NL2037024B1 - Janus kinase (JAK) inhibitors for treating cancer - Google Patents

Abstract

A B S T R A C T The invention relates to inhibitors of JAK proteins for use in the treatment of cancer. In vitro methods for screening of candidate agents as JAK inhibitors, and methods for the selection of a subject suffering from cancer as candidate for a cancer therapy comprising in particular JAK inhibitors are also disclosed.

Description

Title: Janus kinase (JAK) inhibitors for treating cancer

FIELD OF THE INVENTION

[001] This invention pertains in general to the field of medicine, and in particular to the field of cancer treatment. The invention focuses more specifically on pharmacogenomics and personalized medicine.

BACKGROUND OF THE INVENTION

[002] The background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[003] Cancer is a pathological condition in which abnormal cell growth in tissue takes place with the potential to invade or spread to other parts of the body. Every sixth death in the world is due to cancer, making it the second leading cause of death — second only to cardiovascular diseases. Because cancer is one of the leading causes of death, it is one of the world's most pressing problems to make progress against this disease (Max Roser and Hannah Ritchie (2019) - “Cancer” Published online at

OurWorldinData.org. Retrieved from: 'https://ourworldindata.org/cancer' [Online

Resource]). Cancer is a heterogenous diseases and a wide variety of underlying mechanisms have been identified.

[004] The Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway is regarded as an important central communication node in cell function and a myriad of cytokines and growth factors have been identified in the

JAK/STAT signaling pathway. JAK/STAT-mediated downstream events include hematopoiesis, immune fitness, tissue repair, inflammation, apoptosis, and adipogenesis. This makes that the loss or mutation of JAK/STAT components is related to many diseases in humans, and many authors papers have reported the importance of the JAK/STAT signaling pathway in malignancies (i.e., cancer) and autoimmune diseases. Thus, inhibiting the JAK/STAT pathway is promising for treating various diseases, including cancer. Currently, many JAK inhibitors have shown efficacy in clinical settings, and more medications are currently being studied. (see Hu et al. 2021. The JAK/STAT signaling pathway: from bench to clinic. Signal

Transduction and Targeted Therapy (2021) 6:402 ; hiips idol org/10. 1088/54 1392- 021-00781-1, see Rah et al, 2022. JAK/STAT Signaling: Molecular Targets,

Therapeutic Opportunities, and Limitations of Targeted Inhibitions in Solid

Malignancies; Front. Pharmacol. (2002) 13; hitps:didolorg/10.338% phar. 2022 621344).

[005] Various therapeutics have been, and continue to be, developed to modulate the signaling pathway, with varying degrees of success and failures (see Owen et al. JAK-

STAT Signaling: A Double-Edged Sword of Immune Regulation and Cancer

Progression. Cancers. 2019;11:2002. doi: 10.3390/cancers11122002). Differences in drug targets and efficacy aside, these observations are not entirely surprising, given an emerging understanding of the importance of JAK-STAT signaling in both immune regulation and cancer progression.

[006] In one example, in the treatment of cancers, JAK inhibitors have been employed with the aim of indirectly targeting STAT proteins, which aberrant activation is associated with the development and progression of solid tumors (see Qureshy et al. 2020. Targeting the JAK/STAT pathway in solid tumors. J Cancer Metastasis Treat. 2020; 6). However, results suggest that targeting of the JAK/STAT pathway (i.e, mainly by using JAK inhibitors) are not always effective, ruling out effective treatment with such JAK inhibitors in these patients, who see there treatment options reduced.

[007] Compounds aimed to inhibit or modulate JAK proteins for other diseases than cancer diseases may undergo a similar fate. If they are found not to be effective relative to mutated isoforms of these JAK proteins, the compounds are finally set apart in the clinic as they do not provide the required benefit to the patient.

[008] In light of this, new products, compositions, methods and uses in the diagnosis and treatment of cancer would be highly desirable but are not yet readily available. In particular, there is a clear need in the art for reliable, efficient, and reproducible products, compositions, methods and uses that allow to be used in the treatment of cancer. Accordingly, the technical problem underlying the present invention can been seen in the provision of such products, compositions, methods and uses for complying with any of the aforementioned needs, or at least providing the public with a useful choice. The technical problem is solved by the embodiments characterized in the claims and herein below.

SUMMARY OF THE INVENTION

[009] As embodied and broadly described herein, the present invention is directed to the surprising finding that a sub-group of patients suffering from cancer, i.e. certain patients suffering from cancer, predominantly express a newly identified short-version (short isoform) of canonical Janus Kinase 1 (JAK1) protein. The short isoform of JAK1 comprises a (functional) JH1 kinase domain of the JAK1 protein and does not comprise a (functional) JH2 (pseudo)kinase domain of the JAK1 protein. In other words, the short isoform of JAK1 comprising a (functional) JH1 kinase domain of the JAK1 protein does not comprise the JH2 (pseudo)kinase domain of the JAK1 protein, and/or does not comprise a JH2 (pseudo)kinase domain, or part thereof, that is capable of (auto)inhibitory interaction with the JH1 kinase domain of the JAK1 protein.

[010] This short-version results from an alternative mRNA output phenomenon, and gives rise to a pro-tumorigenic (i.e., oncogenic) protein opposed to the acknowledged tumor-suppressing role of the canonical isoform of JAK1 protein. More importantly, this short oncogenic isoform is sensitive to JAK inhibitors previously used in other than cancer clinical conditions, and to JAK inhibitors used for cancer treatment but discharged when a loss of function mutation in JAK proteins was observed.

[011] This alternative mRNA output phenomenon, termed also in this description as

RNA dicing, consists in that some proteins are expressed as different isoforms resulting from mRNA “sub-fractions” capable of translating into reduced subsets of protein domains with a distinctive biological function, even with opposed functions.

Different cell outcomes may result depending on the ratio(s} of the multiple possible isoforms expressed in a cell. The possibility of the existence of all these isoforms of a protein is related with the phenomenon in cells where mRNAs from thousands of genes undergo endonuclease cleavage, producing truncated 5° Uncapped Polyadenylated

Transcripts (5’UPT) with translation potential which significantly diversifies the proteome landscape. This was previously disclosed by the inventors, who disclosed thousands of previously undescribed 5 uncapped and polyadenylated transcripts (5'UPTs) that resist exonucleases due to a highly structured RNA and N6- methyladenosine (m6A) modification at their 5’ termini. The alternative polyadenylation (APA) promotes downstream translation initiation, and a non-canonical protein output with biological impact. (Malka. Y, et al. 2022. Alternative cleavage and polyadenylation generates downstream uncapped RNA isoforms with translation potential. Mol Cell.

2022 Oct 20;82(20):3840-3855.e8. doi: 10.1016/j.molcel.2022.09.036. PMID: 36270248; PMCID: PMC9636002).

[012] For Janus kinase 1 (JAK1), the inventors have surprisingly found an unpredictable event, and which translates into a high impact in clinics. Namely, that it is possible to effectively use JAK inhibitors, even those that would have been previously set apart, to modulate or inhibit signaling normally caused by JAK activity (acting as a tyrosine kinase; in particular JAK1 activity), because these inhibitors will target and effectively reduce the activity of that short and oncogenic isoform of the protein predominantly expressed in cancer.

[013] Thus, in a first aspect the invention provides a Janus Kinase (JAK) inhibitor for use in the treatment of cancer in a subject, wherein the subject comprises a tumor, wherein the tumor is characterized by expression of an isoform of a Janus Kinase 1 (JAK1) protein, wherein the isoform: - atleast comprises a JH1 kinase domain of the JAK1 protein; and - does not comprise a JH2 (pseudo)kinase domain of the JAK1 protein, and/or does not comprise a JH2 (pseudo)kinase domain, or part thereof, that is capable of (auto)inhibitory interaction with the JH1 kinase domain of the JAK1 protein.

[014] This aspect derives, as said, from the unexpected finding by the inventors, detailed in the examples below, and according to which tumor cells isolated from a subgroup of subjects suffering from certain cancers, as well as cells of different cancer cells lines, express an isoform of JAK1, which isoform is non-canonical one, and which cells were sensitive to several tested JAK inhibitors (e.g., CYT387 and CEP-33779).

The administration of these inhibitors caused a significative reduction of the cell viability (log2 fold change).

[015] Contrary to the technical prejudice of administering an inhibitor against any tumor-suppressing protein, the unpredictable finding of the inventors with JAK1 and the existence of several isoforms resulting from an alternative mRNA production in cells, sheds light to new therapeutical approaches, that would have been previously undermined. Moreover, with the example of JAK1, the inventors show the potential applications stemming from comprehending this phenomenon of alternative mRNA production in cells, particularly within the realm of drug treatment. By showcasing how

RNA dicing influences drug tolerance and response, the invention highlights its relevance in the domain of pharmacogenomics and personalized medicine.

