STRATIFICATE AND METHOD TO TREAT A PATIENT SUFFERING
FROM A CANCER
FIELD OF THE INVENTION:
The present invention relates to a method of identifying a patient having or at risk of having or developing a resistance to anti-PD-1 therapy based on the expression level of of the extracellular vesicles adenosine methylated miR-125a-5p (m6A-miR-125a-5p).
BACKGROUND OF THE INVENTION:
Immunotherapy with antibodies disrupting the PD-1/PD-L1 axis provides an important breakthrough in the treatment of cancer as these antibodies have improved the survival outcomes of patients in many cancers types [1], For this reason, the use of antibodies against PD1/PDL1 alone or in combination with other drugs became a gold standard of first line treatment in different advanced/metastatic tumor types. Furthermore in addition to the growing FDA approved indications (e.g. more than ten)), a large number of clinical trials continue to investigate the efficiency of anti-PD-1 therapy alone or in combination with another anticancer therapy in various indications.
Before being involved in the regulation of the immune response and considered one of the most important negative immune checkpoints, PD-1 (Programmed cell Death Protein 1, UniProtKB reference: Q15116) was initially described as a gene associated with apoptosis during thymic T cell selection. Mechanistically, the canonical PD-1 pathway in T cells includes its binding with its ligands CD274/PD-L1 and CD273/PD-L2, the activation of the SHP1/2 signaling pathways and inhibition of PI3K/AKT, MAPK and mTOR pathways to deliver immune escape messages: suppression of T cell proliferation, cytokine production and cytotoxic functions [2], Consequently, the blockade of the PD-1/PD-L1 or PD-L2 axis by antibodies such as Nivolumab (Opdivo), Pembrolizumab (Keytruda) or Cemiplimab (Libtayo) appeared as a promising strategy to restore the antitumor immune response. However, some patients have been completely refractory to anti-PD-1 therapy, and a large part of patients do not present durable remission.
To identify the molecular mechanisms responsible for these phenomena, most studies have focused on immune cells since PD-1 was initially described as being mainly expressed by these cells. Thus, reduced dendritic cells maturation, suboptimal T-cell activation, impaired T- cell trafficking and infiltration, stroma-dependent exclusion, additive negative immune checkpoint on immune cells, presence of immunosuppressive cells have been identified as causes of anti-PD-1 therapy escape [3],
In addition, several authors highlighted that that the PD-1 blockade on the surface of tumors cells could also play a role in the resistance to anti-PD-1 therapy. Kleffel et al. (2015) [4], Li et al. (2017) [5], Du et al. (2018) [6], Pu et al. (2019) [7] and Wang et al. (2020) [8] reported that melanoma, hepatocellular carcinoma, pancreatic ductal adenocarcinoma, lung cancer cells expressed PD-1, respectively. As reported by Yao et al (2018) [9], several databases (TCGA, Human protein Atlas or Cancer Cell Line Encyclopedia (CCLE)) indicate that a large variety of cancer cells express PD-1. Based on these reports, it seems legitimate to question the effect of anti-PD-1 therapy on PD-1 expressing tumor cells. In the context of lung cancer, literature reports that the PD-1 silencing or blockade promoted cell proliferation or colony formation in cellular assay, and tumor growth in in vivo assay (Du et al. (2018) [6] and Wang et al. (2020) [8]). Thus, the use of anti-PD-1 therapy appears paradoxical: on the one hand, anti- PD-1 therapy can provide anti-tumor response by the immune system activation, and on the other hand anti-PD-1 therapy can provide pro-tumor response by increasing the proliferation of tumor cells.
Extracellular vesicles (EVs, i.e. exosomes and microvesicles) and their contents (miRNA, protein, mRNA,...) have been considered as candidate biomarkers for the diagnosis, the monitoring of diseases progression and the prediction of response to cancer therapy [10], Numerous studies show correlations between the proteome and transcriptome of tumor cells and that of EVs circulating in the biological fluids (blood, urine, saliva,...) of patients with different cancers. Moreover, EVs are also considered as actors of cell-cell communication via the transfer of the biological material they contain. Since Valadi et al. (2007) [11] and the description that exosomes transfer mRNA and miRNA between mast cells, a large number of articles have reinforced the major role played by EVs and particularly exosomes in intercellular communication. Our lab also contributed to this by demonstrating that radiotherapy-induced overexpression of exosomal miRNA-378a-3p in cancer cells limits the cytotoxicity of natural killer cells [12], and by reporting that anti-PDl therapy induces lymphocyte-derived exosomal miRNA-4315 release inhibiting Bim-mediated apoptosis of tumor cells [13], Moreover, in these two articles, it appears that the longitudinal study of the expression levels of exomiR-4315 and exomiR-378 during the administration of anticancer treatments can be used as biomarker.
In recent years, epitranscriptomic studies of miRNAs (i.e. the study of reversible chemical modifications occurring within microRNAs (whether mature or not)) have been booming with the development of sequencing methods and/or the adaptation of DNA methylation analysis methods. Indeed, the work of Berulava et al. (2015) [14] with a method coupling m6A-IP and sequencing, Konno et al. (2019) [15] with the detection of methylated miRNA using MALDI-TOF-MS, Pandolfini et al. (2019) [16] with a method coupling m7G-IP and sequencing, Cheray et al. (2020) [17] with BS-miRNA-seq and the use of a method coupling m5C-IP and qPCR-Array, and Carissimi et al. (2021) [18] with the use of BS-miRNA- seq and MAmBA methods have increased the knowledge in the field of epitranscriptomics of miRNA. Moreover, some of these works have highlighted the biomarker character that methylation of some miRs can hold. For example, Konno et al. (2019) [15] report that the adenosine methylation of miR-17-5p is a biomarker for early pancreatic cancer patient from healthy patients. Cheray et al (2020) [17] report that the high level of cytosine methylation of miR-181a-5p was associated with a poor survival of GBM patients. Briand et al. (2020) [19] report that the adenosine methylation of miR-200b-3p was associated with a prognosis of survival for GBM patients. Zhang et al. (2021) report that the level of serum circulating m6A miRNAs can be used as biomarker for cancer detection and the development of non-invasive therapeutic monitoring [20],
To further understand the molecular mechanisms involved in the protumoral effect of anti-PD-1 therapy on lung cancer cells, the inventors propose here an original study aiming at defining whether an epitranscriptomic reprogramming of miRNAs (i.e. their adenosine, cytosine and guanosine methylation, here) in lung cancer cells could be at the origin of the anti- PD-1 therapy-mediated protumoral effect.
SUMMARY OF THE INVENTION:
In this study, the inventors analyzed the adenosine methylation level of miR-125a-5p (m6A-miR-125a-5p) using RNA immunoprecipitation with an anti-methyl-base-antibody followed by qPCR. CLIP and ELISA were used to study the effect of m6A-miR-125a-5p on its ability to be recruited on GW182 and impact PD-1 expression, respectively. They showed that the METTL3 -mediated m6A-miR-125a-5p regulates the expression of IGSF11/VSIG3 which acts as a molecular determinant of the immunogenicity of tumor cells. By deciphering this molecular mechanism, our work also identified anti-IGSFl l antibody -based immunotherapy and METTL3 inhibitor-based therapy as both therapeutic solutions abrogating the anti-PD-1 therapy failure mediated by the miR-125a-5p-METTL3-IGSFl l/VSIG3 axis. Observations in two lung cancer patients treated with anti-PD-1 therapy show that the m6A-miR-125a-5p level is analyzable from EVs/exosomes from longitudinal blood samples. Experiments performed with EVs/exosomes of lung cancer patients treated with anti-PD-1 therapy also provide evidence that the m6A-exomiR-125a-5p level modulates the ISGF11/VSIG3 expression in lung cancer cells and their lysis by IL-2-activated PMBC. These data provide that m6A-miR-125a- 5p level can be used as a biomarker and therapeutic solutions (anti-IGSFl l antibody and METTL3 inhibitor) could potentially address the anti-PDl therapy failure in a context of precision and personalized medicine intended for the right patient, at the right time, with the right therapy.
Thus, the present invention relates to a method of identifying a patient having or at risk of having or developing a resistance to anti-PD-1 therapy based on the expression level of of the extracellular vesicles adenosine methylated miR-125a-5p (m6A-miR-125a-5p). Particularly, the invention is defined by its claims.
DETAILED DESCRIPTION OF THE INVENTION:
Prognostic method
In a first aspect, the invention relates to a method of identifying a patient having or at risk of having or developing a resistance to anti-PD-1 therapy comprising the steps consisting of i) determining the expression level of the extracellular vesicles adenosine methylated miR- 125a-5p (m6A-miR-125a-5p) in a sample from said patient, ii) comparing said expression level with a predetermined reference value and iii) concluding that the patient has or is at risk of having or developing a resistance to the anti-PDl therapy when the expression level of the extracellular vesicles adenosine methylated miR-125a-5p is superior to the predetermined reference value and concluding that the patient has not or is not at risk of having or not develop a resistance to the anti-PDl therapy when the expression level of the extracellular vesicles adenosine methylated miR-125a-5p is inferior to the predetermined reference value.
In other words, the invention relates to a method of stratification of a patient treated by anti-PD-1 therapy.
In a particular embodiment, the presence or the apparition of adenosine methylated miR- 125a-5p means that the patient has or is at risk of having or developing a resistance to anti-PD- 1 therapy.
The invention also relates to a method for predicting an anti-PD-1 therapy response of a patient suffering from a cancer in need thereof, comprising i) determining in a sample obtained from the patient the expression level of the extracellular vesicles adenosine methylated miR- 125a-5p ii) comparing said expression level with a predetermined reference value and iii) concluding that the patient will not respond to the anti-PDl therapy when the expression level of the extracellular vesicles adenosine methylated miR-125a-5p is superior to the predetermined reference value and concluding that the patient will respond to the anti-PDl therapy when the expression level of the extracellular vesicles adenosine methylated miR-125a-5p is inferior to the predetermined reference value.
According to the invention, the methods of the invention are particularly in vitro methods.
In one embodiment, the cancer may be any solid or liquid cancer. Typically, the cancer may be selected from the group consisting of bile duct cancer (e.g. periphilar cancer, distal bile duct cancer, intrahepatic bile duct cancer), bladder cancer, bone cancer (e.g. osteoblastoma, osteochrondroma, hemangioma, chondromyxoid fibroma, osteosarcoma, chondrosarcoma, fibrosarcoma, malignant fibrous histiocytoma, giant cell tumor of the bone, chordoma, lymphoma, multiple myeloma), brain and central nervous system cancer (e.g. meningioma, astocytoma, oligodendrogliomas, glioblastoma, ependymoma, gliomas, medulloblastoma, ganglioglioma, Schwannoma, germinoma, craniopharyngioma), breast cancer (e.g. ductal carcinoma in situ, infiltrating ductal carcinoma, infiltrating, lobular carcinoma, lobular carcinoma in, situ, gynecomastia), Castleman disease (e.g. giant lymph node hyperplasia, angiofollicular lymph node hyperplasia), cervical cancer, colorectal cancer, endometrial cancer (e.g. endometrial adenocarcinoma, adenocanthoma, papillary serous adnocarcinroma, clear cell), esophagus cancer, gallbladder cancer (mucinous adenocarcinoma, small cell carcinoma), gastrointestinal carcinoid tumors (e.g. choriocarcinoma, chorioadenoma destruens), Hodgkin's disease, non-Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer (e.g. renal cell cancer), laryngeal and hypopharyngeal cancer, liver cancer (e.g. hemangioma, hepatic adenoma, focal nodular hyperplasia, hepatocellular carcinoma), lung cancer (e.g. small cell lung cancer, nonsmall cell lung cancer), mesothelioma, plasmacytoma, nasal cavity and paranasal sinus cancer (e.g. esthesioneuroblastoma, midline granuloma), nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma (e.g. embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, pleomorphic rhabdomyosarcoma), salivary gland cancer, skin cancer (e.g. melanoma, nonmelanoma skin cancer), stomach cancer, testicular cancer (e.g. seminoma, nonseminoma germ cell cancer), thymus cancer, thyroid cancer (e.g. follicular carcinoma, anaplastic carcinoma, poorly differentiated carcinoma, medullary thyroid carcinoma, thyroid lymphoma), vaginal cancer, vulvar cancer, and uterine cancer (e.g. uterine leiomyosarcoma). In a particular embodiment, the cancer is a glioblastoma (GBM), a lung cancer, a breast cancer or an ovarian cancer.
In a particular embodiment, the glioblastoma is a glioblastoma multiforme (GBM) and the lung cancer is a lung adenocarcinoma.
Typically, the sample according to the invention may be blood, plasma, serum sample, T cell-derived exosomes or a cancer biopsy.
In a particular embodiment, the term “extracellular vesicles” or “EV” denotes exosomes and microvesicles. Thus, the extracellular vesicles can be exosomal vesicles.
According to the invention, the term ‘exosomal adenosine methylated miR-125a-5p” denotes the presence of the adenosine methylated miR-125a-5p in exosome (or microvesicles) product by cells like T cells.
According to the invention, the term “patient” or “subject” denotes a mammal, such as a rodent, a feline, a canine, and a primate. In some embodiments, the subject is a human. In some embodiments, the subject is a human infant. Particularly, the subject denotes a human with a cancer and particularly a GBM, a lung cancer, a breast cancer or an ovarian cancer.
As used herein, the term “miR-125a-5p” denotes a miRNA gene located on chromosome 19 (19ql3.41) and is accessible in the miRBase database under the ID number: MIMAT0000443.
Particularly, the miR-125a-5p has the nucleic acid sequence of the mature miR-125a-5p is (5’ - 3’) : UCCCUGAGACCCUUUAACCUGUGA (SEQ ID NO: 1).
Particularly, the term adenosine methylated miR-125a-5p denotes that the miR-125-5p is methylated is the adenosine 6 (m6a-miR-125a-5p).
As used herein the term “exosomal adenosine methylated miR-125a-5p” denotes the presence of the adenosine methylated miR-125a-5p in exosomes. In a particular embodiment, the “exosomal adenosine methylated miR-125a-5p” are exosomes containing the adenosine methylated miR-125a-5p derived from T cells exposed to the anti-PD-1 therapy.
