteins, RAF, Kit (or c-Kit), FLT3, CSF-1R, RET, Abl, Itk, LcK, c-FMS, FGFR family proteins, c-Met, P1GF, TNF-a, IFNs, ILs, bFGF, mTOR, or any combination thereof.

86. The kit of any one of claims 84-85, wherein the angiogenesis inhibitor is Afatinib (Gilotrif®), Axitinib (Inlyta®), Bevacizumab (Avastin®), Cabozantinib (Cometriq®), Cetuximab (Erbitux®), Erlotinib (Tarceva®), Everolimus (Afinitor®), Gefitinib (Iressa®), Imatinib (Gleevec®), Lapatinib (Tykerb®), Lenalidomide (Revlimid®), Lenvatinib mesylate (Lenvima®), Necitumumab (Portrazza™), Neratinib (Nerlynx®), Panitumumab (Vectibix®), Pazopanib (Votrient®), Pertuzumab (Perjeta®), Ramucirumab (Cyramza®), Regorafenib (Stivarga®), Sorafenib (Nexavar®), Sunitinib (Sutent®), Thalidomide (Synovir, Thalomid®), Trastuzumab (Ontruzant®), Vandetanib (Caprelsa®), or Ziv-aflibercept (Zaltrap®).

87. The kit of any one of claims 67-83, wherein the HIFla inhibitor is a chemotherapeutic agent; optionally the chemotherapeutic agent comprises an alkylating agent, an antimetabolite, a topoisomerase inhibitor, a mitotic inhibitor, or an antitumor antibiotic.

88. The kit of claim 87, wherein the chemotherapeutic agent is selected from the group consisting of: Altretamine, Bendamustine, Busulfan, Carboplatin, Chlorambucil, Cisplatin, Cyclophosphamide, Dacarbazine, Ifosfamide, Mechlorethamine, Melphalan, Oxaliplatin, Procarbazine, Temozolomide, Thiotepa, Trabectedin, Carmustine, Lomustine, Streptozocin, 5- fluorouracil, 6-mercaptopurine, Azacitidine, Capecitabine, Cladribine, Clofarabine, Cytarabine, Decitabine, Floxuridine, Fludarabine, Gemcitabine, Hydroxyurea, Methotrexate, Nelarabine, Pemetrexed, Pentostatin, Pralatrexate, Thioguanine, Trifluridine/tipiracil combination, Etoposide, Irinotecan, Irinotecan liposomal, Mitoxantrone Teniposide, Topotecan, Cabazitaxel, Docetaxel, Nab-paclitaxel, Paclitaxel, Vinblastine, Vincristine, Vincristine liposomal, Vinorelbine, Daunorubicin, Doxorubicin, Doxorubicin liposomal, Epirubicin, Idarubicin, Mitoxantrone, Valrubicin, Bleomycin, Dactinomycin, Mitomycin-C, Arsenic trioxide, Asparaginase, Eribulin, Ixabepilone, Mitotane, Omacetaxine, Pegaspargase, Procarbazine, Romidepsin, Vorinostat, and a combination therapy comprising folinic acid, 5 -fluorouracil, and irinotecan (FOLFIRI).

89. The kit of any one of claims 67-88, wherein the PLK1 inhibitor is selective and/or specific for PLK1.

90. The kit of any one of claims 67-89, wherein the PLK1 inhibitor is a dihydropteridinone, a pyridopyrimidine, a aminopyrimidine, a substituted thiazolidinone, a pteridine derivative, a dihydroimidazo[l,5-f]pteridine, a metasubstituted thiazolidinone, a benzyl styryl sulfone analogue, a stilbene derivative, or any combination thereof.

91. The kit of any one of claims 67-89, wherein the PLK1 inhibitor is onvansertib, BI2536, Volasertib (BI 6727), GSK461364, AZD1775, CYC140, HMN-176, HMN-214, rigosertib (ON-01910), MLN0905, TKM-080301, TAK-960 or Ro3280; and optionally wherein the PLK1 inhibitor is onvansertib.

92. The kit of claim 91, wherein the instructions comprise instructions for administering onvansertib at 12 mg/m² - 90 mg/m².

93. The kit of any one of claims 67-92, wherein the HIF la inhibitor is bevacizumab or paclitaxel and the PLK1 inhibitor is onvansertib.

94. The kit of any one of claims 67-93, wherein the instructions comprise instructions the subject has received at least one prior treatment for the cancer, and optionally wherein the prior treatment does not comprise the use of a PLK1 inhibitor.

95. The kit of any one of claims 67-94, wherein the instructions comprise instructions the subject was in remission for cancer, and optionally wherein the subject in remission for cancer was in complete remission (CR) or in partial remission (PR).

96. The kit of any one of claims 67-95, further comprising the HIF la inhibitor.