[016] Without being bound to any theory, the inventors propose that RNA dicing may be the result of a particular cell condition, that promotes or favors the expression of these alternative 5'-UPT transcripts to produce certain protein isoforms rather than the canonical ones, or the ones expected due to RNA splicing or common RNA translation. 5 [017] It may also happen that in certain pathological conditions, the balance of expression of several of the possible isoforms is, for any cause (e.g. mutation), shifted to a ratio between these possible isoforms that enhances or feeds back the pathological state. This may be the result, for example, of an increased amount of the pathological inducing isoforms in relation to the non-pathological inducing isoform. The reason why a particular isoform may promote or avoid any pathological state or condition may be the result of the possibility of the differing cell localization, cell function, or protein complex interaction between the possible isoforms.

[018] Janus kinase (JAK) is a family of intracellular, non-receptor tyrosine kinases that transduce cytokine-mediated signals via the previously mentioned JAK-

STAT pathway. The name is taken from the two-faced Roman god of beginnings, endings and duality, Janus, because the JAKs possess two near-identical phosphate- transferring domains. One domain exhibits the kinase activity, while the other negatively regulates the kinase activity of the first.

[019] The JAK proteins family in humans includes four members, namely JAK1,

JAK2, JAK3 and TYK2. The genes that encode for these proteins include several domains all of which are comprised in the considered canonical isoforms of the proteins. The canonical isoform of the JAK proteins includes from N- to C-terminal, the

FERM domain, a protein module involved in localizing proteins to the plasma membrane; the SH2 domain, commonly found in adaptor proteins aiding in the signal transduction of receptor tyrosine kinase pathways; the JH2 (Pseudokinase) domain, a catalytically-deficient pseudoenzyme that decreases the kinase enzymatic activity of

JAK(s) via (auto)inhibitory interaction with the JH1 kinase domain; and the JH1 (Kinase) domain, a module with catalytic function in protein kinases. A schematic representation of the JAK1 domains is illustrated in Fig 1 (c, d).

[020] The expressed (short) isoform in the analyzed tumors from subjects or in the tested cell lines of the examples, comprised the JH1 kinase domain of JAK1 but lacked any JH2 (pseudo)kinase domain or lacked any JH2 (pseudo}kinase domain that was capable of (autojinhibitory interaction with the JH1 kinase domain of the JAK1. This isoform is, as shown by the inventors, the result of the RNA dicing phenomena (i.e., the presence of 5-UPT mRNAs), and, as illustrated in the examples, it is a pro- tumorigenic isoform. Without being bound to any theory, the inventors postulate that its role in carcinogenesis is likely due to its independent expression of and relative expression to the canonical JAK1.

[021] Indeed, the inventors established that in the studied cancers the ratio of the expression of the short isoform according to the invention, abbreviated herewith as

JH1-isoform, and the expression of the canonical JAK1, was higher (relative more

JH1-isoform) as compared to the ration in subjects not suffering from cancer, or in control cell lines. Moreover, they also realized that in more aggressive cancers the ratio JH1-isoform/JAK1 (canonical) was higher.

[022] A major advantage of the JAK inhibitors for use in the treatment of cancer in a subject with a tumor that expresses the pro-tumorigenic isoform (e.g. JH1-isoform) as indicated in this first aspect of the invention, relies on that, strikingly, low doses of JAK inhibitors can be effectively administered to the subject. These doses are, moreover, lower than the ones used nowadays for example to inhibit canonical JAK1 for other indications (i.e., mainly for autoimmune disease). This is noteworthy, because any associated risk of adverse effect linked to the administration of the inhibitors, such as anemia, lymphopenia, neutropenia, opportunistic infections, nausea, headache and acne, may be reduced without compromising the efficacy of the treatment.

[023] The JAK inhibitors tested in the examples described herein and/or performed by the inventors, effectively inhibited the short isoform of JAK1 protein as defined in the first aspect and promoted a reduction of the proliferation of the cells expressing the short isoform of JAK1 protein as defined in the first aspect. The exact mechanism by means of which this is achieved is still under research. All the known JAK{(s) inhibitors target the kinase domains, but it is likely that differential inhibition modes operate depending on whether the JAK1 protein is a canonical JAK one, or an isoform as defined and that results from a 5’UPT, mainly due to differing conformational assemblies between the JAK1 and the inhibitor.

[024] As a consequence of the expression in cells of this sensitive to JAK inhibitors oncogenic isoform, it follows that for the selection of a medical regimen a subject can be screened to detect if this isoform of JAK1 according to the invention is expressed in the tumor cells and, thus, if the subject is susceptible to be treated with a JAK inhibitor in accordance with the invention.

[025] Therefore, in a second aspect the invention relates to an in vitro method for the selection of a subject suffering from cancer as candidate for a cancer therapy, wherein the method comprises determining in a sample comprising tumor material obtained from the subject: - the presence of an isoform of a Janus Kinase 1 (JAK1) protein as defined in the first aspect, namely an isoform that at least comprises a JH1 kinase domain of the JAK1 protein; and does not comprise a JH2 (pseudo}kinase domain of the

JAK1 protein and/or does not comprise a JH2 (pseudo)kinase domain, or part thereof, that is capable of (auto)inhibitory interaction with the JH1 kinase domain of the JAK1 protein; - the presence of a JAK1 gene comprising one or more mutations as defined in the first aspect; and/or - the presence of a RNA molecule that, when translated, provides for an isoform of a Janus Kinase 1 (JAK1) protein as defined in the first aspect, - and wherein if the isoform of a Janus Kinase 1 (JAK1) protein and/or the JAK? gene comprising one or more mutations and/or the RNA molecule is present in the sample, the subject is selected as candidate for the cancer therapy, in particular for cancer therapy using JAK inhibitors.

[026] The surprising finding of the isoform of JAK1 as defined in the first aspect in a tumor and its sensitivity to already known inhibitors also opens the possibility to screen for other compounds (candidates), that can modulate the activity and/or bind to the said isoform, preferably specifically (i.e. wherein modulation of activity and/or binding to the isoform according to the invention is preferential over modulation of activity and/or binding to canonical JAK1 protein), with the aim to provide for compounds useful in therapy.

[027] The invention provides thus, in a third aspect, an in vitro method for screening of candidate agents that: - bind to an isoform of a Janus Kinase 1 (JAK1) protein as defined in the first aspect; and/or - modulate the activity of an isoform of a Janus Kinase 1 (JAK1) protein as defined in the first aspect;

the method comprising the steps of: - contacting the isoform of a Janus Kinase 1 (JAK1) protein as defined in the first aspect with a candidate agent; and - detecting binding of a candidate agent with and/or detecting modulation of the activity of the isoform of the Janus Kinase 1 (JAK1) protein; - optionally, detecting binding of the candidate agent with and/or detecting modulation of the activity of a Janus Kinase 1 (JAK1) protein.

BRIEF DESCRIPTION OF THE DRAWINGS

[028] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

[029] Figure 1: RNA Dicing: Metabolic Mechanism for RNA Processing.(a) Schematic model illustrating how Alternative Polyadenylation (APA) triggers the expression of downstream transcript isoforms. These isoforms, stabilized by RNA structure, serve as templates for uncapped non-canonical protein synthesis in an m6A-dependent manner. Following APA induction there is widespread accumulation and translation of truncated protein isoforms. {(b) Peak calling analysis of m6A-RIPseq data (MacFadden et al.) shows high enrichment of m6A sites in exons around the PROSITE kinase start domain. (c) Schematic representation of the JAK1 domains: FERM - a protein module involved in localizing proteins to the plasma membrane; SH2 - commonly found in adaptor proteins aiding in the signal transduction of receptor tyrosine kinase pathways;

JH2 (Pseudokinase) - a catalytically-deficient pseudoenzyme; JH1 (Kinase) — a module with catalytic function in protein kinases. JH2 induces inhibition. The JH2 pseudokinase domain leads to a dramatic decrease in enzymatic activity. (d) Western blot analysis for JAK1 using antibodies against JH2 domain, JH1 domain, and phosphoJAK1 (JH1) shows expression of different JAK1 isoforms corresponding to full length or diced JAK1 isoforms. Phospho-JAK1 shows detection of only diced JAK1 kinase domain isoform. (e) Knockdown experiment against JAK1 using two different shRNA shows a decrease in full length and the diced phosphorylated kinase domain.{f). Nuclear phosphor-JAK1 on a western blot showing nuclear phosphor-

JAK1 (N) and cytoplasm phosphor-JAK1 (C) as shorter isoforms of 42/44kd.

[030] Figure 2. RNA Dicing plays critical role in JAK1 kinase function and cell proliferation. (a) Design of three constructs of JAK1: WT JAK1 - human JAK1 CDS;

APA-mut JAK1 — codon optimization of nucleotides 990-1176 in the CDS corresponding to the APA region; Codon-optimized JAK1 - substitutions of ~20% of

CDS nucleotides. All constructs contain the open reading frame of WT JAK1. (b)

Codon optimization of JAK1 interferes with JAK1 mRNA dicing, resulting in differential expression of full-length canonical JAK1 and diced phospho-JAK1. (c) RT-qPCR of

Codon-optimized JAK1-expressing cells shows high expression of endogenous WT

JAK1 mainly in diced JAK1 mRNA. (d} Colony formation assay shows reduction in colonies in codon-optimized JAK1-expressing cells.