As used herein, the term “the expression level of the extracellular vesicles adenosine methylated miR-125a-5p” or ‘the level of the extracellular vesicles adenosine methylated miR- 125a-5p” denotes the level of extracellular vesicles adenosine methylated miR-125a-5p compared to the total of a miR-125a-5p (no adenosine methylated).
As used herein, the term “anti-PD-1 therapy denotes the use of at least one antibody anti-PD-1 to treat the cancer of the patient. For example, the antibody anti-PD-1 can be the nivolumab, the pembrolizumab or the cemiplimab. In a particular embodiment, the patient may receive simultaneously, separately or in a sequential manner a standard chemotherapy with the anti-PD-1 therapy.
As used herein, the term “standard chemotherapy” denotes a classical anti-cancer agent selected in the group consisting but not limited to cytarabine, anthracyclines, fludarabine, gemcitabine, capecitabine, methotrexate, taxol, taxotere, mercaptopurine, thioguanine, hydroxyurea, cyclophosphamide, ifosfamide, nitrosoureas, platinum complexes such as cisplatin, carboplatin and oxaliplatin, mitomycin, dacarbazine, procarbizine, etoposide, teniposide, campathecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, L-asparaginase, doxorubicin, epimbicm, 5-fluorouracil, taxanes such as docetaxel and paclitaxel, leucovorin, levamisole, irinotecan, estramustine, etoposide, nitrogen mustards, BCNU, nitrosoureas such as carmustme and lomustine, vinca alkaloids such as vinblastine, vincristine and vinorelbine, imatimb mesylate, hexamethyhnelamine, topotecan, kinase inhibitors, phosphatase inhibitors, ATPase inhibitors, tyrphostins, protease inhibitors, inhibitors herbimycm A, genistein, erbstatin, and lavendustin A. In one embodiment, additional anticancer agents may be selected from, but are not limited to, one or a combination of the following class of agents: alkylating agents, plant alkaloids, DNA topoisomerase inhibitors, anti-folates, pyrimidine analogs, purine analogs, DNA antimetabolites, taxanes, podophyllotoxin, hormonal therapies, retinoids, photosensitizers or photodynamic therapies, angiogenesis inhibitors, antimitotic agents, isoprenylation inhibitors, cell cycle inhibitors, actinomycins, bleomycins, MDR inhibitors and Ca2+ ATPase inhibitors.
Particularly, the standard chemotherapy can be the oxaliplatin, the cisplatin, the temozolomide, the cyclophosphamide, the doxorubicin or the paclitaxel.
In another embodiment, the patient affected with a cancer and particularly a lung cancer can also be treated with a standard treatment consisting of maximal surgical resection, radiotherapy, and concomitant adjuvant standard chemotherapy.
In one embodiment and according to the methods of the invention, the determination of the expression level of the extracellular vesicles adenosine methylated miR-125a-5p of the invention may be determined before or after the beginning of a treatment with the anti-PD-1 therapy of the patient.
The term "determining the expression level of as used above includes qualitative and/or quantitative detection (measuring levels) with or without reference to a control. Typically expression level of the miR of the invention may be measured for example by RNA- immunoprecipitation, Cross-linking immunoprecipitation, qRT-PCR performed and all RNA sequencing methods on the sample. The “reference value” may be a healthy subject, i.e. a subject who does not suffer from any cancer and particularly glioblastoma. Particularly, said control is a not an healthy subject. In another embodiment, the “reference value” may be a subject having a cancer without resistance to anti-PD-1 therapy.
Measuring the expression level of a miR can be performed by a variety of techniques well known in the art. In the case of the invention , before determining the level of the miR of the invention, the exosomes derived from T cells (T cell-derived exosomes) will be isolated and quantified by any technique allowing that (see for example the materials and methods part of the application). Particular, the “ExoQuick” (see the materials and methods part) can be used to isolate the exosomes.
Methods for determining the quantity of miR are well known in the art. For example the nucleic acid contained in the samples (e.g., cell or tissue prepared from the patient) is first extracted according to standard methods, for example using lytic enzymes or chemical solutions or extracted by nucleic-acid-binding resins following the manufacturer's instructions. The extracted miR is then detected by hybridization (e. g., Northern blot analysis, in situ hybridization) and/or amplification (e.g., RT-PCR).
Other methods of Amplification include ligase chain reaction (LCR), transcription- mediated amplification (TMA), strand displacement amplification (SDA) and nucleic acid sequence based amplification (NASBA).
Nucleic acids having at least 10 nucleotides and exhibiting sequence complementarity or homology to the miR of interest herein find utility as hybridization probes or amplification primers. It is understood that such nucleic acids need not be identical, but are typically at least about 80% identical to the homologous region of comparable size, more particularly 85% identical and even more particularly 90-95% identical. In certain embodiments, it will be advantageous to use nucleic acids in combination with appropriate means, such as a detectable label, for detecting hybridization.
Typically, the nucleic acid probes include one or more labels, for example to permit detection of a target nucleic acid molecule using the disclosed probes. In various applications, such as in situ hybridization procedures, a nucleic acid probe includes a label (e.g., a detectable label). A “detectable label” is a molecule or material that can be used to produce a detectable signal that indicates the presence or concentration of the probe (particularly the bound or hybridized probe) in a sample. Thus, a labeled nucleic acid molecule provides an indicator of the presence or concentration of a target nucleic acid sequence (e.g., genomic target nucleic acid sequence) (to which the labeled uniquely specific nucleic acid molecule is bound or hybridized) in a sample. A label associated with one or more nucleic acid molecules (such as a probe generated by the disclosed methods) can be detected either directly or indirectly. A label can be detected by any known or yet to be discovered mechanism including absorption, emission and/ or scattering of a photon (including radio frequency, microwave frequency, infrared frequency, visible frequency and ultra-violet frequency photons). Detectable labels include colored, fluorescent, phosphorescent and luminescent molecules and materials, catalysts (such as enzymes) that convert one substance into another substance to provide a detectable difference (such as by converting a colorless substance into a colored substance or vice versa, or by producing a precipitate or increasing sample turbidity), haptens that can be detected by antibody binding interactions, and paramagnetic and magnetic molecules or materials.
Particular examples of detectable labels include fluorescent molecules (or fluorochromes). Numerous fluorochromes are known to those of skill in the art, and can be selected, for example from Life Technologies (formerly Invitrogen), e.g., see, The Handbook — A Guide to Fluorescent Probes and Labeling Technologies). Examples of particular fluorophores that can be attached (for example, chemically conjugated) to a nucleic acid molecule (such as a uniquely specific binding region) are provided in U.S. Pat. No. 5,866, 366 to Nazarenko et al., such as 4-acetamido-4'-isothiocyanatostilbene-2,2' disulfonic acid, acridine and derivatives such as acridine and acridine isothiocyanate, 5-(2'-aminoethyl) aminonaphthalene- 1 -sulfonic acid (EDANS), 4-amino -N- [3 vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS), N-(4-anilino-l- naphthyl)maleimide, antllranilamide, Brilliant Yellow, coumarin and derivatives such as coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4- trifluoromethylcouluarin (Coumarin 151); cyanosine; 4',6-diarninidino-2-phenylindole (DAPI); 5',5"dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red); 7 -diethylamino -3 (4'-isothiocyanatophenyl)-4-methylcoumarin; diethylenetriamine pentaacetate; 4,4'- diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid; 4,4'-diisothiocyanatostilbene-2,2'- disulforlic acid; 5-[dimethylamino] naphthalene- 1 -sulfonyl chloride (DNS, dansyl chloride); 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL); 4-dimethylaminophenylazophenyl- 4'-isothiocyanate (DABITC); eosin and derivatives such as eosin and eosin isothiocyanate; erythrosin and derivatives such as erythrosin B and erythrosin isothiocyanate; ethidium; fluorescein and derivatives such as 5-carboxyfluorescein (FAM), 5-(4,6diclllorotriazin-2- yDarninofluorescein (DTAF), 2'7'dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), fluorescein, fluorescein isothiocyanate (FITC), and QFITC Q(RITC); 2',7'-difluorofluorescein (OREGON GREEN®); fluorescamine; IR144; IR1446; Malachite Green isothiocyanate; 4- methylumbelliferone; ortho cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red; B- phycoerythrin; o-phthaldialdehyde; pyrene and derivatives such as pyrene, pyrene butyrate and succinimidyl 1 -pyrene butyrate; Reactive Red 4 (Cibacron Brilliant Red 3B-A); rhodamine and derivatives such as 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, rhodamine green, sulforhodamine B, sulforhodamine 101 and sulfonyl chloride derivative of sulforhodamine 101 (Texas Red); N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine; tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid and terbium chelate derivatives. Other suitable fluorophores include thiol-reactive europium chelates which emit at approximately 617 mn (Heyduk and Heyduk, Analyt. Biochem. 248:216-27, 1997; J. Biol. Chem. 274:3315-22, 1999), as well as GFP, LissamineTM, diethylaminocoumarin, fluorescein chlorotriazinyl, naphthofluorescein, 4,7-dichlororhodamine and xanthene (as described in U.S. Pat. No. 5,800,996 to Lee et al.) and derivatives thereof. Other fluorophores known to those skilled in the art can also be used, for example those available from Life Technologies (Invitrogen; Molecular Probes (Eugene, Oreg.)) and including the ALEXA FLUOR® series of dyes (for example, as described in U.S. Pat. Nos. 5,696,157, 6, 130, 101 and 6,716,979), the BODIPY series of dyes (dipyrrometheneboron difluoride dyes, for example as described in U.S. Pat. Nos. 4,774,339, 5,187,288, 5,248,782, 5,274,113, 5,338,854, 5,451,663 and 5,433,896), Cascade Blue (an amine reactive derivative of the sulfonated pyrene described in U.S. Pat. No. 5,132,432) and Marina Blue (U.S. Pat. No. 5,830,912).
In addition to the fluorochromes described above, a fluorescent label can be a fluorescent nanoparticle, such as a semiconductor nanocrystal, e.g., a QUANTUM DOTTM (obtained, for example, from Life Technologies (QuantumDot Corp, Invitrogen Nanocrystal Technologies, Eugene, Oreg.); see also, U.S. Pat. Nos. 6,815,064; 6,682,596; and 6,649, 138). Semiconductor nanocrystals are microscopic particles having size-dependent optical and/or electrical properties. When semiconductor nanocrystals are illuminated with a primary energy source, a secondary emission of energy occurs of a frequency that corresponds to the handgap of the semiconductor material used in the semiconductor nanocrystal. This emission can he detected as colored light of a specific wavelength or fluorescence. Semiconductor nanocrystals with different spectral characteristics are described in e.g., U.S. Pat. No. 6,602,671. Semiconductor nanocrystals that can he coupled to a variety of biological molecules (including dNTPs and/or nucleic acids) or substrates by techniques described in, for example, Bruchez et al., Science 281 :20132016, 1998; Chan et al., Science 281 :2016-2018, 1998; and U.S. Pat. No. 6,274,323. Formation of semiconductor nanocrystals of various compositions are disclosed in, e.g., U.S. Pat. Nos. 6,927, 069; 6,914,256; 6,855,202; 6,709,929; 6,689,338; 6,500,622; 6,306,736; 6,225,198; 6,207,392; 6,114,038; 6,048,616; 5,990,479; 5,690,807; 5,571,018; 5,505,928; 5,262,357 and in U.S. Patent Puhlication No. 2003/0165951 as well as PCT Puhlication No. 99/26299 (published May 27, 1999). Separate populations of semiconductor nanocrystals can he produced that are identifiable based on their different spectral characteristics. For example, semiconductor nanocrystals can he produced that emit light of different colors hased on their composition, size or size and composition. For example, quantum dots that emit light at different wavelengths based on size (565 mn, 655 mn, 705 mn, or 800 mn emission wavelengths), which are suitable as fluorescent labels in the probes disclosed herein are available from Life Technologies (Carlshad, Calif.).
Additional labels include, for example, radioisotopes (such as 3 H), metal chelates such as DOTA and DPTA chelates of radioactive or paramagnetic metal ions like Gd3+, and liposomes.
Detectable labels that can he used with nucleic acid molecules also include enzymes, for example horseradish peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, beta-galactosidase, beta-glucuronidase, or beta-lactamase.
Alternatively, an enzyme can he used in a metallographic detection scheme. For example, silver in situ hyhridization (SISH) procedures involve metallographic detection schemes for identification and localization of a hybridized genomic target nucleic acid sequence. Metallographic detection methods include using an enzyme, such as alkaline phosphatase, in combination with a water-soluble metal ion and a redox-inactive substrate of the enzyme. The substrate is converted to a redox-active agent by the enzyme, and the redoxactive agent reduces the metal ion, causing it to form a detectable precipitate. (See, for example, U.S. Patent Application Puhlication No. 2005/0100976, PCT Publication No. 2005/ 003777 and U.S. Patent Application Publication No. 2004/ 0265922). Metallographic detection methods also include using an oxido-reductase enzyme (such as horseradish peroxidase) along with a water soluble metal ion, an oxidizing agent and a reducing agent, again to form a detectable precipitate. (See, for example, U.S. Pat. No. 6,670,113).
Probes made using the disclosed methods can be used for nucleic acid detection, such as ISH procedures (for example, fluorescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH) and silver in situ hybridization (SISH)) or comparative genomic hybridization (CGH). In situ hybridization (ISH) involves contacting a sample containing target nucleic acid sequence (e.g., genomic target nucleic acid sequence) in the context of a metaphase or interphase chromosome preparation (such as a cell or tissue sample mounted on a slide) with a labeled probe specifically hybridizable or specific for the target nucleic acid sequence (e.g., genomic target nucleic acid sequence). The slides are optionally pretreated, e.g., to remove paraffin or other materials that can interfere with uniform hybridization. The sample and the probe are both treated, for example by heating to denature the double stranded nucleic acids. The probe (formulated in a suitable hybridization buffer) and the sample are combined, under conditions and for sufficient time to permit hybridization to occur (typically to reach equilibrium). The chromosome preparation is washed to remove excess probe, and detection of specific labeling of the chromosome target is performed using standard techniques.