[031] Figure 3. Nonsense/frameshift mutation have different effect on JAK1 expression due to modular expression of canonical and diced isoforms. (a) JAK1 hotspot frameshift mutations occur at position 430 (P430Rfs) and mainly at position 860 (K860Nfs}, which are located between the JH2 and JH1 coding regions. (b} Z- scores from 108 base editing proliferation screens inducing nonsense stop codon mutations are plotted for each gRNA. Screen z-scores are calculated independently for each base editor and plotted together for comparison.{c) Nonsense mutations are separated for each corresponding protein domain mRNA region. Induction of a premature stop codon results in an increased proliferation rate, except for insertions of stop codons at the kinase domain.

[032] Figure 4. JAK1 frameshift mutations in endometrial cancer affect the expression of the full-length JAK1 isoform but not the expression of the diced JAK1 kinase domain. (a) RNA-seq data from TCGA-UCEC of 70 patients with WT JAK1 and 7 patients with

JAK1fs mutation. Average RNA-seq read coverage deviation of patients/normal adjacent tissue (24 samples) shows no difference in RNA distribution in WT JAK1 patients and a sharp decrease in JH2, followed by an increase in JH1 domain expression in JAK1fs patients, indicating independent JH1 RNA expression. (b) Mass spectrometry of EC (CPTAC cohort) of JAK1fs patients shows an identical trend of domain peptide coverage to RNA-seq domain coverage, indicating independent JAK1 kinase domain isoform in JAK1fs patients. (c) Mass spectrometry analysis of CAL51 cell line harboring homozygous JAK1fs mutation at position 860 shows 50% in-frame peptide coverage downstream of the frameshift position. (d) Western blot of MCF7 and

ISHIKAWA cell lines shows diced phospho JAK1 expression in WT expressing JAK1

MCF7 and ISHIKAWA cells harboring homozygous frameshift mutation at position 60.

Full-length JAK1 is detected only in MCF7 cells. (e) Cell growth of cells expressing

WT JAK1 or codon-optimized JAK1 constructs was measured using cell counting. Full- length JAK1 expression significantly reduced cell growth. (f) Colony formation assay shows a reduction in colonies in both JAK1 full-length expressing construct cells.

[033] Figure 5. Cells with JAK1fs mutations exhibit high intolerance to JAK inhibitors due to the expression of the oncogenic diced JAK1 kinase and complete KD of the canonical full length tumor suppressor. (a) RNA and protein expression of JAK1 (434 and 221 cell lines, respectively) showed no correlation with the efficacy of CYT387 treatment at various doses. (b) PRISM Repurposing Secondary Screen of JAK inhibitors CYT387 in multiple EC cell lines, Breast, Prostate, and Ovarian cancers with

WT/homozygous JAK1fs mutations. (c) Out of 17 EC cell lines that were treated with

CYT387 (2.4 uM), cells carrying JAK1 mutation were found to be among the most sensitive to this treatment. This included 9 cell lines with WT JAK1, 3 cell lines with a heterozygous mutation, and 5 cell lines with a heterozygous damaging mutation in

JAK1.

[034] Figure 6. The PRISM Repurposing Secondary Screen for JAK inhibitors

CYT387 (indicated by the upper box) and CEP-33779 (indicated by the lower box) demonstrates high sensitivity in multiple cancer cell lines, and to cells carrying JKA 1fs mutations, to these inhibitors. Endometrial Cancer cell lines (WT JAK1 cell lines;

MFE280, SNU1077, SNU685, JHUEM2, AN3CA, HEC1B, HEC1A, SNGM, HECS,

HEC59. JAK1fs cell line; HEC265, ISHIKAWAHERAKLIOO2ZER, HEC151, MFE319,

MFE296, HEC108. Breast (WT JAK1 cell lines; BT549, HCC1143, CAMA1, MCF7,

HCC38, CAL120, MDAMB231, MDAMB438, EFM192A, HCC1937, HCC1419,

HCC1428, BT474, MDAMB468, ZR751, MDAMB175VII, HDQP1, T47D. JAK1fs cell line; CAL51), Prostate (WT JAK1 cell lines; PC3, DU145. JAK1fs cell line; 22RV1).

DESCRIPTIONDefinitions

[035] A portion of this disclosure contains material that is subject to copyright protection (such as, but not limited to, diagrams, device photographs, or any other aspects of this submission for which copyright protection is or may be available in any jurisdiction}. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or patent disclosure, as it appears in the Patent Office patent file or records, but otherwise reserves all copyright rights whatsoever.

[036] Various terms relating to the methods, compositions, uses and other aspects of the present invention are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art to which the invention pertains, unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definition provided herein. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein.

[037] For purposes of the present invention, the following terms are defined below.

[038] As used herein, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. The indefinite articles “a” and “an” are synonymous with “at least one” or “one or more”.

[039] As used herein, the term “amount” is used interchangeably with the term “dose”.

[040] As used herein, the term “and/or” indicates that one or more of the stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases.

[041] As used herein, the term "at least" a particular value means that particular value or more. For example, "at least 2" is understood to be the same as "2 or more" i.e., 2, 3,4,5,6,7,8,9, 10, 11,12, 13, 14, 15, ..., etc.

[042] As used herein, “comprising” or “to comprise” is construed as being inclusive and open ended, and not exclusive. Specifically, the term and variations thereof mean the specified features, steps or components are included. These terms are not to be interpreted to exclude the presence of other features, steps or components. It also encompasses the more limiting “to consist of”.

[043] As used herein, “conventional techniques” or “methods known to the skilled person” refer to a situation wherein the methods of carrying out the conventional techniques used in methods as disclosed herein will be evident to the skilled worker.

The practice of conventional techniques in molecular biology, biochemistry, cell culture, genomics, sequencing, medical treatment, pharmacology, immunology and related fields are well-known to those of skill in the art and are discussed, in various handbooks and literature references.

[044] As used herein the term “nucleic acid” or “polynucleotide” refers to any polymers or oligomers of (contiguous) nucleotides. The nucleic acid may be DNA or

RNA, or a mixture thereof, and may exist permanently or transitionally in single- stranded or double-stranded form, including homoduplex, heteroduplex, and hybrid states. The present invention also contemplates any deoxyribonucleotide, ribonucleotide or peptide nucleic acid component, and any chemical variants thereof, such as methylated, hydroxymethylated or glycosylated forms of these bases, and the like. The polymers or oligomers may be heterogeneous or homogenous in composition and may be isolated from naturally occurring sources or may be artificially or synthetically produced.

[045] As used herein, “protein” or “polypeptide” are used interchangeably and refer to molecules consisting of a chain of amino acids, without reference to a specific mode of action, size, 3-dimensional structure or origin. A “fragment” or “portion” of a protein may thus still be referred to as a “protein.” When a fragment is referred to it preferably includes a fragment of the protein that is a functional fragment and, thus, that can perform the function of the whole protein. A protein as defined herein and as used in any method as defined herein may be an isolated protein. An “isolated protein” is used to refer to a protein which is no longer in its natural environment, for example in vitro or in a recombinant host cell.

[046] As used herein, “sequence”, “amino acid sequence” or “(poly)nucleotide sequence” refers to the order of amino acids, nucleotides of, or within a polypeptide or nucleic acid/polynucleotide. In other words, any order of amino acids or nucleotides may be referred to as a sequence (amino acids sequence, nucleotide sequence).

[047] As used herein, the term “canonical” when referring to a protein, namely to JAK proteins, refers to the isoform that comprises all the (functional) protein domains common to the JAK protein family and including the FERM, SH2, JH2 and JH1 domains, also termed herein as the long-isoform. In general a canonical protein is identified by several criteria, mainly including experimental data on its functional role; data about its expression in different tissues of an organism; and existence of the same combination of exons in orthologous proteins and in different curated databases.

[048] The term "subject" or "patient" as used herein, refers to mammals. Mammalian species that can benefit from the disclosed methods of treatment include, but are not limited to, humans, non-human primates such as apes; chimpanzees; monkeys, and orangutans, domesticated animals, including dogs and cats, as well as livestock such as horses, cattle, pigs, sheep, and goats, or other mammalian species including, without | imitation, mice, rats, guinea pigs, rabbits, hamsters, and the like.

[049] As used herein, the term “pharmaceutical composition” refers to a composition formulated in pharmaceutically-acceptable or physiologically-acceptable compositions for administration to a cell or subject. The compositions of the invention may be administered in combination with other agents as well, provided that the additional agents do not adversely affect the ability of the composition to deliver the intended therapy. The pharmaceutical composition often comprises, in addition to a pharmaceutical active agent (i.e., the nucleic acid construct, vector or host cell or viral particle), one or more pharmaceutical acceptable carriers (or excipients).

[050] As used herein, the term “therapeutically-effective amount" or “effective amount” refers to the amount of a nucleic acid construct, vector, host cell, viral particle, or any other product as disclosed herein, which is effective for producing an effective and desired (therapeutic) effect in a subject at a reasonable benefit/risk ratio applicable and within the context of the treatment of the invention.

[051] As used herein, the terms “treatment” and “treating” refer to therapeutic treatment. The object of the treatment is to at least slow down the disease condition.

Those in need of the treatment include those already with the disease condition.

[052] As used herein “identity” or “sequence identity” refers to the degree of relatedness between two or more amino acid sequences, or two or more nucleic acid sequences (polynucleotide sequences), as determined by comparing the sequences.

The comparison of sequences and determination of sequence identity may be accomplished using a mathematical algorithm; those skilled in the art will be aware of computer programs available to align two sequences and determine the percent identity between them. The skilled person will appreciate that different algorithms may yield slightly different results.

[053] Thus, the “percent identity” between a query nucleic acid sequence and a subject nucleic acid sequence is the “identities” value, expressed as a percentage, that is calculated by, for example, the BLASTN algorithm when a subject nucleic acid sequence has 100 % query coverage with a query nucleic acid sequence after a pair- wise BLASTN alignment is performed. Such pairwise BLASTN alignments between a query nucleic acid sequence and a subject nucleic acid sequence are performed by using the default settings of the BLASTN algorithm available on the National Center for Biotechnology Institute's website with the filter for low complexity regions turned off. Importantly, a query nucleic acid sequence may be described by a nucleic acid sequence identified in one or more claims herein.

[054] Similarly, the “percent identity” between a query amino acid sequence and a subject amino acid sequence is the “identities” value, expressed as a percentage, that is calculated by the BLASTP algorithm when a subject amino acid sequence has 100 % query coverage with a query amino acid sequence after a pair-wise BLASTP alignment is performed. Such pairwise BLASTP alignments between a query amino acid sequence and a subject amino acid sequence are performed by using the default settings of the BLASTP algorithm available on the National Center for Biotechnology

Institute's website with the filter for low complexity regions turned off. Importantly, a query amino acid sequence may be described by an amino acid sequence identified in one or more claims herein.

[055] The query sequence may be 100 % identical to the subject sequence, or it may include up to a certain integer number of amino acid or nucleotide alterations as compared to the subject sequence such that the % identity is less than 100%. For example, the query sequence is at least 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identical to the subject sequence. Such alterations include at least one amino acid deletion, substitution (including conservative and non-conservative substitution), or insertion, and wherein said alterations may occur at the amino- or carboxy-terminal positions of the query sequence or anywhere between those terminal positions, interspersed either individually among the amino acids or nucleotides in the query sequence or in one or more contiguous groups within the query sequence.

[056] As used herein “candidate agent” or “agent” refer to a molecule that may be screened for, or be identified as, binding and/or modulating activity of a target activity (e.g. binding or modulating of the activity of a JAK protein, e.g. the JAK1 isoform as defined in the first aspect of the invention). Such agent may, for example, be an inhibitor or enhancer of the activity and may find use in a variety of applications, including as therapeutic agents, as agricultural chemicals, and so on. The screening methods will typically be assays which provide for qualitative/quantitative measurements of the activity in the presence of a particular candidate agent. For example, the assay could be an assay which measures the JAK (e.g. JAK1) kinase activity in the presence and absence of a candidate agent. The screening method may be an in vitro or in vivo format, and both formats are readily developed by those of skill in the art.

[057] (Candidate) agents may be obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts or purified compounds are available or may be produced. Additionally, natural or synthetically produced libraries and compounds can be prepared using conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries.

[058] Using the above screening methods, a variety of different therapeutic agents may be identified. Such agents may target an enzyme itself, or an expression regulator factor thereof. Such agents may be inhibitors or promoters of the targeted activity, where inhibitors are those agents that result in at least a reduction of activity as compared to a control and enhancers result in at least an increase in activity as compared to a control. Such agents may be used in a variety of (therapeutic) applications.

[059] As used herein, the term “determining”, for example determining activity, production, and/or amounts includes measuring, analyzing, estimating, following, and the like of such activity, production, and/or amounts, for example, using conventional means and/or techniques. Likewise, the term “providing”, for example, providing a cell includes preparing, isolating, obtaining, and the like, of such cell.

Detailed description

[060] The invention is defined herein, and in particular in the accompanying claims.

[061] It is contemplated that any method, use, or composition described herein can be implemented with respect to any other method, use or composition described herein. Embodiments discussed in the context of methods, use and/or compositions of the invention may be employed with respect to any other method, use or composition described herein. Thus, an embodiment pertaining to one method, use or composition may be applied to other methods, uses and compositions of the invention as well.

[062] Any references in the description to methods of treatment refer to the compounds, pharmaceutical compositions, and medicaments of the present invention for use in a method for treatment of the human {or animal) body by therapy.

[063] As embodied and broadly described herein, the present invention is directed to the surprising finding that a short-version {short isoform) of the canonical JAK1 protein is expressed in cells, tumor cells, obtained from (a subgroup of) cancer patients, and these short isoforms/tumor cells were surprisingly sensitive to JAK inhibitors. The said expressed isoform(s) are oncogenic isoforms (pro-tumorigenic), and appear to have lost the role to function as tumor-suppressors, a role attributed to the canonical JAK1 protein. This non-canonical JAK1 isoform according to the invention is also termed in this description as short-version of JAK1 short isoform, or JH1-form. The JAK1 short isoform comprises a (functional) JH1 kinase domain of the JAK1 protein and does not comprise a JH2 (pseudo)kinase domain of the JAK1 protein, and/or does not comprise a JH2 (pseudo)kinase domain, or part thereof, that is capable of (auto)inhibitory interaction with the JH1 kinase domain of the JAK1 protein. The non-canonical isoform according to the invention results from the herewith termed and previously disclosed

RNA dicing phenomena. The invention provides for the repurposing of known JAK modulating {e.g., inhibitors) compounds, as well as for the possibility to use JAK1 isoforms expressed as the result of this alternative RNA processing phenomena in cells to test for and identify new candidate compounds useful in therapy, such a cancer therapy. Preferably the screening is for inhibitors of the JAK1 isoforms according to the invention, even more preferably the screening is for inhibitors of the JAK1 isoforms that are specific for the JAK1 isoforms according to the invention (i.e. that preferentially inhibit the JAK1 isoforms according to the invention over canonical JAK1 (or JAK) protein).

[064] Moreover, considering that one of the roles acknowledged for the canonical isoform of JAK1 protein is as a tumor-suppressor protein, the proposal of inhibitors of the same which have been demonstrated to be effective in the treatment of cancers is unexpected and counter-intuitive.

JAK inhibitors for use in the prevention and/or treatment of a tumor

[065] As previously indicated, a first aspect of the invention relates to JAK inhibitors for use in the treatment of cancer in a subject, wherein the subject comprises a tumor,

wherein the tumor is characterized by expression (in cells) of an isoform of a Janus

Kinase 1 (JAK1) protein, wherein the isoform: - at least comprises a JH1 kinase domain of the JAK1 protein; and - does not comprise a JH2 (pseudo)kinase domain of the JAK1 protein and/or does not comprise a JH2 (pseudo)kinase domain, or part thereof, that is capable of (auto)inhibitory interaction with the JH1 kinase domain of the JAK1.

[066] When in this description is said that a tumor in a patient is characterized by the expression of a non-canonical and/or oncogenic isoform of JAK1, it means that one or more cells of the tumoral mass, as well as any cell derived therefrom, express the said oncogenic or pro-tumorigenic isoform of JAK1 according to the invention, i.e. comprising the JH1 kinase domains and not comprising any functional JH2 (pseudo)kinase domain capable of inhibiting the activity of the JH1 domain. This isoform of JAK1 according to the invention is non-canonical since it does not comprise all the commonly found (functional) domains in a protein of the JAK family, while in addition it is oncogenic, which means that it favors, triggers or supports the appearance and/or growth of tumorous tissues (i.e., tumors).

[067] The isoform according to the invention displays kinase activity due to the presence of the JH1 domain, and this kinase activity is considered as an uncontrolled kinase activity since the isoform according to the invention does nat contain any functional domain (e.g. JH2 domain) being capable of inhibiting or regulating the kinase activity of the JH1 kinase domain in the isoform according to the invention. In other words, the isoform according to the invention does not have the autoinhibitory capability present in the canonical JAK1 protein (due to the presence of a functional

JH2-domain), and which can modulate the level and/or final function of the protein.

[068] Cells in an organism may express several of the possible isoforms derivable from a codifying region of a gene. Thus, in the sense of the invention, the expression of the one or more isoforms in a particular cell may be defined by the particular ratio between the several isoforms. As said, in cells of a tumor that express the non- canonical and oncogenic isoform according to the invention, the ratio of the expression of this isoform (also referred to as JH1-isoform) in relation to the canonical isoform (JAK1) is higher than in a cell from a non-tumoral tissue.

[069] From this one can derive the provision of the ratio of the expression of the short isoform according to the invention, abbreviated herewith as JH1-isoform, and the expression of the canonical JAK1, the ratio being expressed for example as JH1- isoform/JAK1 (canonical), as a biomarker of a tumor in a subject, preferably as a biomarker indicative of the aggressivity of the tumor. The aggressivity is to be understood as the fast grow, spread or formation of a tumor, and/or as a stage of cancer of or above grade 2. In other words, also provided is an in vitro method for the diagnosis of a subject suffering from cancer, the method comprising determining in a sample comprising tumor material obtained from the subject: (i) the presence and amount of a (short) isoform of JAK1 protein (JH1-isoform) as defined according to the invention; (ij the presence and amount of a canonical isoform of JAK1 protein JAK1 (canonical); and (ii) obtaining a ratio, for example the ratio JH1-isoform/JAK1 (canonical); wherein optionally the subject is diagnosed of suffering from cancer, and preferably of a stage grade, by comparing the ratio with that of a reference.