For example, a biotinylated probe can be detected using fluorescein-labeled avidin or avidin-alkaline phosphatase. For fluorochrome detection, the fluorochrome can be detected directly, or the samples can be incubated, for example, with fluorescein isothiocyanate (FITC)- conjugated avidin. Amplification of the FITC signal can be effected, if necessary, by incubation with biotin-conjugated goat antiavidin antibodies, washing and a second incubation with FITC- conjugated avidin. For detection by enzyme activity, samples can be incubated, for example, with streptavidin, washed, incubated with biotin-conjugated alkaline phosphatase, washed again and pre-equilibrated (e.g., in alkaline phosphatase (AP) buffer). For a general description of in situ hybridization procedures, see, e.g., U.S. Pat. No. 4,888,278.
Numerous procedures for FISH, CISH, and SISH are known in the art. For example, procedures for performing FISH are described in U.S. Pat. Nos. 5,447,841; 5,472,842; and 5,427,932; and for example, in Pirlkel et al., Proc. Natl. Acad. Sci. 83:2934-2938, 1986; Pinkel et al., Proc. Natl. Acad. Sci. 85:9138-9142, 1988; and Lichter et al., Proc. Natl. Acad. Sci. 85:9664-9668, 1988. CISH is described in, e.g., Tanner et al., Am. l. Pathol. 157: 1467-1472, 2000 and U.S. Pat. No. 6,942,970. Additional detection methods are provided in U.S. Pat. No. 6,280,929.
Numerous reagents and detection schemes can be employed in conjunction with FISH, CISH, and SISH procedures to improve sensitivity, resolution, or other desirable properties. As discussed above probes labeled with fluorophores (including fluorescent dyes and QUANTUM DOTS®) can be directly optically detected when performing FISH. Alternatively, the probe can be labeled with a nonfluorescent molecule, such as a hapten (such as the following nonlimiting examples: biotin, digoxigenin, DNP, and various oxazoles, pyrrazoles, thiazoles, nitroaryls, benzofurazans, triterpenes, ureas, thioureas, rotenones, coumarin, courmarin-based compounds, Podophyllotoxin, Podophyllotoxin-based compounds, and combinations thereof), ligand or other indirectly detectable moiety. Probes labeled with such non-fluorescent molecules (and the target nucleic acid sequences to which they bind) can then be detected by contacting the sample (e.g., the cell or tissue sample to which the probe is bound) with a labeled detection reagent, such as an antibody (or receptor, or other specific binding partner) specific for the chosen hapten or ligand. The detection reagent can be labeled with a fluorophore (e.g., QUANTUM DOT®) or with another indirectly detectable moiety, or can be contacted with one or more additional specific binding agents (e.g., secondary or specific antibodies), which can be labeled with a fluorophore.
In other examples, the probe, or specific binding agent (such as an antibody, e.g., a primary antibody, receptor or other binding agent) is labeled with an enzyme that is capable of converting a fluorogenic or chromogenic composition into a detectable fluorescent, colored or otherwise detectable signal (e.g., as in deposition of detectable metal particles in SISH). As indicated above, the enzyme can be attached directly or indirectly via a linker to the relevant probe or detection reagent. Examples of suitable reagents (e.g., binding reagents) and chemistries (e.g., linker and attachment chemistries) are described in U.S. Patent Application Publication Nos. 2006/0246524; 2006/0246523, and 2007/ 01 17153.
It will he appreciated by those of skill in the art that by appropriately selecting labelled probe-specific binding agent pairs, multiplex detection schemes can he produced to facilitate detection of multiple target nucleic acid sequences (e.g., genomic target nucleic acid sequences) in a single assay (e.g., on a single cell or tissue sample or on more than one cell or tissue sample). For example, a first probe that corresponds to a first target sequence can he labelled with a first hapten, such as biotin, while a second probe that corresponds to a second target sequence can be labelled with a second hapten, such as DNP. Following exposure of the sample to the probes, the bound probes can he detected by contacting the sample with a first specific binding agent (in this case avidin labelled with a first fluorophore, for example, a first spectrally distinct QUANTUM DOT®, e.g., that emits at 585 mn) and a second specific binding agent (in this case an anti-DNP antibody, or antibody fragment, labelled with a second fluorophore (for example, a second spectrally distinct QUANTUM DOT®, e.g., that emits at 705 mn). Additional probes/binding agent pairs can he added to the multiplex detection scheme using other spectrally distinct fluorophores. Numerous variations of direct, and indirect (one step, two step or more) can he envisioned, all of which are suitable in the context of the disclosed probes and assays. Probes typically comprise single-stranded nucleic acids of between 10 to 1000 nucleotides in length, for instance of between 10 and 800, more particularly of between 15 and 700, typically of between 20 and 500. Primers typically are shorter single-stranded nucleic acids, of between 10 to 25 nucleotides in length, designed to perfectly or almost perfectly match a nucleic acid of interest, to be amplified. The probes and primers are “specific” to the nucleic acids they hybridize to, i.e. they particularly hybridize under high stringency hybridization conditions (corresponding to the highest melting temperature Tm, e.g., 50 % formamide, 5x or 6x SCC. SCC is a 0.15 M NaCl, 0.015 M Na-citrate).
The nucleic acid primers or probes used in the above amplification and detection method may be assembled as a kit. Such a kit includes consensus primers and molecular probes. A particular kit also includes the components necessary to determine if amplification has occurred. The kit may also include, for example, PCR buffers and enzymes; positive control sequences, reaction control primers; and instructions for amplifying and detecting the specific sequences.
In a particular embodiment, the methods of the invention comprise the steps of providing total miR extracted from cumulus cells and subjecting the miR to amplification and hybridization to specific probes, more particularly by means of a quantitative or semi- quantitative RT-PCR.
In another particular embodiment, the expression level is determined by DNA chip analysis. Such DNA chip or nucleic acid microarray consists of different nucleic acid probes that are chemically attached to a substrate, which can be a microchip, a glass slide or a microsphere-sized bead. A microchip may be constituted of polymers, plastics, resins, polysaccharides, silica or silica-based materials, carbon, metals, inorganic glasses, or nitrocellulose. Probes comprise nucleic acids such as cDNAs or oligonucleotides that may be about 10 to about 60 base pairs. To determine the expression level, a sample from a test subject, optionally first subjected to a reverse transcription, is labelled and contacted with the microarray in hybridization conditions, leading to the formation of complexes between target nucleic acids that are complementary to probe sequences attached to the microarray surface. The labelled hybridized complexes are then detected and can be quantified or semi-quantified. Labelling may be achieved by various methods, e.g. by using radioactive or fluorescent labelling. Many variants of the microarray hybridization technology are available to the man skilled in the art (see e.g. the review by Hoheisel, Nature Reviews, Genetics, 2006, 7:200-210).
Expression level of a gene may be expressed as absolute expression level or normalized expression level. Typically, expression levels are normalized by correcting the absolute expression level of a gene by comparing its expression to the expression of a gene that is not a relevant for determining the cancer stage of the patient, e.g., a housekeeping gene that is constitutively expressed. Suitable genes for normalization include housekeeping genes such as the actin gene ACTB, ribosomal 18S gene, GUSB, PGK1 and TFRC. According to the invention the housekeeping genes used were GAPDH, GUSB, TBP and ABL1. This normalization allows the comparison of the expression level in one sample, e.g., a patient sample, to another sample, or between samples from different sources.
Typically, a "threshold value", "threshold level", “reference value” or "cut-off value" can be determined experimentally, empirically, or theoretically. A threshold value can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skilled in the art. Particularly, the person skilled in the art may compare the expression levels of the miR of the invention obtained according to the method of the invention with a defined threshold value.
Particularly, said threshold value is the mean expression level of the miR of the invention of a population of healthy individuals. As used herein, the term "healthy individual" denotes a human which is known to be healthy, i.e. which does not suffer from a cancer and in particular from a glioblastoma and does not need any medical care.
Typically, the skilled person in the art may determine the expression level of the miR of the invention in a biological sample, particularly a biopsy of a glioblastoma cancer for example, of 100 individuals known to be healthy or not. The mean value of the obtained expression levels is then determined, according to well-known statistical analysis, so as to obtain the mean expression level of the miR of the invention. Said value is then considered as being normal and thus constitutes a threshold value. By comparing the expression levels of the miR of the invention to this threshold value, the physician is then able to classify and prognostic the cancer.
Accordingly, the physician would be able to adapt and optimize appropriate medical care of a patient in a critical and life-threatening condition suffering from cancer. The determination of said prognosis is highly appropriate for follow-up care and clinical decision making.
The present invention also relates to kits useful for the methods of the invention, comprising means for detecting the miR of the invention.
Therapeutic method
A second aspect of the invention relates to a IGSF11 inhibitor and/or METTL3/ KHDRBS3 pathway inhibitor, and/or a Hur inhibitor and/or a Elkl inhibitor for use in the treatment of a cancer in a subject identified as having or which will have or develop a resistance to anti-PD-1 therapy according to the invention.
As used herein, the terms IGSF11, METTL3, KHDRBS3, Hur or Elkl are designated by the terms “the targets of the invention”.
As used herein, the terms “IGSF11 inhibitor, METTL3/ KHDRBS3 pathway inhibitor, Hur inhibitor and Elkl inhibitor” are designated by the terms “the inhibitors of the invention”.
In other word, the invention relates to a IGSF11 inhibitor and/or METTL3/ KHDRBS3 pathway inhibitor and/or a Hur inhibitor and/or a Elkl inhibitor for use in the treatment of a subject which will not respond to an anti-PD-1 therapy according to the invention.
Thus, the invention also relates to a method of identifying a patient having or at risk of having or developing a resistance to anti-PD-1 therapy comprising the steps consisting of i) determining the expression level of the extracellular vesicles miRNA-4315 in a sample from said patient, ii) comparing said ilexpression level with a predetermined reference value and iii) concluding that the patient has or is at risk of having or develop a resistance to the anti-PDl therapy when the expression level of the extracellular vesicles adenosine methylated miR-125a- 5p is superior to the predetermined reference value and concluding that the patient has not or is not at risk of having or not develop a resistance to the anti-PDl therapy when the expression level of the extracellular vesicles adenosine methylated miR-125a-5p is inferior to the predetermined reference value and wherein a IGSF11 inhibitor and/or METTL3/KHDRBS3 pathway inhibitor and/or a Hur inhibitor and/or a Elkl inhibitor will be administrated to the patient which has or which is at risk of having or developing a resistance to the anti-PDl therapy.
In other word, the invention relates to a method for predicting an anti-PD-1 therapy response of a patient suffering from a cancer in need thereof, comprising i) determining in a sample obtained from the patient the expression level of the extracellular vesicles adenosine methylated miR-125a-5p ii) comparing said expression level with a predetermined reference value and iii) concluding that the patient will not respond to the anti-PDl therapy when the expression level of the extracellular vesicles adenosine methylated miR-125a-5p is superior to the predetermined reference value and concluding that the patient will respond to the anti-PDl therapy when the expression level of the extracellular vesicles adenosine methylated miR-125a- 5p is inferior to the predetermined reference value and wherein a IGSF11 inhibitor and/or METTL3/KHDRBS3 pathway inhibitor and/or a Hur inhibitor and/or a Elkl inhibitor will be administrated to the patient which will not respond to the anti-PDl therapy. As used herein, the term “IGSFH” has its general meaning in the art and denotes an immunoglobulin (Ig) superfamily member that is preferentially expressed in brain and testis. Its Entrez accession number is: 152404 and its Uniprot accession number is: Q5DX21.
As used herein, the term “IGSF11 inhibitor” denotes a molecule or compound which can inhibit its function like or a molecule or compound which destabilizes IGSF11. The term “IGS11 inhibitor” also denotes an inhibitor of the expression of the gene coding for the protein. IGSF11 inhibitor include for example small molecules or antibodies against IGSF11 (see for example https://iomx.com/pipeline).
As used herein, the term “METTL3” for N6-adenosine-methyltransferase 70 kDa subunit has its general meaning in the art and denotes an enzyme which is involved in the post- transcriptional methylation of internal adenosine residues in eukaryotic mRNAs, forming N6- methyladenosine (m6A).Its Entrez accession number is: 56339 and its Uniprot accession number is: Q86U44.
As used herein, the term “KHDRBS3” for KH domain-containing, RNA-binding, signal transduction-associated protein 3 has its general meaning in the art and denotes a protein which interacts with SIAH1, splicing protein Sam68 and oncogene metadherin in prostate cancer cells. Its Entrez accession number is: 10656 and its Uniprot accession number is: 075525.
As used herein, the term “METTL3/KHDRBS3 pathway inhibitor” denotes all molecule or compound which can inhibit or prevent the formation of the complex METTL3/KHDRBS3 and thus inhibit its function. METTL3/KHDRBS3 pathway inhibitor can thus be an METTL3 inhibitor or an KHDR3 inhibitor.
As used herein, the term “METTL3 inhibitor” denotes a molecule or compound which can inhibit its function of methyltransferase or a molecule or compound which destabilizes METTL3. The term “METTL3 inhibitor” also denotes an inhibitor of the expression of the gene coding for the protein. METTL3 inhibitor include for example small molecules or antibodies against METTL3 (see for example https://www.stormtherapeutics.com/science/pipeline/).
As used herein, the term “KHDRBS3 inhibitor” denotes a molecule or compound which can inhibit its function of interaction or a molecule or compound which destabilizes KHDRBS3. The term “KHDRBS3 inhibitor” also denotes an inhibitor of the expression of the gene coding for the protein. KHDRBS3 inhibitor include for example small molecules or antibodies against METTL3.
As use herein, the term “HuR” for human antigen R also known as ELAV-like protein 1 has its general meaning in the art and denotes RNA-binding domains and binds cis-acting AU-rich elements. One of its best-known functions is to stabilize mRNAs in order to regulate gene expression. Its Entrez accession number is: 1994 and its Uniprot accession number is: Q15717.
As used herein, the term “HuR inhibitor” denotes a molecule or compound which can inhibit its function of stabilization of the mRNAs or a molecule or compound which destabilizes HuR. The term “HuR inhibitor” also denotes an inhibitor of the expression of the gene coding for the protein. HuR inhibitor include for example small molecules or antibodies against HuR.