[070] The proposed JAK inhibitors for use in the treatment of cancer may act, thus, on a JAK1 isoform that comprises a JH1 kinase domain of the JAK1 protein, and that does not comprise a JH2 (pseudo)kinase domain of the JAK1 protein; and/or on a

JAK1 isoform that comprises a JH1 kinase domain of the JAK1 protein and that does not comprises a JH2 (pseudo)kinase domain, or part thereof, that is capable of (auto)inhibitory interaction with the JH1 kinase domain of the JAK1 protein.

[071] In a preferred embodiment of the said JAK inhibitor for use according to the first aspect, the isoform of a JAK1 does not comprise a JH2 (pseudo)kinase domain of the

JAK1 protein.

[072] In another preferred embodiment of the first aspect, the tumor is characterized by the presence of a JAK1 gene comprising one or more mutations, wherein the mutation is selected from a frameshift mutation, a splice-variant mutation, a point mutation, a nonsense mutation, or a combination thereof, and preferably wherein this one or more mutation leads to the expression of the isoform according to the invention.

[073] In a more preferred embodiment, in the JAK inhibitor for use according to the first aspect, optionally in combination with any embodiments disclosed above or below, the tumor is characterized by the presence of a JAK1 gene comprising one or more mutations, wherein at least one mutation is a frameshift mutation.

[074] Frameshift mutations (also called a framing error or a reading frame shift) are mutations caused by indels (insertions or deletions) of a number of nucleotides in a

DNA sequence that is not divisible by three. Due to the triplet nature of gene expression by codons, the insertion or deletion can change the reading frame (the grouping of the codons), resulting in a completely different translation from the original.

[075] Several frameshift mutations have been detected or predicted in the sequence of the gene codifying for the JAK1 protein. In yet another more preferred embodiment according to this first aspect, the tumor is characterized by the presence of a JAK1 gene comprising one or more mutations, wherein at least one mutation is selected from the group consisting of a K860Nfs frameshift mutation, a P430R frameshift mutation, a L431V frameshift mutation, a K142R frameshift mutation and a 1143D frameshift mutation.

[076] In another embodiment, the tumor is characterized by the presence of a JAK1 gene comprising one or more mutations, wherein at least one mutation is a nonsense mutation, said nonsense mutation preferably occurring in a position upstream to a nucleic acid sequence codifying for the JH1 kinase domain, and wherein said nonsense mutation provides for the expression of a JAK1 isoform as defined according tothe invention. In this embodiment, the tumor is characterized by the presence of any nonsense mutation, that is, a mutation that introduces a (premature) stop codon, occurring upstream of the region coding for the kinase JH1 domain. Preferably the nonsense mutation occurs upstream of the position codifying for the aminio acid represented by amino acid 875 in the amino acid sequence with the accession number

P23458 (version 2 of 2008-11-25 v2) in the UniProt KB database, and wherein the said nonsense mutation(s) provide(s) for the expression of a JAK1 isoform according to the invention that comprises a JH1 kinase domain and lacks any functional JH2 (pseudo)kinase domain having inhibitory (i.e., autoinhibitory) function over the JH1 kinase domain. In this context, upstream is to be understood according to the common/ordinary meaning, which by convention, defines upstream and downstream as the 5' to 3' direction respectively in which RNA transcription takes place. The terms upstream and downstream are also applied to a polypeptide sequence, where upstream refers to a region N-terminal and downstream to residues C-terminal of a reference point.

[077] The JAK inhibitor for use according to any of the previous claims, is in a preferred embodiment a JAK inhibitor of an isoform (according to the invention) of a

JAK1 protein that displays uncontrolled kinase activity. As said, this uncontrolled kinase activity is provided in a particular embodiment, because the JAK1 isoform according to the invention comprises a JH1 domain and lacks any functional JH2 domain having inhibitory (i.e., autoinhibitory) function over the JH1 kinase domain.

[078] In an embodiment, the subject is a mammalian, particularly a human, and the isoform of JAK1 is consequently a mammalian, and particularly a human, isoform according to the invention of JAK1.

[079] The JAK inhibitor for use according to any of the previous claims, is in another embodiment, a JAK inhibitor of an isoform of a JAK1 protein that comprises or consists of an amino acid sequence that is at least 85 % identical, preferably 90 % - 100 % identical to the amino acid sequence SEQ ID NO: 1.

[080] This sequence SEQ ID NO: 1 comprises amino acid 876 to amino acid 1154 of the human sequence displayed in the UniProt KB database with the access number

P23458 (version 2 of 2008-11-25 v2). The nucleic acid sequence codifying for the same is depicted in SEQ ID NO: 18, derived from JAK1 JH1 domain nucleic acid sequence (chr1:65298906-65432187 (hg19)). The sequences are listed below:

SEQ ID NO: 16:

AAGAGGATCCGTGACTTGGGAGAGGGCCACTTTGGGAAGGTTGAGCTCTG

CAGGTATGACCCCGAAGGGGACAATACAGGGGAGCAGGTGGCTGTTAAAT

CTCTGAAGCCTGAGAGTGGAGGTAACCACATAGCTGATCTGAAAAAGGAA

ATCGAGATCTTAAGGAACCTCTATCATGAGAACATTGTGAAGTACAAAGG

AATCTGCACAGAAGACGGAGGAAATGGTATTAAGCTCATCATGGAATTTC

TGCCTTCGGGAAGCCTTAAGGAATATCTTCCAAAGAATAAGAACAAAATA

AACCTCAAACAGCAGCTAAAATATGCCGTTCAGATTTGTAAGGGGATGGA

CTATTTGGGTTCTCGGCAATACGTTCACCGGGACTTGGCAGCAAGAAATG

TCCTTGTTGAGAGTGAACACCAAGTGAAAATTGGAGACTTCGGTTTAACC

AAAGCAATTGAAACCGATAAGGAGTATTACACCGTCAAGGATGACCGGGA

CAGCCCTGTGTTTTGGTATGCTCCAGAATGTTTAATGCAATCTAAATTTT

ATATTGCCTCTGACGTCTGGTCTTTTGGAGTCACTCTGCATGAGCTGCTG

ACTTACTGTGATTCAGATTCTAGTCCCATGGCTTTGTTCCTGAAAATGAT

AGGCCCAACCCATGGCCAGATGACAGTCACAAGACTTGTGAATACGTTAA

AAGAAGGAAAACGCCTGCCGTGCCCACCTAACTGTCCAGATGAGGTTTAT

CAACTTATGAGGAAATGCTGGGAATTCCAACCATCCAATCGGACAAGCTT

TCAGAACCTTATTGAAGGATTTGAAGCACTTTTAAAATAA

Translated protein, SEQ ID NO: 1:

KRIRDLGEGHFGKVELCRYDPEGDNTGEQVAVKSLKPESGGNHIADLKKEIEILRNLY

HENIVKYKGICTEDGGNGIKLIMEFLPSGSLKEYLPKNKNKINLKQQLKYAVQICKGMD

YLGSRQYVHRDLAARNVLVESEHQVKIGDFGLTKAIETDKEYYTVKDDRDSPVFWYA

PECLMQSKFYIASDVWSFGVTLHELLTYCDSDSSPMALFLKMIGPTHGQMTVTRLVN

TLKEGKRLPCPPNCPDEVYQLMRKCWEFQPSNRTSFQNLIEGFEALLK

[081] SEQ ID NO: 1 comprises, thus, a (functional) JH1 domain of JAK1 and, preferably, it does not comprise a JH2 (pseudo)kinase domain of the JAK1 protein and/or does not comprise a JH2 (pseudo)kinase domain, or part thereof, that is capable of (auto)inhibitory interaction with the JH1 kinase domain of the JAK1 protein.

In embodiments, the JAK1 isoform according to the invention does not comprise a functional JH2 (pseudo)kinase domain (i.e. does not comprise a JH2 domain capable of (auto)inhibitory interaction with the JH1 kinase domain of the JAK1 protein).

[082] In a preferred embodiment, the JAK inhibitor is an inhibitor of an isoform of a

JAK1 protein according to the invention and that consists of amino acid sequence SEQ

ID NO: 1, or comprises SEQ ID NO:1.

[083] The invention also encompasses an isolated protein that comprises or consists of an amino acid sequence that is at least 85 % identical, preferably 90 % - 100 % identical to the amino acid sequence SEQ ID NO: 1. The isolated protein is characterized by comprising a (functional) JH1 domain of JAK1 and the absence of a functional JH2 (pseudo)kinase domain of the JAK1 protein capable of (auto)inhibitory interaction with the JH1 kinase domain of the JAK1 protein.

[084] Also encompassed are isolated nucleic acid sequences, either DNA or RNA, that codify for a protein that comprises or consists of an amino acid sequence that is at least 85 % identical, preferably 90 % - 100 % identical to the amino acid sequence

SEQ ID NO: 1, and as described above. These nucleic acid sequences may, in some embodiments be comprised in vectors, preferably expression vectors commonly used in the field, and obtained using conventional techniques. The said vectors and/or isolated protein and/or isolated nucleic acid sequences that codify for the protein may, in another embodiment be comprised in a host cell or in a viral particle. Host cells and/or viral particles will express the encoded protein (related to the short isoform of

JAK1 according to the invention) and may be used for screening and/or testing purposes, for example, such as disclosed herein.

[085] When in this description JAK inhibitors are mentioned, they are to be understood in its broadest sense and to encompass those compounds or compositions that inhibit at least JAK1 (any isoform), as well as inhibitors that inhibit one or more of other members of the JAK protein family, thus one or more of JAK 2, JAK 3 and TYK 2, i.e., pan-JAK inhibitors.

[086] In yet another embodiment of the JAK inhibitor for use according to the first aspect, the JAK inhibitor is an inhibitor of at least JAK1, preferably an inhibitor capable of inhibiting kinase activity of the JH1 kinase domain of JAK1. The JAK1 inhibitors used in the invention inhibit the kinase activity of the JAK1 isoforms according to the invention.

[087] Therefore, the inhibitors include compounds that inhibit or modulate the activity of a JH1 kinase domain as comprised in the JAK1 isoform according to the invention are (and/or as comprised in canonical JAK1 protein). This may include new or known compounds not yet classified as JAK1 inhibitors.

[088] Several JAK inhibitors are known in the art, and they have been used or tested in clinical trials for the therapy of several pathologies such as in rheumatoid arthritis, psoriasis, graft-versus-host disease, Bowel and Crohn's diseases, alopecia, vitiligo, or

Lupus as well as cancer (e.g., myelofibrosis, myeloproliferative neoplasm, polycythemia vera, pancreatic cancer, lung cancer, leukemias, and melanomas). JAK inhibitors generally differ in specificity (selectivity) for each JAK member of the family.

The differences in specificities for JAK are the basis for the different trials: JAK2 specificity for myeloproliferative neoplasms and certain malignant disorders, and JAK1 and JAK3 specificity for inflammation and auto-immune diseases (see Vainchenker W, Leroy E, Gilles L et al. JAK inhibitors for the treatment of myeloproliferative neoplasms and other disorders [version 1; referees: 2 approved]

F1000Research 2018, 7(F1000 Faculty Rev):82 (doi: 10.12688/f1000research.13167)).

[089] Most of the JAK inhibitors that are selective or more specific for JAK1 have been tested for other diseases than cancer. Tofacitinib, which is a JAK1/3 inhibitor is

FDA approved for treatment of rheumatoid arthritis and ulcerative colitis.

[090] In an embodiment, the JAK inhibitor is a JAK1 inhibitor, or a inhibitor that is selective or more specific for JAK1 as compared to JAK2, or as compared to JAKS, or as compared to JAK2 and JAK3. In an embodiment, the JAK inhibitor is a JAK1 inhibitor and a JAK2 inhibitor, or an inhibitor that is selective or more specific for JAK1 and JAKZ as compared to JAKS.

[091] In another embodiment of the first aspect, the JAK inhibitor is selected from the group consisting of Momelotinib (CYT387), CEP-33779 (CAS No. 1257704-57-6),

Ruxolitinib, ltacitinib, Tofacitinib, Oclacitinib, Baricitinib, Peficitinib, Upadacitinib,

Fedratinib, Delgocitinib, Filgotinib, Abrocitinib, Pacritinib, Deucravacitinib, Ritlecitinib,

AZD1480 (CAS No. 935666-88-9), Gandotinib, Upadacitinib, Gusacitinib, Cerdulatinib, and combinations thereof. A preferred JAK inhibitor is momelotinib.

[092] Momelotinib (CYT387) is a JAK1/2 inhibitor with high selectivity for JAK1.

Several preclinical studies in solid tumor models have investigated the impact of momelotinib on the JAK/STAT pathway. Momelotinib has been shown to increase sensitivity of ovarian cancer to chemotherapy in in vitro and in vivo preclinical models.

In combination with paclitaxel, momelotinib inhibited tumor growth, suppressed STAT3 activation, reduced expression of the stem cell marker OCT4, significantly increased the time to recurrence, and decreased tumor burden. Similarly, in GBM preclinical models, momelotinib in combination with temozolomide inhibited STAT3 activation, decreased cell growth, increased apoptosis, and inhibited tumor growth compared to temozolomide monotherapy. In colorectal cancer cells, momelotinib inhibited STATS activation, decreased cell growth, and increased cell death. These promising preclinical results across several types of solid tumors support further investigation of momelotinib as a therapeutic agent (see Qureshy et al. 2020. Targeting the JAK/STAT pathway in solid tumors. J Cancer Metastasis Treat. 2020 ; 6). However, some of the clinical trials have been stopped (see Vainchenker, supra).

[093] Data in the examples herein and in experiments performed by the inventors demonstrate that momelotinib (CYT387) enhanced the antiproliferative activity in cells with a frameshift mutation in the gene codifying for JAK1, and which cells express high levels of the non-canonical isoform of JAK1 according to the invention and that comprises the JH1 domain of JAK1 but does not comprise the JH2 (pseudojkinase domain.

[094] With the example of JAK1 protein, the inventors have demonstrated that several isoforms of a protein, resulting from the RNA dicing phenomenon, may behave totally opposed, at such extent that some isoforms are tumor-suppressor isoforms, meanwhile others are pro-tumorigenic. The existence of known modulators, preferably inhibitors of the tumor-now suppressing isoforms find a new application, thus, in the treatment of cells, tumors or patients, expressing those pro-tumorigenic isoforms.

[095] In a particular embodiment of the first aspect of the invention, in which JAK1 inhibitors are for use in the treatment of a tumor, the tumor is an epithelial tissue tumor.

[096] This epithelial tissue tumor (i.e., carcinoma) is preferably selected from the group consisting of a breast tumor or a tumor in the breast, an endometrial tumor or a tumor in the endometrium, an ovarian tumor or a tumor in the ovarian, a prostate tumor or a tumor in the prostate, a pancreatic tumor or a tumor in the pancreas, myelofibrosis, a lung tumor or a tumor in the lung.

[097] When in this description a “tumor in a tissue” is referred to, for example a tumor in the breast, it is encompassed either a tumor that originates in the breast epithelial tissue, thus, itis a primary tumor, as well as a tumor that originates in other tissue and that becomes a secondary tumor in the breast.

[098] In another embodiment of the first aspect, the JAK1 inhibitor is for use in the prevention and/or treatment of a non-epithelial tissue tumor. Preferred non-epithelial tissue tumors are selected from the group consisting of a lymphoma, a melanoma, and a leukemia.

Screening of candidate subjects as tumor treatment-respondent

[099] The invention also encompasses an in vitro method for the selection of a subject suffering from cancer as candidate for a cancer therapy, wherein the method comprises determining in a sample comprising tumor material obtained from the subject: - the presence of an isoform of a JAK1 protein as defined in the first aspect according to the invention, and in any one of its embodiments (i.e., a JAK1 isoform that comprises a JH1 kinase domain of the JAK1 protein, and that does not comprise a JH2 (pseudo)kinase domain of the JAK1 protein; and/or on a JAK1 isoform that comprises a JH1 kinase domain of the JAK1 protein and that does not comprises a JH2 (pseudo)kinase domain, or part thereof, that is capable of (auto}inhibitory interaction with the JH1 kinase domain of the JAK1 protein}; - the presence of a JAK1 gene comprising one or more mutations as defined in the first aspect and in any one of its embodiments; and/or - the presence of a RNA molecule that, when translated, provides for an isoform of a JAK1 protein as defined in the first aspect and in any one of its embodiments, - and wherein if the JAK1 isoform according to the invention and/or the JAK? gene comprising one or more mutations and/or the RNA molecule is present in the sample, the subject is selected as candidate for the cancer therapy

[100] In an embodiment of the in vitro method for the selection of a subject suffering from cancer as candidate for a cancer therapy, the therapy comprises the administering of at least one JAK inhibitor to the subject. Examples of the one or more inhibitors have been listed above. . Preferably the cancer therapy comprises treatment of the patient with a JAK inhibitor, for example a JAK1 inhibitor, as disclosed herein.

Preferably the JAK inhibitor as disclosed herein is a pharmaceutically acceptable JAK inhibitor.

[101] As the skilled person in the art will recognize, additional chemotherapeutic compounds and/or cycles of ionizing radiation may, in another embodiment, be used in combination with the one or more of the JAK inhibitors in the treatment of cancer.

[102] Several isolated samples may be used an in vitro method for the selection of a subject suffering from cancer as candidate for a cancer therapy, which are those commonly used for each of the cancer types.

[103] In an embodiment of the in vitro method according to this second aspect, the sample is selected from one or more of a tumor biopsy, a fluid sample with tumor cells (e.g. uterine fluid), blood, saliva, tears, etc.

[104] In another embodiment of the in vitro method according to the second aspect, the method further comprises determining if the isoform of the Janus Kinase 1 (JAK1) may result from an endonuclease cleavage of a mRNA molecule that produces a 5’

Uncapped Polyadenylated Transcript (5’UPT) encoding the isoform of the JAK1 protein.