As use herein, the term “Elkl” for ETS Like-1 has its general meaning in the art and denotes a transcription activator. It is classified as a ternary complex factor (TCF), a subclass of the ETS family, which is characterized by a common protein domain that regulates DNA binding to target sequences. Elkl plays important roles in various contexts, including long-term memory formation, drug addiction, Alzheimer's disease, Down syndrome, breast cancer, and depression. Its Entrez accession number is: 2002 and its Uniprot accession number is: Pl 9419.
As used herein, the term “Elkl inhibitor” denotes a molecule or compound which can inhibit its function of activation of the transcription or a molecule or compound which destabilizes Elkl. The term “Elkl inhibitor” also denotes an inhibitor of the expression of the gene coding for the protein. Elkl inhibitor include for example small molecules or antibodies against HuR. Particularly, ERK inhibitor like Ravoxertinib can be used since Elkl is a member of the MEKZERK pathway.
According to the invention, the METTL3 inhibitor can be the Sinefungin, the UZHla [see 24] or the STM2457 [see 30],
According to the invention, the HuR inhibitor can be the CMLD-2 [see Muralidharan R. et al, 2017],
In another embodiment, the invention also relates to an anti- miR-125a-5p (antago-miR) for use in the treatment of a cancer in a subject identified as having or which will have or develop a resistance to anti-PD-1 therapy according to the invention.
In other word, the invention relates to an anti- miR-125a-5p (antago-miR) for use in the treatment of a subject which will not respond to an anti-PD-1 therapy according to the invention.
As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).
The invention also relates to i) an anti-PD-1 therapy and ii) a IGSF11 inhibitor and/or METTL3 inhibitor and/or a Hur inhibitor and/or a Elkl inhibitor, as a combined preparation for simultaneous, separate or sequential for use in the treatment of a cancer in a subject identified as having or which will have or develop a resistance to anti-PD-1 therapy according to the invention.
The invention also relates to i) an anti-PD-1 therapy and ii) a IGSF11 inhibitor and/or METTL3 inhibitor and/or a Hur inhibitor and/or a Elkl inhibitor, as a combined preparation for simultaneous, separate or sequential for use in the treatment of a cancer in a subject which will not respond to the anti-PDl therapy according to the invention.
The invention also relates to i) an anti-PD-1 therapy, ii) a IGSF11 inhibitor and/or METTL3 inhibitor and/or a Hur inhibitor and/or a Elkl inhibitor and iii) standard chemotherapy according to the invention, as a combined preparation for simultaneous, separate or sequential for use in the treatment of a cancer in a subject identified as having or which will have or develop a resistance to anti-PD-1 therapy according to the invention.
The invention also relates to i) an anti-PD-1 therapy, ii) a IGSF11 inhibitor and/or METTL3 inhibitor and/or a Hur inhibitor and/or a Elkl inhibitor and iii) standard chemotherapy according to the invention, as a combined preparation for simultaneous, separate or sequential for use in the treatment of a cancer in a subject which will not respond to the anti-PDl therapy according to the invention.
In a particular embodiment, the subject can also be treated by radiotherapy or by a radiotherapeutic agent.
As used herein, “radiotherapy” may consist of gamma-radiation, X-ray radiation, electrons or photons, external radiotherapy or curitherapy.
As used herein, the term “radiotherapeutic agent”, is intended to refer to any radiotherapeutic agent known to one of skill in the art to be effective to treat or ameliorate cancer, without limitation. For instance, the radiotherapeutic agent can be an agent such as those administered in brachytherapy or radionuclide therapy. Such methods can optionally further comprise the administration of one or more additional cancer therapies, such as, but not limited to, chemotherapies, and/or another radiotherapy.
In one embodiment and according to the method of treatment, the cancer may be any solid or liquid cancer. Typically, the cancer may be selected from the group consisting of bile duct cancer (e.g. periphilar cancer, distal bile duct cancer, intrahepatic bile duct cancer), bladder cancer, bone cancer (e.g. osteoblastoma, osteochrondroma, hemangioma, chondromyxoid fibroma, osteosarcoma, chondrosarcoma, fibrosarcoma, malignant fibrous histiocytoma, giant cell tumor of the bone, chordoma, lymphoma, multiple myeloma), brain and central nervous system cancer (e.g. meningioma, astocytoma, oligodendrogliomas, glioblastoma, ependymoma, gliomas, medulloblastoma, ganglioglioma, Schwannoma, germinoma, craniopharyngioma), breast cancer (e.g. ductal carcinoma in situ, infiltrating ductal carcinoma, infiltrating, lobular carcinoma, lobular carcinoma in, situ, gynecomastia), Castleman disease (e.g. giant lymph node hyperplasia, angiofollicular lymph node hyperplasia), cervical cancer, colorectal cancer, endometrial cancer (e.g. endometrial adenocarcinoma, adenocanthoma, papillary serous adnocarcinroma, clear cell), esophagus cancer, gallbladder cancer (mucinous adenocarcinoma, small cell carcinoma), gastrointestinal carcinoid tumors (e.g. choriocarcinoma, chorioadenoma destruens), Hodgkin's disease, non-Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer (e.g. renal cell cancer), laryngeal and hypopharyngeal cancer, liver cancer (e.g. hemangioma, hepatic adenoma, focal nodular hyperplasia, hepatocellular carcinoma), lung cancer (e.g. small cell lung cancer, non-small cell lung cancer), mesothelioma, plasmacytoma, nasal cavity and paranasal sinus cancer (e.g. esthesioneuroblastoma, midline granuloma), nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma (e.g. embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, pleomorphic rhabdomyosarcoma), salivary gland cancer, skin cancer (e.g. melanoma, nonmelanoma skin cancer), stomach cancer, testicular cancer (e.g. seminoma, nonseminoma germ cell cancer), thymus cancer, thyroid cancer (e.g. follicular carcinoma, anaplastic carcinoma, poorly differentiated carcinoma, medullary thyroid carcinoma, thyroid lymphoma), vaginal cancer, vulvar cancer, and uterine cancer (e.g. uterine leiomyosarcoma).
In a particular embodiment, the glioblastoma is a GBM, a lung cancer, a breast cancer or an ovarian cancer.
In one embodiment, the inhibitors of the invention may be a low molecular weight compound, e. g. a small organic molecule (natural or not). The term "small organic molecule" refers to a molecule (natural or not) of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e. g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 10000 Da, more preferably up to 5000 Da, more preferably up to 2000 Da and most preferably up to about 1000 Da.
In one embodiment, the inhibitors of the invention are antibodies. Antibodies or directed against the targets of the invention can be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others. Various adjuvants known in the art can be used to enhance antibody production. Although antibodies useful in practicing the invention can be polyclonal, monoclonal antibodies are preferred. Monoclonal antibodies against the targets of the invention can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique originally described by Kohler and Milstein (1975); the human B-cell hybridoma technique (Cote et al., 1983); and the EBV- hybridoma technique (Cole et al. 1985). Alternatively, techniques described for the production of single chain antibodies (see e.g., U.S. Pat. No. 4,946,778) can be adapted to produce antitargets of the invention single chain antibodies. Coumpounds useful in practicing the present invention also include anti- targets of the invention antibody fragments including but not limited to F(ab')2 fragments, which can be generated by pepsin digestion of an intact antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab and/or scFv expression libraries can be constructed to allow rapid identification of fragments having the desired specificity to the targets of the invention.
Humanized anti- targets of the invention antibodies and antibody fragments therefrom can also be prepared according to known techniques. "Humanized antibodies" are forms of nonhuman (e.g., rodent) chimeric antibodies that contain minimal sequence derived from nonhuman immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (CDRs) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Methods for making humanized antibodies are described, for example, by Winter (U.S. Pat. No. 5,225,539) and Boss (Celltech, U.S. Pat. No. 4,816,397).
Then, for this invention, neutralizing antibodies of the targets of the invention are selected.
In one embodiment, the compound according to the invention is an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena S.D., 1999. Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al., 1996).
Then, for this invention, neutralizing aptamers of the targets of the invention are selected.
In one embodiment, the compound according to the invention is a polypeptide. In a particular embodiment the polypeptide is an antagonist of the targets of the invention and is capable to prevent the function of the targets of the invention. Particularly, the polypeptide can be a mutated protein or a similar protein without the function of the targets of the invention. The polypeptides of the invention may be produced by any suitable means, as will be apparent to those of skill in the art. In order to produce sufficient amounts of polypeptide or functional equivalents thereof for use in accordance with the present invention, expression may conveniently be achieved by culturing under appropriate conditions recombinant host cells containing the polypeptide of the invention. Preferably, the polypeptide is produced by recombinant means, by expression from an encoding nucleic acid molecule. Systems for cloning and expression of a polypeptide in a variety of different host cells are well known. When expressed in recombinant form, the polypeptide is preferably generated by expression from an encoding nucleic acid in a host cell. Any host cell may be used, depending upon the individual requirements of a particular system. Suitable host cells include bacteria mammalian cells, plant cells, yeast and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells. HeLa cells, baby hamster kidney cells and many others. Bacteria are also preferred hosts for the production of recombinant protein, due to the ease with which bacteria may be manipulated and grown. A common, preferred bacterial host is E coli. In specific embodiments, it is contemplated that polypeptides used in the therapeutic methods of the present invention may be modified in order to improve their therapeutic efficacy. Such modification of therapeutic compounds may be used to decrease toxicity, increase circulatory time, or modify biodistribution. For example, the toxicity of potentially important therapeutic compounds can be decreased significantly by combination with a variety of drug carrier vehicles that modify biodistribution. In example adding dipeptides can improve the penetration of a circulating agent in the eye through the blood retinal barrier by using endogenous transporters. A strategy for improving drug viability is the utilization of water-soluble polymers. Various water-soluble polymers have been shown to modify biodistribution, improve the mode of cellular uptake, change the permeability through physiological barriers; and modify the rate of clearance from the body. To achieve either a targeting or sustained-release effect, water- soluble polymers have been synthesized that contain drug moieties as terminal groups, as part of the backbone, or as pendent groups on the polymer chain. Polyethylene glycol (PEG) has been widely used as a drug carrier, given its high degree of biocompatibility and ease of modification. Attachment to various drugs, proteins, and liposomes has been shown to improve residence time and decrease toxicity. PEG can be coupled to active agents through the hydroxyl groups at the ends of the chain and via other chemical methods; however, PEG itself is limited to at most two active agents per molecule. In a different approach, copolymers of PEG and amino acids were explored as novel biomaterials which would retain the biocompatibility properties of PEG, but which would have the added advantage of numerous attachment points per molecule (providing greater drug loading), and which could be synthetically designed to suit a variety of applications. Those of skill in the art are aware of PEGylation techniques for the effective modification of drugs. For example, drug delivery polymers that consist of alternating polymers of PEG and tri -functional monomers such as lysine have been used by VectraMed (Plainsboro, N. J.). The PEG chains (typically 2000 daltons or less) are linked to the a- and e-amino groups of lysine through stable urethane linkages. Such copolymers retain the desirable properties of PEG, while providing reactive pendent groups (the carboxylic acid groups of lysine) at strictly controlled and predetermined intervals along the polymer chain. The reactive pendent groups can be used for derivatization, cross-linking, or conjugation with other molecules. These polymers are useful in producing stable, long-circulating pro-drugs by varying the molecular weight of the polymer, the molecular weight of the PEG segments, and the cleavable linkage between the drug and the polymer. The molecular weight of the PEG segments affects the spacing of the drug/linking group complex and the amount of drug per molecular weight of conjugate (smaller PEG segments provides greater drug loading). In general, increasing the overall molecular weight of the block co-polymer conjugate will increase the circulatory half-life of the conjugate. Nevertheless, the conjugate must either be readily degradable or have a molecular weight below the threshold-limiting glomular filtration (e.g., less than 60 kDa). In addition, to the polymer backbone being important in maintaining circulatory half-life, and biodistribution, linkers may be used to maintain the therapeutic agent in a pro-drug form until released from the backbone polymer by a specific trigger, typically enzyme activity in the targeted tissue. For example, this type of tissue activated drug delivery is particularly useful where delivery to a specific site of biodistribution is required and the therapeutic agent is released at or near the site of pathology. Linking group libraries for use in activated drug delivery are known to those of skill in the art and may be based on enzyme kinetics, prevalence of active enzyme, and cleavage specificity of the selected disease-specific enzymes. Such linkers may be used in modifying the protein or fragment of the protein described herein for therapeutic delivery.
In another embodiment, the inhibitors of the invention are inhibitors of gene expression of the targets of the invention.
Small inhibitory RNAs (siRNAs) can also function as inhibitors of expression for use in the present invention. Gene expression can be reduced by contacting a subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that the gene expression of the targets of the invention are specifically inhibited (i.e. RNA interference or RNAi). Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequence is known (e.g. see for example Tuschl, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, GJ. (2002); McManus, MT. et al. (2002); Brummelkamp, TR. et al. (2002); U.S. Pat. Nos. 6,573,099 and 6,506,559; and International Patent Publication Nos. WO 01/36646, WO 99/32619, and WO 01/68836).
Ribozymes can also function as inhibitors of gene expression for use in the present invention. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of G-CSF mRNA sequences are thereby useful within the scope of the present invention. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays. Both antisense oligonucleotides and ribozymes useful as inhibitors of gene expression can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase phosphoramadite chemical synthesis. Alternatively, anti-sense RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Various modifications to the oligonucleotides of the invention can be introduced as a means of increasing intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2'-O-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone.
Antisense oligonucleotides, siRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide siRNA or ribozyme nucleic acid to the cells and preferably cells expressing G-CSF. Preferably, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide siRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus; adenovirus, adeno-associated virus; SV40- type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art. Preferred viral vectors are based on non-cytopathic eukaryotic viruses in which non-essential genes have been replaced with the gene of interest. Non- cytopathic viruses include retroviruses (e.g., lentivirus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell lined with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles are provided in Kriegler, 1990 and in Murry, 1991. Preferred viruses for certain applications are the adeno-viruses and adeno-associated viruses, which are double-stranded DNA viruses that have already been approved for human use in gene therapy. The adeno- associated virus can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species. It further has advantages such as, heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hemopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions. Reportedly, the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection. In addition, wild-type adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event. The adeno-associated virus can also function in an extrachromosomal fashion.