[105] For the determining whether the JAK1 isoform results from a 5’ UPT transcript, bioinformatic tools the skilled person in the art will know, as well as biochemical conventional assays can be employed.

[106] In yet another embodiment, the in vitro method according to the second aspect of the invention further comprises contacting a sample comprising tumor cell material obtained from a subject that is selected as candidate for the cancer therapy, with one or more anti-tumor agents, preferably one or more JAK inhibitors, and determining a change in proliferation of the tumor cells compared to a control in absence of said anti- tumor agents, preferably JAK inhibitors, wherein if proliferation is reduced in the presence of said anti-tumor agents, preferably JAK inhibitors, the subject is selected as candidate for a cancer therapy wherein the therapy comprises administering of at least one anti-tumor agent, preferably JAK inhibitor, to the subject, preferably wherein the at least one anti-tumor agent, preferably JAK inhibitor demonstrated a change in proliferation of the tumor cells, in particular a reduction in proliferation.

[107] Once a subject is identified, according to the method according to the invention, as candidate for the cancer therapy, preferably for a cancer therapy that comprises administering of at least one anti-tumor agent, preferably a JAK inhibitor, the said anti- tumor agent, preferably JAK inhibitor, may be conveniently administered to a patient and/or formulated in a pharmaceutical compositions before administration.

[108] The pharmaceutical compositions comprise, together with one or more pharmaceutically acceptable carriers and/or excipients, a therapeutically effective amount of an anti-tumor agent, preferably JAK inhibitor.

[109] Surprisingly, the therapeutically effective amount, optionally administered as active principle in a pharmaceutical composition, of a JAK inhibitor for use in the treatment of cancer in a subject whose tumor expresses a JAK1 protein as defined in the first aspect, is in some embodiments lower than the therapeutically effective amount of the said JAK inhibitor commonly used for other than cancer indications.

Screening of candidate compounds

[110] The invention also relates to an in vitro method for screening of candidate agents that: - bind to an isoform of a JAK1 isoform as defined in the first aspect and in any one of its embodiments; and/or - modulate the activity of an isoform of a Janus Kinase 1 (JAK1) protein as defined in the first aspect and in any one of its embodiments. The method comprises the steps of:

- contacting the isoform of a Janus Kinase 1 (JAK1) protein (isoform) as defined in the first aspect and in any one of its embodiments with a candidate agent; and - detecting binding of a candidate agent with and/or detecting modulation of the activity of the isoform of the Janus Kinase 1 (JAK1) protein as defined in the first aspect and in any one of its embodiments; - optionally, detecting binding of the candidate agent with and/or detecting modulation of the activity of a Janus Kinase 1 (JAK1)} protein, such as for example of the canonical

JAK1 protein or of an isoform other than the one as defined in the first aspect.

[111] In an embodiment of the in vitro method for the screening according to this third aspect, it comprises the step of detecting binding of the candidate agent with and/or detecting modulation of the activity of a Janus Kinase 1 (JAK1) protein. This embodiment allows to determine if the candidate is also able to bind/modulate, for example, a canonical

JAK1 protein as well.

[112] In a preferred embodiment of the in vitro method for the screening according to this third aspect, the step of detecting modulation of the activity of the isoform of the Janus

Kinase 1 (JAK1) protein comprises detecting modulation of the tumorigenic activity, preferably by means of cell assays, and/or the detection of markers, of the isoform of the

Janus Kinase 1 (JAK1) protein compared to the tumorigenic activity in absence of the candidate agent, and/or wherein detecting modulation of the activity of the isoform of the

Janus Kinase 1 (JAK1) protein comprises detecting kinase activity of the isoform of the

Janus Kinase 1 (JAK1) protein compared to the kinase activity of the isoform of the Janus

Kinase 1 (JAK1) protein activity in absence of the candidate agent.

[113] In another embodiment, the method for the screening comprises the steps of: - detecting binding of the candidate agent with and/or detecting modulation of the activity of a Janus Kinase 1 (JAK1) protein, preferably canonical JAK1 protein; and — comparing the detected binding and /or modulation of the activity of a Janus

Kinase 1 (JAK1) protein of the first step with the binding and /or modulation of the activity of the Janus Kinase 1 (JAK1) isoform as defined in the first aspect and in any one of its embodiments.

[114] This embodiment allows to characterize the selectivity and/or specificity of the candidate for the Janus Kinase 1 (JAK1) protein as defined in the first aspect in relation to another Janus Kinase 1 (JAK1 } protein, preferably canonical JAK1 protein. Compounds that are selective to or specific for the JAK1 isoform according to the invention, as compared to other JAK1 protein isoforms, such as canonical JAK1 protein, are useful in treatment of conditions, such as cancer, in which it is preferred to be able to selectively and/or specifically inhibit the JAK1 isoform according to the invention as compared to any other JAK1 isoforms, in particular as compared to canonical JAK1 protein.

[115] The terms “selectivity” and “specificity” when referred to a compound or candidate in this description are used as synonymous, and they relate to the preferential binding of the candidate and/or modulating of the activity of a JAK1 protein (canonical or non- canonical isoform as the one disclosed in the first aspect) by that candidate. A skilled person understands and can recognize such preferential binding and/or modulation, in particular inhibition of the JAK1 isoform according to the invention.

[116] The invention also provides for a method for the identification of selective modulators of a JH1 domain of a JAK1 protein, preferably inhibitors of a JH1 domain of a JAK1 protein, wherein a compound or composition already known as modulator (e.g. as inhibitor) of a JAK protein, preferably of a JAK1 protein, is tested as candidate agent that: - binds to an isoform of a JAK1 protein as defined in the first aspect; and/or - that modulates the activity of an isoform of a JAK1 protein as defined in the first aspect; wherein the method comprises the steps of: - contacting the isoform of a JAK1 protein as defined in the first aspect and in any one of its embodiments, with a JAK modulator (e.g. JAK inhibitor), preferably with a

JAK1 modulator (e.g. JAK1 inhibitor); and - detecting binding of said candidate agent with and/or detecting modulation of the activity of the isoform of the JAK1 protein as defined in the first aspect; - optionally, detecting binding of the candidate agent with and/or detecting modulation of the activity of a Janus Kinase 1 (JAK1) protein, such as for example of the canonical

JAK1 protein or of an isoform other than the one as defined in the first aspect.

[117] In another embodiment of the in vitro method for screening of candidate agents according to the invention, these candidates are of any nature and they are, in a more particular embodiment, selected from one or more of RNA, peptides, small-molecules.

[118] Inventors have realized that the non-canonical and oncogenic JAK1 protein isoform as previously disclosed is mainly located or able to enter the nucleus of the cells to perform its function. Thus, in an embodiment, the candidates are molecules, of any nature, that can enter the nucleus and there to block, to modulate or to interfere in the function of the JAK1 oncogenic isoform as disclosed. In another embodiment,

the candidates are molecules, of any nature, that can block the entry into the nucleus of the non-canonical and oncogenic JAK1 protein isoform as previously disclosed.

[119] The modulating of the activity of an isoform of a JAK1 includes either the capability of inhibiting or stimulating the isoform, preferably the capability to inhibit the activity of the isoform by any method (i.e., competitive, non-competitive, allosteric, etc.). In yet another embodiment, the modulating may be achieved either in vivo or in vitro.

[120] In an embodiment of the in vitro method for screening of candidate agents, one or more of the steps of contacting the isoform of a JAK1 protein as defined in the first aspect and detecting binding of a candidate agent with and/or detecting modulation of the activity of the said isoform, are carried out by means of conventional in vitro assays (or pot-assays) using conventional techniques. In an alternative or complementary embodiment, one or more of these steps (i.e., contacting the isoform and/or detecting the binding and/or detecting the modulation of the activity of the isoform) are carried out in silico with modeling and simulation software predictive tools, also commonly used and widely known in the field of bioinformatics.

[121] Once a candidate agent is identified to bind to an isoform of a JAK1 protein as defined in the first aspect and in any one of its embodiments; and/or to modulate the activity of an isoform of a JAK1 protein as defined in the first aspect and in any one of its embodiments, the candidate may be conveniently formulated in pharmaceutical compositions to be administered to a subject suffering from cancer wherein the subject comprises a tumor, wherein the tumor is characterized by expression of an isoform of a Janus Kinase 1 (JAK1) protein, wherein the isoform at least comprises a

JH1 kinase domain of the JAK1 protein; and does not comprise a JH2 (pseudo)kinase domain of the JAK1 protein and/or does not comprise a JH2 (pseudo)kinase domain, or part thereof, that is capable of (auto)inhibitory interaction with the JH1 kinase domain of the JAK1 protein.

[122] The pharmaceutical compositions comprise, together with one or more pharmaceutically acceptable carriers and/or excipients, a therapeutically effective amount of an agent that binds to an isoform of a JAK1 protein as defined in the first aspect; and/or that modulates the activity of an isoform of a JAK1 protein as defined in the first aspect.

[123] The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art (including the contents of the references cited herein), readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present invention.

Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein.