Other vectors include plasmid vectors. Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g. Sambrook et al., 1989. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigenencoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid. Some commonly used plasmids include pBR322, pUC18, pUC19, pRC/CMV, SV40, and pBlueScript. Other plasmids are well known to those of ordinary skill in the art. Additionally, plasmids may be custom designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA. Plasmids may be delivered by a variety of parenteral, mucosal and topical routes. For example, the DNA plasmid can be injected by intramuscular, eye, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally. It may also be administered into the epidermis or a mucosal surface using a gene-gun. The plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and microencap sul ati on .
In a particular embodiment, the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequence is under the control of a heterologous regulatory region, e.g., a heterologous promoter. The promoter can also be, e.g., a viral promoter, such as CMV promoter or any synthetic promoters.
In order to test the functionality of a putative inhibitor of the target of the invention, a test is necessary. For that purpose, to identify inhibitors of the target analysis of the level of the methylation of the exosomal adenosine methylated miR-125a-5p by qPCR or memiRIP (Methylated miRNA Immuno Precipitation).
Another object of the invention relates to a method for treating cancer comprising administrating to a subject identified as having or which will have or develop a resistance to anti-PD-1 therapy according to the invention a therapeutically effective amount of IGSF11 inhibitor and/or METTL3/KHDRBS3 pathway inhibitor and/or a Hur inhibitor and/or a Elkl inhibitor.
Therapeutic composition
Another object of the invention relates to a therapeutic composition comprising a IGSF11 inhibitor and/or METTL3/KHDRBS3 pathway inhibitor and/or a Hur inhibitor and/or a Elkl inhibitor according to the invention for use in the treatment of cancer in a subject identified as having or which will have or develop a resistance to anti-PD-1 therapy according to the invention.
In still another object of the invention relates to a therapeutic composition comprising a IGSF11 inhibitor and/or METTL3/KHDRBS3 pathway inhibitor and/or a Hur inhibitor and/or a Elkl inhibitor according to the invention for use in the treatment of cancer in a subject will not respond to an anti-PD-1 therapy according to the invention.
Any therapeutic agent of the invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.
"Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The form of the pharmaceutical compositions, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the patient, etc.
The pharmaceutical compositions of the invention can be formulated for a topical, oral, intranasal, parenteral, intraocular, intravenous, intramuscular or subcutaneous administration and the like.
Particularly, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
The doses used for the administration can be adapted as a function of various parameters, and in particular as a function of the mode of administration used, of the relevant pathology, or alternatively of the desired duration of treatment.
In addition, other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; time release capsules; and any other form currently can be used.
Pharmaceutical compositions of the present invention may comprise a further therapeutic active agent. The present invention also relates to a kit comprising a compound according to the invention and a further therapeutic active agent.
In one embodiment said therapeutic active agent may be an anti-cancer agent.
Anti-cancer agents may be Melphalan, Vincristine (Oncovin), Cyclophosphamide (Cytoxan), Etoposide (VP- 16), Doxorubicin (Adriamycin), Liposomal doxorubicin (Doxil) and Bendamustine (Treanda).
Others anti-cancer agents may be for example cytarabine, anthracyclines, fludarabine, gemcitabine, capecitabine, methotrexate, taxol, taxotere, mercaptopurine, thioguanine, hydroxyurea, cyclophosphamide, ifosfamide, nitrosoureas, platinum complexes such as cisplatin, carboplatin and oxaliplatin, mitomycin, dacarbazine, procarbizine, etoposide, teniposide, campathecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, L-asparaginase, doxorubicin, epimbicm, 5-fluorouracil, taxanes such as docetaxel and paclitaxel, leucovorin, levamisole, irinotecan, estramustine, etoposide, nitrogen mustards, BCNU, nitrosoureas such as carmustme and lomustine, vinca alkaloids such as vinblastine, vincristine and vinorelbine, imatimb mesylate, hexamethyhnelamine, topotecan, kinase inhibitors, phosphatase inhibitors, ATPase inhibitors, tyrphostins, protease inhibitors, inhibitors herbimycm A, genistein, erbstatin, and lavendustin A. In one embodiment, additional anticancer agents may be selected from, but are not limited to, one or a combination of the following class of agents: alkylating agents, plant alkaloids, DNA topoisomerase inhibitors, anti-folates, pyrimidine analogs, purine analogs, DNA antimetabolites, taxanes, podophyllotoxin, hormonal therapies, retinoids, photosensitizers or photodynamic therapies, angiogenesis inhibitors, antimitotic agents, isoprenylation inhibitors, cell cycle inhibitors, actinomycins, bleomycins, MDR inhibitors and Ca2+ ATPase inhibitors.
Additional anti-cancer agents may be selected from, but are not limited to, cytokines, chemokines, growth factors, growth inhibitory factors, hormones, soluble receptors, decoy receptors, monoclonal or polyclonal antibodies, mono-specific, bi-specific or multi-specific antibodies, monobodies, polybodies.
Additional anti-cancer agent may be selected from, but are not limited to, growth or hematopoietic factors such as erythropoietin and thrombopoietin, and growth factor mimetics thereof.
In the present methods for treating cancer the further therapeutic active agent can be an antiemetic agent. Suitable antiemetic agents include, but are not limited to, metoclopromide, domperidone, prochlorperazine, promethazine, chlorpromazine, trimethobenzamide, ondansetron, granisetron, hydroxyzine, acethylleucine monoemanolamine, alizapride, azasetron, benzquinamide, bietanautine, bromopride, buclizine, clebopride, cyclizine, dunenhydrinate, diphenidol, dolasetron, meclizme, methallatal, metopimazine, nabilone, oxypemdyl, pipamazine, scopolamine, sulpiride, tetrahydrocannabinols, thiefhylperazine, thioproperazine and tropisetron. In a preferred embodiment, the antiemetic agent is granisetron or ondansetron.
In another embodiment, the further therapeutic active agent can be an hematopoietic colony stimulating factor. Suitable hematopoietic colony stimulating factors include, but are not limited to, filgrastim, sargramostim, molgramostim and epoietin alpha.
In still another embodiment, the other therapeutic active agent can be an opioid or nonopioid analgesic agent. Suitable opioid analgesic agents include, but are not limited to, morphine, heroin, hydromorphone, hydrocodone, oxymorphone, oxycodone, metopon, apomorphine, nomioiphine, etoipbine, buprenorphine, mepeddine, lopermide, anileddine, ethoheptazine, piminidine, betaprodine, diphenoxylate, fentanil, sufentanil, alfentanil, remifentanil, levorphanol, dextromethorphan, phenazodne, pemazocine, cyclazocine, methadone, isomethadone and propoxyphene. Suitable non-opioid analgesic agents include, but are not limited to, aspirin, celecoxib, rofecoxib, diclofinac, diflusinal, etodolac, fenoprofen, flurbiprofen, ibuprofen, ketoprofen, indomethacin, ketorolac, meclofenamate, mefanamic acid, nabumetone, naproxen, piroxicam and sulindac.
In yet another embodiment, the further therapeutic active agent can be an anxiolytic agent. Suitable anxiolytic agents include, but are not limited to, buspirone, and benzodiazepines such as diazepam, lorazepam, oxazapam, chlorazepate, clonazepam, chlordiazepoxide and alprazolam.
In yet another embodiment, the further therapeutic active agent can be a checkpoint blockade cancer immunotherapy agent.
Typically, the checkpoint blockade cancer immunotherapy agent is an agent which blocks an immunosuppressive receptor expressed by activated T lymphocytes, such as cytotoxic T lymphocyte-associated protein 4 (CTLA4) and programmed cell death 1 (PDCD1, best known as PD-1), or by NK cells, like various members of the killer cell immunoglobulin- like receptor (KIR) family, or an agent which blocks the principal ligands of these receptors, such as PD-1 ligand CD274 (best known as PD-L1 or B7-H1).
Typically, the checkpoint blockade cancer immunotherapy agent is an antibody.
In some embodiments, the checkpoint blockade cancer immunotherapy agent is an antibody selected from the group consisting of anti-CTLA4 antibodies, anti-PDl antibodies, anti-PDLl antibodies, anti-PDL2 antibodies, anti-TIM-3 antibodies, anti-LAG3 antibodies, anti-IDOl antibodies, anti-TIGIT antibodies, anti-B7H3 antibodies, anti-B7H4 antibodies, anti- BTLA antibodies, and anti-B7H6 antibodies.
The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.
FIGURES:
Figure 1: Adenosine methylation of miR-125a-5p regulates its repressive function toward VSIG3/IGSF11 and influences the cellular immunogenicity. A. Graphs illustrate the impact of Sinefungin (SFG) on the expression and adenosine methylation level of miR-125a- 5p, on the interaction of this miRNA with GW182 and on the protein and mRNA expression levels of IGSF11 in H1975NivoSat cells. H1975 cells were used as control. B. Immunogenicity of H1975NivoSat cells was compared with the one of H1975NivoSat cells treated with Sinefungin or anti-IGSFl 1 through the analysis of their PBMC-induced lysis. H1975 cells were used as control. Figure 2: METTL3/KHDRBS3 promotes the adenosine methylation of miR-125a- 5p. A. The transfection impact of H1975NivoSat cells with siRNA control (siRNA-A) and siRNA directed against KHDSBR3 and METTL3 on the expression level of miR-125a-5p and 41 l-5p (RT-qPCR), KHDRBS3 and METTL3 transcripts (RT-qPCR), the level of enrichment in m6A of miR-125a-5p and 41 l-5p. B. Impact of chemical (Ravoxertinib, lOOnM, 72h) and biological (siRNA) inactivation of Elkl on the expression level of the KHDRBS3 transcript and on the enrichment level of Elkl on the KHDRBS3 promoter. DMSO and siRNA-A were used as control.
Figure 3: HuR blocks the recruitment of m6A-miR-125a-5p to GW182. A. Analysis of the impact of siRNA invalidation of HuR on the adenosine methylation and expression levels of miR-125a-5p, on the miR125a-5p/GW 182 interaction and on the expression level of IGSF 11. B. Analysis of miR-125a-5p/HuR interaction within H1975, H1975NivoSat and METTL3 inhibitor (SFG) treated cells.
Figure 4. Investigation on the adenosine methylation of exosomal miR-125a-5p in two lung cancer patients. A. Graphs illustrate the fact that modification of adenosine methylation of the miR-125a-5p can be analyzed using exosomal miR-125a-5p. Three anti-PD- 1 therapies were used in to investigate this point in H1975 cells. B. The expression and adenosine methylation levels of miR-125a-5p were analyzed using exosomes from longitudinal blood samples taken from patient#A between 2 MRIs assessing the fate of his tumor. C. The expression and adenosine methylation levels of miR-125a-5p were analyzed using exosomes from longitudinal blood samples taken from patient#B between 2 MRIs assessing the fate of his tumor. D. Analysis of the impact of exosomes obtained from several blood samples of patients #A and #B on the expression level of IGSF11 in H1975 cells.
EXAMPLE:
Material & Methods
Cell culture
A549 (ATCC#CCL-185), H1975 (ATCC#CRL-5908), H358 (ATCC#CRL-5807) and H1650 (ATCC#CRL-5883), cells were cultured in RMPI medium supplemented with 10% of fetal bovine serum, 1% penicillin-streptomycin, and 2 mM L-glutamine. All cells were cultivated in a 5% CO2 incubator at a temperature of 37 °C.
Cytometry analysis
Directly-labeled monoclonal antibodies were used for flow cytometry APC-mouse IGglK isotype control (#555751, BD Biosciences, France) and APC-mouse anti human CD279 (#558694, BD Biosciences, France). 200 000 cells of each cell lines were labeled at room temperature in PBS/1% BSA, washed twice in PBS and analyzed on a BD ACCURI C6 cytometer (France). Cancer lines were gated according to their forward-scatter and side-scatter properties and excluding debris and doublets.
PD-1 receptor occupancy (saturation) analysis
PD-1 receptor occupancy (saturation) by Nivolumab was investigated using in cell ELISA method. Tumor cells (4000 cells/well) were plated in 96 wells plate for 24h. Cells were fixed using 8% paraformaldehyde solution (15min, room temperature). After four washes with IX phosphate-buffered saline (PBS), Nivolumab (#A1307, Biovision/CliniScience, France) were next incubated at indicated concentration for 3h at room temperature. After four extensive washes, anti-PD-l-HRP (0.5pg/ml, #10377-MM07-H, Interchim, France) was incubated for 2h at room temperature and signal was detected using Step Ultra TMB-ELISA substrate solution according to the manufacturer’s instructions (#34028, ThermoFisher Scientific, France). Saturation percentage was calculated relatively to experiment performed with a control antibody (#DDXCH0P-100, Novus, France). miRNA Extraction miRNA extractions were performed using miRNeasy mini kit (#217004, Qiagen, France) on a QIAcube instrument according to the manufacturer’s instructions (Qiagen, France). qPCR of miRNA
For miRNA expression analysis and detection from products of RNA immunoprecipitation (RIP) performed with anti-m6A antibody, miRNA was reverse transcribed using a miScript II RT kit (#218161, Qiagen, France) and analyzed by qPCR with the miScript SYBR Green PCR kit (#218076, Qiagen, France) using the specific miScript primer assays (#218300, Qiagen, France) according to the manufacturer’s instructions. Rotor- Gene Q was used as real-time thermocycler (Qiagen, France). mRNA extraction and qPCR
RNA extractions were performed using RNeasy kit (#74116, Qiagen, France) and QIAcube according to the manufacturer’s instructions (Qiagen, France). Reverse transcription was then performed with QuantiTect Reverse Transcription Kit (#205311, Qiagen, France) and PCR was performed with QuantiFast SYBR Green PCR Kit (#204156, Qiagen, France) and Rotor-Gene Q (Qiagen, France). Primers used were the QuantiTect Primers assay (#249900, Qiagen, France) and more precisely RPLP0 (QT00075012), KHDRBS3 (QT00065296), METTL3 (QT00036540) and HuR (QT00037856). Methylation level of miRNAs
For RNA immunoprecipitation (RIP), two rounds using 5 pg of anti-m6A (#ab208577, Abeam, France), anti-m7G (#6655, Biovison, USA) and anti-m5C (#61255, Active Motif, France) antibodies and 5 pg of small RNA were performed. The reaction was carried out using a Dynabeads protein G IP kit with some modifications (#10007D, ThermoFisher Scientific, France) such as described by Berulava et al (2015)[14] and Briand et al. (2021)[19], As a control, IP was performed using IgG (#ab 18443, Abeam, France) instead of anti-m6A antibody. miRNAs obtained from RIP were reverse transcribed using mi Script II RT kit (#218161, Qiagen, France) and analyzed using the mi Script miRNA PCR array human cancer pathway kit (#331221, Qiagen, France) according to the manufacturers’ instructions. Fold enrichment was next calculated using Ct value obtained from qRT-PCR performed with input miR, IP-IgG, and IP-m6A, and the 2-AACt formula. siRNA transfection siRNA directed against METTL3, KHDRBS3 and HuR (#sc-92172, #sc-40922, and sc- 35619 Santa Cruz, France) were used in this article. siRNA-A (#sc-37007, Santa Cruz, France) i.e. a scrambled sequence devoid of specific degradation of any cellular message was used as control. In a six wells culture plate, 2.105 cells were incubated for 24h at 37°C in CO2 incubator. Then, 60pmol of siRNA were incubated on cells for 7h at 37°C in a CO2 incubator. Without removing the siRNA mixture, we next added 1 ml of normal growth medium containing 2 times the normal serum and antibiotics concentration, for 24h. Then, cells were expanded for 48h in normal culture medium. Thus, analyses were realized about 72h after the siRNA incubation.
Immunoprecipitation
After 2 cold PBS IX washes (10ml), three 100mm dish culture at confluence 75% (2.109 cells) were incubated with 500 pl of lysis buffer (20mM TrisHcl pH 7.4, 150mM de NaCl, ImM EDTA, 1% Triton + Protease cocktail inhibitor) for 5 minutes on ice. Then, cells were scrapped and centrifuged in 1.5ml Eppendorf tube (4°C, 15 min, 14000g). After a transfer in a new tube, supernatant is used for protein dosage (#23225, Pierce BCA Protein Assay Kit, ThermoScientific, France). At this step supernatant can be stored at -80°C. Then, we performed of pre-clean step. For that, 20 pL of magnetic beads (#10004D, DynaBeads, ThermoScientific, France) were washed twice with 1 ml of lysis buffer in a 1.5ml Eppendorf tube. After the last wash, 500 pg of lysate were added to magnetic beads for 2h at 4°C on a revolver tube mixer (Labnet international, France) to saturate aspecific interactions. After magnetic separation, the lysate is then collected to be incubated (overnight, 4°C, revolver tube mixer) with the antibody (4 pg of anti-METTL3 (#abl95352, Abeam, France) or IgG as control (#2729S, Cell Signaling/Ozyme, France). Then, lysate-antibody mixture was incubated (2h, 4°C, revolver tube mixer) with 20 pl of saturated magnetic beads (as previously described). Four washes were next performed (two with lysis buffer and two lysis buffer devoid of triton). Finally, beads were resuspended in lOOpL of Buffer NH4HCO3 at 50mM pH=8 and stored for future use. miRNA pull-down assay
Cellular extracts were performed by incubating lOmin on ice 2.106 cells in cell lysis buffer (85nM KC1, 0.5% NP40, 5mM HEPES, pH 7.4, (three volume of buffer for one cell volume). After incubation, mixture was sonicated in a Bioruptor/Diagenode (15 cycles, high, 30s on/off). After a centrifugation (lOmin, 14 000g, 4°C) supernatant was transferred in a fresh tube to be quantified (Qubit Method, ThermoFisher, France).
Streptavidin M280 Dynabeads (#11205D, ThermoFisher, France) were washed 3 times in binding/washing buffer (10 mM Tris - HC1, pH 7.5, 1 mM EDTA, 2M NaCl), twice with 50 /z L of 0.1 NaOH/0.05 M NaCl buffer, once with 50 /z L of 0.1 M NaCl. Beads were finally resuspended in 50 pL of 0.1 M NaCl and transfered in a new DNA low binding tube. Then, 800 pg of protein were incubated with 50 pL of beads for 15 min at room temperature.
Then, tube was placed on magnetic stand for 2-3 min and supernatant was collected for future use.
In parallel, 500pmol of biotinylated mimic miRNA were then incubated with beads for 15 min at room temperature under gentle rotation. Then, tube was placed on magnetic stand for 2-3 min to collect miRNA-immobilized beads. After 2 washes in binding/washing buffer, miRNA-immobilized beads were resuspended in 100 pL of Protein binding buffer (20 mM Hepes (pH 7.3), 50 mM KCL, 10% glycerol, 5 mM MgC12. Add the following reagents before use: 1/1000 vol. of 1 M Dithiothreitol (DTT), Phosphatase inhibitor (100X), Proteinase inhibitor (100X), RNase Inhibitor (25-1000 U/mL, for RNA application).
Supernatant was then added and the mixture was incubated 30 min at room temperature.
Tube was next placed on magnetic stand for 1 min and supernatant was discard. After 3 washes, elution was performed using 20 pl of 4x Laemmli buffer (#1610747, BioRad, France). After an incubation for 10 min at 95°C., samples were loaded onto a SDS-PAGE gel and proteins were detected by Western blotting
Western blot
After a denaturation step (dry-bath/95°C/10min), samples are placed in the wells of a 4- 15% Mini -PROTEAN electrophoresis gel (#4568084, Bio-Rad, France), and the electrophoresis (90V) is performed in the presence of Tris/Glycine/SDS Electrophoresis Buffer (#1610732EDU, Bio-Rad, France). Transfer (30V, 90min RT) is performed on a PVDF membrane (Trans-Blot Turbo Mini PVDF, #1704156, Bio-Rad, France) in the presence of cold Tris/Glycine transfer buffer (#1610734EDU, BioRad, France). The membrane is then saturated with the saturation solution (5% milk, PBS IX). Primary and secondary antibodies (METTL3#MA5-27527, ThermoFischer, France; KHDRBS3#ab68515, Abeam, France, RBMX# PA5-99433, ThermoFischer, France; HuR# 39-0600, ThermoFischer, France; HRP- anti-Rabbit IgG (#111-035-006, Jackson Immunology) and HRP-anti-Mouse IgG (#115-036- 75, Jackson Immunology)) are incubated in milkl%-PBS IX. The HRP signal is revealed using ChemiDoc imager (Bio-Rad, France) and the ECL-Clarity kit (#1705061, Bio-Rad, France).
Chromatin Immunoprecipitation (ChIP) analyses
ChIP were performed using ChlP-IT Express kit (Active Motif, France) according to the manufacturer’s instructions. The cross-linking step was performed by treating the cells with 37% formaldehyde solution for 15 min at room temperature. Sonication was performed with the Bioruptor Plus (8 cycles 30sec ON/90sec OFF) (Diagenode, France). The QuantiFast SYBR Green PCR Kit and Rotor-Gene Q (Qiagen, France) were used to perform the qPCR. Antibodies used were: Anti-IgG (#ab2410, Abeam, France) and anti-Elkl (sc365876x, Santa Cruz, France).
Cell Cytotoxicity Assay
This assay permit to estimate the lysis of desired target cells (Hl 975 here) via an effector immune cell of choice (PBMC activated by IL2, here). In this assay, the lysis of H1975 cells is determined by labeling these cells with fluorescent molecule, co-incubating it with Deactivated PBMC, then measuring the release of the labeled molecule in the supernatant. DELFIA EuTDA Cell Cytotoxicity (#AD0116, Perkin Elmer, France) reagents were used as previously described (Briand et al. 2019)[21], For PBMC isolation, whole blood was diluted with an equal amount of IX phosphate-buff ered saline (PBS) and placed on top of Ficoll-Paque PLUS (#17-1440-02, VWR, France) in tubes for centrifugation at room temperature for 20min at 1020g. PBMCs were next carefully collected from the interface layer between the blood plasma and Ficoll solution. The collected PBMCs were twice washed with IX PBS, then cultured in RPMI1640 medium containing 10% FBS, 0.1 nM human IL-2 (Diaclone Research, France), and 55 pM P-mercaptoethanol for 24 h. BATDA-labeled target cells and PBMC effector cells (effector to target cell ratio: 10: 1) were incubated (2h, 37°C, 5% CO2) together to allow redirected lysis. The supernatant containing TDA released by dead cells was incubated with Europium solution to allow stable formation of a highly fluorescent Europium-TDA chelate (EuTDA). The amount of EuTDA was quantified by analysis of the relative fluorescence intensity (FLsample) at 520 nm. Spontaneous fluorescence intensity was determined with target cells without effector cells. Fluorescence intensity of maximal lysis (FLmax) was determined using target cells and DELFIA® lysis buffer. The percentage of specific cell lysis was calculated as 100 * (FLsample - FLSpon)/(FLmax - FLSpon).
RT-qPCR analysis
RNA extract is performed using RNeasy Mini QIAcube Kit and QIAcube (#1038703, Qiagen, France). RT-qPCRs are performed using QuantiTect Reverse Transcription Kit (#205313, Qiagen, France), QuantiFast SYBR Green PCR Kit (#204057, Qiagen, France), QuantiTect Primer Assays (#249900, Qiagen, France) and Rotor-Gene Q as real-time thermocycler (Qiagen, France). Reference gene RPLP0 was used, with the 2-AACt relative quantification method.
CLIP
CLIP assays were performed using a RIP-assay kit (#RN1005 Clini Science, France) from 10 million per sample of UV-crosslinked cells (150 mJ/cm2 of UVA (Bio-link, Vilber, France)) according to the manufacturer’s instructions. IP assays were performed in the presence of 15 pg of anti-GW182 (#RNP033P, CliniScience, France) overnight at 4°C.
Isolating exosomal miRNA from blood
From the blood sample collected in K + EDTA tubes, 4-5 ml of blood was centrifugated for 10 min/1900 g/4 °C. Supernatants were carefully transferred into a new centrifuge tubes and centrifuged a second time at 16000g for 10 min at 4 °C to remove cellular debris. Plasmas were aliquoted in 2 ml tubes and were frozen at -80°C until use. 1 ml of plasma was processed for the isolation of miRNA using the ExomiRNeasy Midi kit (#77144, Qiagen, France) according to the manufacturers instructions.
Exosome isolation
As described in our previous articlesfl 3]-[12], ExoQuick kits (#EXOTC10A1 and #EXOCG50A1, Ozyme, France) were used according to the manufacturer’ s instructions. Exosome total protein concentration was determined using the Bradford assay (Bio-Rad Laboratories, France), and exosomes were stored at - 80 °C until use. As already mentioned, Nanosight experiment was performed to validate the use of the “exosome” term due to the isolation of EVs with a size predominantly between 80 and 120 nm, i.e. values commonly characterizing exosomes. Consequently, the term “ exosome” is used in this article.
Patients data Plasma was collected from patients treated at the “Institut de Cancerologie de 1’Ouest” (ICO, http://www.ico-cancer.fr). All patients recruited gave signed, informed consent. All the samples collected and the associated clinical information were registered in the database (N° DC-2018-3321) validated by the French research ministry. Biological resources were stored at the “Centre de Ressources Biologiques-Tumorotheque (CRB)” (Institut de Cancerologie de 1’Ouest, Saint-Herblain, F44800, France)[22], All clinical and radiologic data were collected from the medical electronic records stored at the Institut de Cancerologie de 1’Ouest.
Statistical analysis and results
Except when indicated, data are representative of the mean and standard deviation calculated from three independent experiments. Significance of the differences in means ± standard deviations was calculated using the Student-t test. The significance of correlation between two parameters was calculated using Pearson’s test, p < 0.05 was used as a criterion for statistical significance.
Results
Nivolumab saturation of PD-1 in H1975 lung cancer cells affects the methylation status of miRNAs and the cellular immunogenicity
To identify a cellular model on which to study the impact of anti-PD-1 therapy on miRNA methylation levels, we analyzed PD-1 expression on the surface of a panel of four lung cancer cell lines (A549, H1975, HT358 and HT1650). Flow cytometry analysis showed that all lung cancer cell lines expressed PD-1 on their surface and that H1975 cell line was the cell line expressing the most PD-1 on its surface (data not shown). We thus retained this cell line to carry out the continuation of our experiments. In parallel, we then arbitrarily chose to work with Nivolumab, an anti-PD-1 very frequently used in the treatment of lung cancer (more than 290 clinical trials listed on the ClinicalTrails.gov website with the “lung cancer and Nivolumab” key words). According to our saturation experimental design (data not shown), we noted that 0.1 pg/ml Nivolumab saturated 80% of PD-1 expressed on the surface of H1975 lung cancer cells (H1975NivoSat) (data not shown). We thus retained this concentration to carry out the continuation of our experiments.
Changes in miRNA expression level were analyzed using qPCR miR array and changes in adenosine (m6A), cytosine (m5C) and guanosine (m7G) methylation levels were analyzed using RNA immunoprecipitation with an anti-methyl-base-antibody followed by qPCR miR array (n=84). Among all the miRNA measured by the miR Array, the expression fold change between H1975 and H1975NivoSat cells appeared homogeneous on the whole (fold change varies within the range -5/+5) (data not shown). By contrast, when considering only methylated miRNA (data not shown), a few of them could be distinguished from the group since they increased out of the range. A gain of cytosine methylation (m5C) was observed in 7 miRNAs in H1975NivoSat: hsa-miR-27a-3p, hsa-miR-126-3p, hsa-miR-132-3p, hsa-miR-181c-5p, hsa- miR-184, hsa-miR-200c-3p, and hsa-miR-210-3p data not shown). Such specific modification of guanosine methylated miRNA could not be identified in H1975NivoSat cells (data not shown).
TAM2.0 bioinformatic tool was next used to determine the functional implications of miRNAs having a gain of cytosine and adenosine methylation [23], Thus, we observed that miRNAs having a gain of cytosine and adenosine methylation were mainly associated with the immune response function (data not shown). In other words, this analysis suggests that the immune response towards H1975NivoSat cells could be modified. To investigate this hypothesis, we performed a cell cytotoxicity assay in which the H1975 and H1975NivoSat lysis is performed by IL2-activated PBMC. Our data indicated that the IL-2-activated PBMC- induced lysis of H1975NivoSat cells was lower than the one of H1975 cells (data not shown).
Adenosine methylation of miR-125a-5p regulates its repressive function toward VSIG3/IGSF11 and influences the cellular immunogenicity
We then extended our study by investigating how variations in cytosine and adenosine methylation of miRNAs could modulate the immunogenicity of H1975NivoSat cells. For this purpose, we hypothesized that the previous observed gain of miRNAs methylation could affect the expression of immune response actors. Among a panel of 8 immune actors B7H3/CD276 (Q5ZPR3), B7H4/VTCN1 (Q7Z7D3), GAL9/HOM-HD-21 (000182), TNFRSF14/CD270 (Q92956), PD-1/CD279 (Q15116), PD-L1/CD274 (Q9NZQ7), PD-L2/CD273 (Q9BQ51) and IGSF11/VSIG3 (Q5DX21), RT-qPCR indicated that only IGSFl lmRNA level was increased in H1975NivoSat in comparison with H1975 (data not shown).
Among the 156 miRNAs identified by the miRDB website as regulators of IGSF11 (Supplementary File 1), only two miRNAs, miR-125a-5p and miR-181c-5p, were also included in the list of 12 miRNAs having a gain of adenosine/cytosine methylation (data not shown).
Based on this finding, we next analyzed whether the gain of adenosine and cytosine methylation of miR-125a-5p and miR181c-5p could be responsible for the increase in IGSF11 expression. For this purpose, two sets of experiments were performed.
First, we analyzed the ability of unmethylated and methylated miR-125a-5p and miR181c-5p to regulate the IGSF1 ImRNA expression. Forthat, we designed unmethylated and methylated mimic miRs in which the positions of adenosine and cytosine methylation were predicted by the presence of DRCAH and CG sequences. Interestingly, mutation of adenosine at position 9 and 17 and cytosine at position 15 in miR-125a-5p and miR-181c-5p abolished the METTL3/METTL14 and DNMT3A/AGO4-mediated methylation of these miRs (data not shown). Thus, it was observed that neither the mimic-miR181c-5p transfection of nor its cytosine methylated form altered IGSF1 ImRNA expression (data not shown). On the contrary, the transfection of the unmethylated mimic-miR-125a-5p down-regulated the IGSF1 ImRNA expression, while similar quantity of adenosine-methylated mimic-miR-125a-5p did not alter IGSF1 ImRNA expression (data not shown). Under these conditions, we also observed that the quantity of miR-125a-5p co-immunoprecipitated with GW182 remained unchanged in experiments performed with adenosine-methylated mimic-miR-125a-5p in comparison with control condition, while the quantity of miR-125a-5p co-immunoprecipitated with GW182 strongly increased in experiments performed with unmethylated mimic-miR-125a-5p (data not shown). The results of this first set of experiments support the idea that miR-125a-5p down- regulates the IGSF11 expression, while the adenosine-methylated form of this miR loses this ability.
Secondly, we analyzed the Sinefungin (SFG) effect, an inhibitor of METTL3, the main actor responsible for adenosine methylation of miRNA, on the adenosine methylation of miR- 125a-5p and the IGSF11 expression. Thus, we observed that SFG treatment i) decreased the level of m6a-miR-125a-5p without changing the expression level of miR-125a-5p, ii) restored the miR-125a-5p enrichment on GW182 that had been decreased by Nivolumab saturation and iii) decreased the IGSF1 ImRNA and IGSF11 expressions (Figure 1 A). The fact that Nivolumab increased the adenosine methylation of miR-125a-5p and the IGSF11 expression was also observed in two other models based on the consideration of H1650 and A549 cells (data not shown). This second set of data supports the idea that the adenosine methylation level of miR- 125a regulates the IGSF11 expression.
Finally, we observed that SFG and anti-IGSFl l have the ability to restore an IL-2- activated PBMC-induced lysis cell of H1975NivoSat to a level comparable to the one observed in H1975 cells. (Figure IB). In other terms this last result reinforces the fact that IGSF11 plays a crucial role on the low level of IL-2-activated PBMC-induced lysis of H1975NivoSat.
METTL3/KHDRBS3 promotes the adenosine methylation of miR-125a-5p
To explain the molecular mechanism involved in the gain of adenosine methylation of miR-125a-5p, we first investigated a putative change in METTL3 or FTO expression since these proteins are main enzymes governing the adenosine methylation level of miRNA. ELISA indicated that METTL3 and FTO expression were the same in H1975 and H1975NivoSat cells (data not shown).
We next hypothesized that the gain of adenosine methylation of miR-125a-5p seen in H1975NivoSat cells could be due to a process that we called RNA binding protein-directed miRNA methylation. In this process, the miRNA methylation could be due to the formation of complex including METTL3 and a RNA binding protein (RBP) having the ability to bind miR- 125a-5p in sequence specific manner. To investigate this hypothesis, we first in silico searched for a RBP with the ability to bind miR-125a-5p. The database of RNA-binding protein (http://rbpdb.ccbr.utoronto.ca/index.php) identified six RBP (QKI, AC01, YBX1, RBMX/HNRNPG, KHDRBS3 and ELAVLl/HuR) likely to bind miR-125a-5p (data not shown). By integrating this list with the list of “METTL3 interactors” (according to BioGrid), only the KHDSBR3 protein appears in common (data not shown). Based on this analysis, we then hypothesized that METTL3 could form a complex with KHDRBS3 to adenosinemethylate the miR-125a-5p. IP experiments indicated that KHDRBS3 was coimmunoprecipitated with METTL3 only in H1975NivoSat cells (data not s hown).
We next performed siRNA-mediated METTL3 and KHDRBS3 down-regulation in order to question if METTL3 and KHDRBS3 cooperate to methylate miR-125a-5p. Thus, we observed that siRNA-directed against KHDRBS3 decreased the adenosine methylation of miR- 125a-5p without affecting its expression level (Figure 2A). Under these conditions, the expression and adenosine methylation level of miR-411-5p (a miRNA that includes the DRACH sequence of adenosine methylation but not the KHDRBS3 binding sequence (data not shown) remained unchanged (Figure 2A).
We also noted that siRNA-directed against METTL3 decreased the adenosine methylation of both miR-125a-5p and miR-411-5p without modifying their expression levels (Figure 2A). These last data support the idea that METTL3 and KHDRBS3 form a complex responsible for the adenosine methylation of miR-125a-5p.
It was previously reported that cells treated with Nivolumab have a higher activated ERK level than the cells treated with a control antibody [8], ERK activating the transcriptional activity of Elkl [24] and KHDRBS3 is a Elkl -regulated gene (according to the Harmonizome website (data not shown) To explain the METTL3/KHDRBS3 co-immunoprecipitation seen in H1975NivoSat cells and not in H1975 cells, we thus hypothesized that this could be due to the KHDRBS3 overexpression following to the activation of the Nivolumab/PD-l/ERK/Elkl signaling. To incriminate this pathway in our cells, experiments were performed in presence of ERK inhibitor (Ravoxertinib, #HY-15947, MedChem Express, France) and/or siRNA directed against Elkl. As expected, chemical and biologic inhibition of Elkl decreased the KHDSBR3 expression (Figure 2B). The direct involvement of Elkl on the KHDRBS3 expression was here supported by the fact that Elkl enrichment on the KHDRBS3 promoter was more important in H1975NivoSat than in H1975 and decreased in presence of Elkl inhibitor or siRNA (Figure 2B). Thus, these results together support the idea that the presence of METTL3/KHDRBS3 complex in H1975NivoSat cells is due to the activation of the Nivolumab/PD- 1/ERKZElkl signaling.
HuR blocks the recruitment of m6A-miR-125a-5p to GW182
To explain why the adenosine-methylated form of miR-125a-5p (m6a-miR-125a-5p) is not recruited by GW182, we hypothesized that this could be due to steric blockade induced by the recruitment of an "adenosine-methylated-binding protein (m6A-binding protein)". Based on literature, we established a list of 35 m6A-binding proteins (data not shown). By integrating this list with the list of RBP binding miR-125a-5p, we identified two proteins that we suspected to play the role of steric blocker: HuR and RBMX (data not shown). In order to determine whether these proteins have the ability to bind m6A-miR-125a-5p, a pull-down experiment was performed. Western blot performed from this experiment shows that RBMX remains in the flow through when subjected to a pull-down with a biotinylated miR-125a-3p mimic attached to avidin-sepharose beads, while HuR physically interacts with the biotinylated miR-125a-3p mimic (data not shown). We then completed our study by analyzing the impact of siRNA- induced down regulation of HuR on the level of adenosine methylation of miR-125a-5p, the recruitment of this miR by GW-182 and HuR and the IGSF11 expression. After observing that siRNA-HuR decreased the expression level of HuRmRNA, our data associate this context with the maintenance of the adenosine methylation and expression levels of miR-125a-5p, the increase miR-125a-5p recruitment by GW182 and the decrease of IGSF11 expression (Figure 3 A). In parallel, we also observed that HuR recruited miR-125a-5p in H1975NivoSat cells but not in H1975 cells (Figure 3B). Our investigations also show that treatment of H1975NivoSat cells with SFG (a METTL3 inhibitor decreasing the adenosine methylation level of miR-125a- 5p and increasing the recruitment of this miR by GW182 (Figure 1A) reduced the recruitment of miR-125a-5p by HuR (Figure 3B). Thus, all these results support the idea that HuR recruited the adenosine-methylated form of miR-125a-3p to block its recruitment with GW182. Investigation on the adenosine methylation of exosomal miR-125a-5p in two lung cancer patients
To determine whether the adenosine methylation level of miR-125a-5p could be associated with positive or negative response of lung cancer patients treated with anti-PD-1 therapy, we questioned that the adenosine methylation level of exosomal miR-125a-5p (m6A- exomiR- 125 a- 5 p) .
In vitro, we observed an increase of m6A-miR-125a-5p level in exosomes issued from H1975NivoSat cells, without observing changes in expression level of exomiR-125a-5p (Figure 4A). We also addressed this question for the two others antibodies used in patients for anti-PDl therapy used in clinic: Pembrolizumab (#A1306, Biovision/CliniScience, France) and Cemiplimab (#A2249, Biovision/CliniScience, France). Our data indicated that the three anti- PDl therapies tested increased the adenosine methylation of exomiR-125a-5p. Under this condition, we also observed an increase of IGSF11 expression (data not shown).
Based on this finding, we analyzed the m6A-exomiR-125a-5p and exomiR-125a-5p levels from longitudinal blood samples from two lung cancer patients treated with anti-PDl therapy.
Patient#A, was a 69-year-old male, former smoker, treated for a lung adenocarcinoma TTF1+ without EGFR, KRAS or BRAF mutation or ALK or ROS1 translocation but an expression of 100% of PDL1 on tumor cell. This patient showed synchronous bone, lung and brain metastases. He received pembrolizumab in first line setting from 04/24/2018 to 06/11/2020 (35 cycles) and presented, as best response, a partial response (-42%) according to RECIST 1.1. obtained at the first evaluation (8 weeks). The patient is always on follow up and presents until now no disease progression. Our analyses showed a rapid decrease of m6A- exomiR-125a-5p level without changes in the expression level of exomiR-125a-5p (Figure 4B). Patient#B, was a 53-year-old male, current smoker, with a lung adenocarcinoma TTF1+ PDL1 0% without EGFR KRAS or BRAF mutation or ALK and ROS1 translocation but STK11 mutation. The tumor was initially locally advanced (IIIC). The patient received in first line carboplatine pemetrexed bevacizumab from august 2018 to november 2018 with a partial response allowing the realization of a closing mediastinal radiotherapy. Three months after the end of radiotherapy, the CT scan showed the apparition of bilateral lung metastases and multiple brain metastasis. A second line with Nivolumab was started on june 2019. The first evaluation after 4 cycles showed a progression according to RECIST 1.1 criteria confirmed on a second CT Scan one month later. Nivolumab was stopped after 6 cycles. Our analyses showed a rapid increase of m6A-exomiR-125a-5p level without changes in the expression level of exomiR- 125a-5p (Figure 4C). The report of these two cases provides proofs of concept that the anti-PD- 1 therapy-mediated modulation of adenosine methylation level of miR-125a-5p can be analyzed by considering exomiR-125a-5p in longitudinal blood samples of lung cancer patients.
Finally, we analyzed the effect of patient-derived exosomes on the IGSF11 expression of Hl 975 cells.
For patient#A, exosomes from D63 and D105 blood samples decreased IGFS11 expression in H1975 cells more strongly than did exosomes from D2 blood samples (Figure 4D). The ability of patient#A exosomes to induce the decrease of IGSF11 expression in H1975 cells is even higher as it contains low adenosine methylated exomiR-125a-5p. The fact that transfection of D105 exosomes with an anti-miR-125a-5p results in the loss of repression of IGSF11 reinforces the idea that miR-125a-5p was the exosomal actor of this repression.
For patient#B, exosomes from D42 and D80 blood samples increased IGFS11 expression in H1975 cells compared with exosomes from DO blood samples (Figure 4D). In other words, a high amount of adenosine-methylated exomiR-125a-5p in exosomes inhibits their ability to trigger IGSF11 repression in Hl 975 cells. The dual fact that transfection of miR- 125a-5p into D80 exosomes decreases IGSF11 expression while transfection of miR-125a-5p- m6A does not, confirms the role played by adenosine methylation of miR-125a-5p in its repressive function towards IGSF11. Thus, these experiments performed with patient-derived exosomes reinforce the interest to consider the adenosine methylation of miR-125a-5p on the miR-125a-5p-mediated regulation of IGSF11 expression.
Conclusion:
This study identifies the miR-125a-5p/IGSFl 1 as an axis involved in the resistance to anti-PD-1 therapy and the adenosine methylation of this miRNA as a crucial mechanism regulating this axis. The inventors also show that the adenosine methylation of exosomal miR- 125a-5p could be a potential biomarker of anti-PD-1 therapy failure of lung cancer patients. In addition, this study shows that anti-IGSFl l - and METTL3 inhibitor-based therapies could be used to abrogate this mechanism of anti-PD-1 therapy failure.
REFERENCES:
Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure. 1. Xie, T.; Zhang, Z.; Qi, C.; Lu, M.; Zhang, X.; Li, J.; Shen, L.; Peng, Z. The Inconsistent and Inadequate Reporting Of Immune-Related Adverse Events in PD-1/PD-L1 Inhibitors: A Systematic Review of Randomized Controlled Clinical Trials. Oncologist 2021, doi: 10.1002/onco.13940.
2. Patsoukis, N.; Wang, Q.; Strauss, L.; Boussiotis, V.A. Revisiting the PD-1 Pathway. Sci Adv 2020, 6, eabd2712, doi: 10.1126/sciadv.abd2712.
3. Kim, J.M.; Chen, D.S. Immune Escape to PD-L1/PD-1 Blockade: Seven Steps to Success (or Failure). Ann Oncol 2016, 27, 1492-1504, doi: 10.1093/annonc/mdw217.
4. Kleffel, S.; Posch, C.; Barthel, S.R.; Mueller, H.; Schlapbach, C.; Guenova, E.;
Elco, C.P.; Lee, N.; Juneja, V.R.; Zhan, Q.; et al. Melanoma Cell-Intrinsic PD-1 Receptor Functions Promote Tumor Growth. Cell 2015, 162, 1242-1256, doi:10.1016/j. cell.2015.08.052.
5. Li, H.; Li, X.; Liu, S.; Guo, L.; Zhang, B.; Zhang, J.; Ye, Q. Programmed Cell Death-1 (PD-1) Checkpoint Blockade in Combination with a Mammalian Target of Rapamycin Inhibitor Restrains Hepatocellular Carcinoma Growth Induced by Hepatoma Cell-Intrinsic PD- 1. Hepatology 2017, 66, 1920-1933, doi: 10.1002/hep.29360.
6. Du, S.; McCall, N.; Park, K.; Guan, Q.; Fontina, P.; Ertel, A.; Zhan, T.; Dicker, A.P.; Lu, B. Blockade of Tumor-Expressed PD-1 Promotes Lung Cancer Growth. Oncoimmunology 2018, 7, el408747, doi: 10.1080/2162402X.2017.1408747.
7. Pu, N.; Gao, S.; Yin, H.; Li, J.-A.; Wu, W.; Fang, Y.; Zhang, L.; Rong, Y.; Xu, X.; Wang, D.; et al. Cell-Intrinsic PD-1 Promotes Proliferation in Pancreatic Cancer by Targeting CYR61/CTGF via the Hippo Pathway. Cancer Lett 2019, 460, 42-53, doi:10.1016/j.canlet.2019.06.013.
8. Wang, X.; Yang, X.; Zhang, C.; Wang, Y.; Cheng, T.; Duan, L.; Tong, Z.; Tan, S.; Zhang, H.; Saw, P.E.; et al. Tumor Cell-Intrinsic PD-1 Receptor Is a Tumor Suppressor and Mediates Resistance to PD-1 Blockade Therapy. Proc Natl Acad Sci U S A 2020, 117, 6640- 6650, doi: 10.1073/pnas.1921445117.
9. Yao, H.; Wang, H.; Li, C.; Fang, J.-Y.; Xu, J. Cancer Cell-Intrinsic PD-1 and Implications in Combinatorial Immunotherapy. Front Immunol 2018, 9, 1774, doi:10.3389/fimmu.2018.01774.
10. Zhou, E.; Li, Y.; Wu, F.; Guo, M.; Xu, J.; Wang, S.; Tan, Q.; Ma, P.; Song, S.; Jin, Y. Circulating Extracellular Vesicles Are Effective Biomarkers for Predicting Response to Cancer Therapy. EBioMedicine 2021, 67, 103365, doi: 10.1016/j.ebiom.2021.103365. 11. Valadi, H.; Ekstrom, K.; Bossios, A.; Sjostrand, M.; Lee, J. J.; Lotvall, J.O. Exosome-Mediated Transfer of MRNAs and MicroRNAs Is a Novel Mechanism of Genetic Exchange between Cells. Nat Cell Biol 2007, 9, 654-659, doi: 10.1038/ncbl596.
12. Briand, J.; Gamier, D.; Nadaradjane, A.; Clement-Colmou, K.; Potiron, V.; Supiot, S.; Bougras-Cartron, G.; Frenel, J.-S.; Heymann, D.; Vallette, F.M.; et al. Radiotherapy- Induced Overexpression of Exosomal MiRNA-378a-3p in Cancer Cells Limits Natural Killer Cells Cytotoxicity. Epigenomics 2020, 12, 397-408, doi: 10.2217/epi-2019-0193.
13. Guyon, N.; Gamier, D.; Briand, J.; Nadaradjane, A.; Bougras-Cartron, G.; Raimbourg, J.; Campone, M.; Heymann, D.; Vallette, F.M.; Frenel, J.-S.; et al. Anti-PDl Therapy Induces Lymphocyte-Derived Exosomal MiRNA-4315 Release Inhibiting Bim- Mediated Apoptosis of Tumor Cells. Cell Death Dis 2020, 11, 1048, doi: 10.1038/s41419-020- 03224-z.
14. Berulava, T.; Rahmann, S.; Rademacher, K.; Klein-Hitpass, L.; Horsthemke, B.
N6-Adenosine Methylation in MiRNAs. PLoS ONE 2015, 10, eO 118438, doi : 10.1371/joumal.pone.Ol 18438.
15. Konno, M.; Koseki, J.; Asai, A.; Yamagata, A.; Shimamura, T.; Motooka, D.; Okuzaki, D.; Kawamoto, K.; Mizushima, T.; Eguchi, H.; et al. Distinct Methylation Levels of Mature MicroRNAs in Gastrointestinal Cancers. Nat Commun 2019, 10, 3888, doi: 10.1038/s41467-019-l 1826-1.
16. Pandolfini, L.; Barbieri, I.; Bannister, A.J.; Hendrick, A.; Andrews, B.; Webster,
N.; Murat, P.; Mach, P.; Brandi, R.; Robson, S.C.; et al. METTL1 Promotes Let-7 MicroRNA Processing via M7G Methylation. Mol. Cell 2019, 74, 1278-1290. e9, doi:10.1016/j.molcel.2019.03.040.
17. Cheray, M.; Etcheverry, A.; Jacques, C.; Pacaud, R.; Bougras-Cartron, G.; Aubry, M.; Denoual, F.; Peterlongo, P.; Nadaradjane, A.; Briand, J.; et al. Cytosine Methylation of Mature MicroRNAs Inhibits Their Functions and Is Associated with Poor Prognosis in Glioblastoma Multiforme. Mol. Cancer 2020, 19, 36, doi: 10.1186/sl2943-020-01155-z.
18. Carissimi, C.; Laudadio, I.; Lorefice, E.; Azzalin, G.; De Paolis, V.; Fulci, V. Bisulphite MiRNA-Seq Reveals Widespread CpG and Non-CpG 5 -(Hydroxy )Methyl -Cytosine in Human MicroRNAs. RNA Biol 2021, 1-10, doi: 10.1080/15476286.2021.1927423.
19. Briand, J.; Serandour, A.A.; Nadaradjane, A.; Bougras-Cartron, G.; Heymann, D.; Ory, B.; Vallette, F.M.; Cartron, P.-F. N6-Adenosine Methylation of MiRNA-200b-3p Influences Its Functionality and Is a Theranostic Tool. Mol Ther Nucleic Acids 2020, 22, 72- 83, doi: 10.1016/j.omtn.2020.08.010. 20. Zhang, B.; Chen, Z.; Tao, B.; Yi, C.; Lin, Z.; Li, Y.; Shao, W.; Lin, J.; Chen, J. M6A Target MicroRNAs in Serum for Cancer Detection. Mol Cancer 2021, 20, 170, doi:10.1186/sl2943-021-01477-6.
21. Briand, J.; Joalland, M.-P.; Nadaradjane, A.; Bougras-Cartron, G.; Olivier, C.; Vallette, F.M.; Perruche, S.; Cartron, P.-F. Diuron Modulates the DNA Methylation Status of the ILT7 and TRAIL/TNFSF10 Genes and Decreases the Killing Activity of Plasmacytoid Dendritic Cells. Environ Sci Eur 2019, 31, 35, doi: 10.1186/sl2302-019-0219-8.
22. Heymann, D.; Kerdraon, O.; Verriele, V.; Verhille, E.; Veron, V.; Vitre, M.; Delmas, F.; Henry, C.; Gouy, Y.; Amiand, M.; et al. Centre de Ressources Biologiques- Tumorotheque: Bioresources and Associated Clinical Data Dedicated to Translational Research in Oncology at the Institut de Cancerologie de 1’Ouest, France. Open Journal of Bioresources 2020, 7, 5, doi: 10.5334/ojb.62.
23. Li, J.; Han, X.; Wan, Y.; Zhang, S.; Zhao, Y.; Fan, R.; Cui, Q.; Zhou, Y. TAM 2.0: Tool for MicroRNA Set Analysis. Nucleic Acids Res 2018, 46, W180-W185, doi: 10.1093/nar/gky509.
24. Gille, H.; Kortenjann, M.; Thomae, O.; Moomaw, C.; Slaughter, C.; Cobb, M.H.; Shaw, P.E. ERK Phosphorylation Potentiates Elk- 1 -Mediated Ternary Complex Formation and Transactivation. EMBO J 1995, 14, 951-962, doi: 10.1002/j.1460-2075.1995.tb07076.x.
25. Moroz-Omori, E.V.; Huang, D.; Kumar Bedi, R.; Cheriyamkunnel, S.J.; Bochenkova, E.; Dolbois, A.; Rzeczkowski, M.D.; Li, Y.; Wiedmer, L.; Caflisch, A. METTL3 Inhibitors for Epitranscriptomic Modulation of Cellular Processes. ChemMedChem 2021, doi : 10.1002/cmdc.202100291.
26. Watanabe, T.; Suda, T.; Tsunoda, T.; Uchida, N.; Ura, K.; Kato, T.; Hasegawa, S.; Satoh, S.; Ohgi, S.; Tahara, H.; et al. Identification of Immunoglobulin Superfamily 11 (IGSF11) as a Novel Target for Cancer Immunotherapy of Gastrointestinal and Hepatocellular Carcinomas. Cancer Sci 2005, 96, 498-506, doi: 10.1111/j.1349-7006.2005.00073.x.
27. Ghouzlani, A.; Rafii, S.; Karkouri, M.; Lakhdar, A.; Badou, A. The Promising IgSFl 1 Immune Checkpoint Is Highly Expressed in Advanced Human Gliomas and Associates to Poor Prognosis. Front Oncol 2020, 10, 608609, doi: 10.3389/fonc.2020.608609.
28. Alarcon, C.R.; Lee, H.; Goodarzi, H.; Halberg, N.; Tavazoie, S.F. N6- Methyladenosine Marks Primary MicroRNAs for Processing. Nature 2015, 519, 482-485, doi:10.1038/naturel4281.
29. Selberg, S.; Blokhina, D.; Aatonen, M.; Koivisto, P.; Siltanen, A.; Mervaala, E.; Kankuri, E.; Karelson, M. Discovery of Small Molecules That Activate RNA Methylation through Cooperative Binding to the METTL3-14-WTAP Complex Active Site. Cell Rep 2019, 26, 3762-3771. e5, doi: 10.1016/j.celrep.2019.02.100.
30. Bedi, R.K.; Huang, D.; Eberle, S.A.; Wiedmer, L.; Sledz, P.; Caflisch, A. Small- Molecule Inhibitors of METTL3, the Major Human Epitranscriptomic Writer. ChemMedChem 2020, 15, 744-748, doi:10.1002/cmdc.202000011.
31. Yankova, E.; Blackaby, W.; Albertella, M.; Rak, J.; De Braekeleer, E.; Tsagkogeorga, G.; Pilka, E.S.; Aspris, D.; Leggate, D.; Hendrick, A.G.; et al. Small-Molecule Inhibition of METTL3 as a Strategy against Myeloid Leukaemia. Nature 2021, 593, 597-601, doi : 10.1038/s41586-021 -03536-w.
32. Koyama, S.; Akbay, E.A.; Li, Y.Y.; Herter-Sprie, G.S.; Buczkowski, K.A.; Richards, W.G.; Gandhi, L.; Redig, A.J.; Rodig, S.J.; Asahina, H.; et al. Adaptive Resistance to Therapeutic PD-1 Blockade Is Associated with Upregulation of Alternative Immune Checkpoints. Nat Commun 2016, 7, 10501, doi : 10.1038/ncomms 10501.
33. Grimaldi, A.M.; Marincola, F.M.; Ascierto, P.A. Single versus Combination Immunotherapy Drug Treatment in Melanoma. Expert Opin Biol Ther 2016, 16, 433-441, doi:10.1517/14712598.2016.1128891.
34. Kaludov, N.K.; Wolffe, A.P. MeCP2 Driven Transcriptional Repression in Vitro: Selectivity for Methylated DNA, Action at a Distance and Contacts with the Basal Transcription Machinery. Nucleic Acids Res 2000, 28, 1921-1928, doi: 10.1093/nar/28.9.1921.
35. Prokhortchouk, E.; Hendrich, B. Methyl-CpG Binding Proteins and Cancer: Are
MeCpGs More Important than MBDs? Oncogene 2002, 21, 5394-5399, doi: 10.1038/sj. one.1205631.
36. Prokhortchouk, A.; Hendrich, B.; Jorgensen, H.; Ruzov, A.; Wilm, M.; Georgiev, G.; Bird, A.; Prokhortchouk, E. The P120 Catenin Partner Kaiso Is a DNA Methylation-Dependent Transcriptional Repressor. Genes Dev 2001, 15, 1613-1618, doi: 10.1101/gad.198501.
Muralidharan Ranganayaki, Meghna Mehta, Rebaz Ahmed, Sudeshna Roy, Liang Xu, Jeffrey Aube, Allshine Chen, Yan Daniel Zhao, Terence Herman, Rajagopal Ramesh, and Anupama Munshicorresponding author. HuR-targeted small molecule inhibitor exhibits cytotoxicity towards human lung cancer cells. Sci Rep. 2017; 7: 9694.