[124] All references cited herein, including journal articles or abstracts, published or corresponding patent applications, patents, or any other references, are entirely incorporated by reference herein, including all data, tables, figures, and text presented in the cited references. Additionally, the entire contents of the references cited within the references cited herein are also entirely incorporated by references.

[125] It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance presented herein, in combination with the knowledge of one of ordinary skill in the art.

[126] It will be understood that all details, embodiments, and preferences discussed with respect to one aspect of embodiment of the invention is likewise applicable to any other aspect or embodiment of the invention and that there is therefore not need to detail all such details, embodiments, and preferences for all aspect separately.

[127] Having now generally described the invention, the same will be more readily understood through reference to the following examples which is provided by way of illustration and is not intended to be limiting of the present invention. Further aspects and embodiments will be apparent to those skilled in the art.

EXAMPLES

[128] Traditionally, genes have been perceived as blueprints for distinct proteins, yet mounting evidence reveals that individual proteins possess multifunctional biological roles, including varied potentials for localization, interaction, and other functions.

These phenomena are partially explained by RNA metabolism processes like splicing or post-translational modifications such as phosphorylation but remain largely undefined for most proteins.

[129] The functionality of a single protein is mediated by a collection of domain subunits, each regulating specific biological functions within what is traditionally viewed as a homogeneous entity. This multifunctionality, however, contrasts with the structured organization of protein domains and their defined roles, raising questions about our conventional understanding of protein functionality and suggesting a more complex interplay between genes and their protein isoforms.

[130] Recent advancements in mRNA translation studies have revealed extensive translation events beyond canonical open reading frames (ORFs). Proteomic analyses, enhanced by high-resolution mass spectrometry, show that the diversity of proteins expressed in cells significantly exceeds predictions based on gene count.

This suggests a greater complexity in protein synthesis and function, potentially resolving some paradoxes in genome biology.

[131] Previously, we reported a widespread phenomenon where mRNAs from thousands of genes undergo endonuclease cleavage, producing truncated 5

Uncapped Polyadenylated Transcripts (5'UPT) with translation potential which significantly diversifies the proteome landscape (Malka et al., 2022, supra). Here, we show that cleavage within the ORF results in mRNA sub-fractions capable of translating into reduced subsets of protein domains with a distinctive biological function. Focusing on JAK1 kinase, we demonstrate that metabolic cleavage plays a critical function generating SUPT correspond for the translation of the C-terminal kinase domain, leading to an enhanced activation mode and alternative localization.

This process which we term 'RNA DICING', provides a new conceptual model by which metabolism of MRNA molecules provides a critical biological function and redefine misconception of MRNA molecule as a monolithic biological subunit, to a modular system.

[132] The materials and methods for the Examples below are all listed at the end of this section

Example 1. Dynamic Expression of Protein Domains Through RNA Dicing.

Example with JAK1.

[133] Our previous studies demonstrated that endonuclease cleavage by alternative polyadenylation (APA) machinery can generate uncapped, autonomous RNA fragments with translational potential. This process, implicated in multiple regulatory layers, includes APA-mediated cleavage, RNA structure for stability, and m6A modifications facilitating cap-independent translation (Fig.1a). Notably, increased APA usage, prevalent in various biological contexts, amplifies RNA dicing, thereby favoring the production of 5’ untranslated regions (5'UPTs) over canonical mRNA variants.

MRNA truncation within the open reading frame (ORF) limits translation to corresponding truncated protein domains. Deletion assays studies across numerous genes revealed altered isoform functions based on domain combinations. We hypothesize that RNA dicing selectively excludes certain protein domains, creating new isoforms with potentially novel biological properties. We analyzed multi-omics datasets, including APA sites, uncapped 5’end RNAseq, m6A RIPseq, and ribosome profiling following harringtonine treatment, to identify translation initiation sites. Next, we expand our search for subdomain expression, focusing on the kinase protein family, and explored RNA dicing as a mechanism for localized translation of specific domains. Multiple studies have shown that kinase catalytic domains can exhibit functionality when independently expressed using domains physical deletion assay.

Our analysis of mSA-RIPseq data and N-terminal Mass Spectrometry (MS) which we predict to potentially mark internal non-canonical translation sites revealed m6A peaks preceding kinase domains, and not in non-kinase regions of the same genes, suggesting independent kinase expression post-RNA dicing (Fig. 1 b). To conduct a detailed analysis, we focused on the JAK1 kinase, a member of the JAK kinase family.

These intracellular, non-receptor tyrosine kinases are pivotal in signaling transduction for various cytokines and growth factors. The JAK family consists of four members, each characterized by four major domains. The JH1 domain is the active kinase domain responsible for enzymatic activity. In contrast, the JH2 domain functions as a pseudokinase, regulating JH1's activity. The other two domains, FERM and SH2, are crucial for binding to cytokine receptors and regulating kinase activity (Fig. 1c). An in- depth multi-omics analysis of JAK1, a member of the JAK kinase family, revealed a conserved polyadenylation signal in exon 8, aligning with the C-terminal of the FERM domain (Data not shown). Active APA usage at this site was confirmed through 3’ polyadenylation sequencing. TEX treatment and 5' uncapped RNAseq confirmed the presence of uncapped variants post-APA cleavage which is expected following internal

APA usage. As previously described, uncapped RNA can avoid exonuclease degradation due to a highly structured at its 5’end. To study the RNA structure at the proximity to the dicing site, we performed an icSHAPE analysis which indicated a highly structured 5'end. In addition, cytoplasmic icSHAPE analysis showed higher structural integrity than nuclear fractions, suggesting post-transcriptional cleavage (Data not shown). To evaluate the diced 5’UPT and its potential ORF, we used Long- read Nanopore sequencing which further supported the existence of long 5' UPTs, which decreased following TEX treatment. m8A RIPseq identified modification sites within the JAK1 CDS, and Ntem MS analysis indicated non-canonical translation starting downstream of the APA site (Data not shown). Next, we performed western blot analyses using antibodies targeting either the JH1 kinase domain or an upstream region (aa 551-776) corresponding to the JH2 domain. The JH2-specific antibody revealed the expected full-length JAK1 protein at 120 kDa (Fig. 1d}. In contrast, the

JH1 antibody detected additional bands around 37 kDa. Given that JAK1 phosphorylation occurs at the JH1 domain, we further probed cell extracts with different phospho-JAK1 antibody, which identified the same two bands around 37/39 kDa corresponding to the non-phosphorylated JH1 variants (Fig. 1d). To ensure the specificity of these antibodies in detecting JAK1 isoforms, we treated cells with shRNAs targeting either the JH1 or JH2 domains of JAK1. Consistent with our western blot results, the JH2-targeted shRNA predominantly reduced full-length JAK1 expression, while the JH1-targeted shRNA primarily affected the truncated kinase domain (Fig. 1e). Our findings indicate that RNA dicing of JAK1 leads to the production of truncated mRNA, potentially translating into localized and independent JH1 kinase domains.

Example 2. Critical Role of RNA Dicing in Determining JAK1 Isoform Functions

[134] Kinases, including JAK1, are known for their multifunctionality, including dynamic cellular localization, varied interaction capabilities, and even opposing effects. JAK1 is found in both the cell membrane and nucleus, with its nuclear presence linked to rapid cell cycle progression and tumorigenesis. Interestingly, early studies reported phospho-JAK1 has been detected in the nuclear fraction while full length JAK1 was observed in the cytoplasmic fraction. In addition, the nuclear phosphor-JAK1 has been show on a western blot as shorter isoforms of 42/44kd which we confirmed here (Fig.1f). This suggests distinct functions for the full-length and truncated JH1 kinase domains of JAK1.

[135] To investigate JAK1's metabolic potential and the biological output of independent JH1 domain expression in MCF7 cell line, we designed three constructs: wild-type (WT) JAK1, APA-mutant JAK1 (optimized at the polyadenylation signal), and

Codon-optimized JAK1 (20% nucleotide sequence change across the entire JAK1

CDS, maintaining WT amino acid sequence) (Fig.2a). qRT-PCR before and after TEX treatment showed that APA-mutant and Codon-optimized constructs significantly reduced JAK1 mRNA dicing and as a result 5’UPT expression (Data not shown). On a protein level, western blot analyses indicated increased full-length JAK1 expression and reduced JH1 kinase domain presence in these constructs, further supports dicing dependency for JH1 expression (Fig. 2 b). Interestingly, both APA-mutant and Codon- optimized constructs showed higher levels of non-phosphorylated truncated JAK1 which we postulate derive from the endogenous expression of JAK1 (Fig. 2 b}. qRT-

PCR targeting regions upstream and downstream to dicing site of endogenous JAK1

MRNA revealed that Codon-optimized construct induction increased endogenous

JAK1 mRNA, particularly the truncated JH1 domain (Fig. 2c), indicating a dynamic interplay between full-length and JH1 domain expressions which was induced JAK1 dicing and JH1 expression when canonical full length JAK1 was increased artificially.

To further address the dynamic expression between canonical JAK1 and JH1 diced

JAK1, we induced the cells with IFNy which activate JAK-STAT pathway via JAK phosphorylation. Interestingly, we observed higher phosphorylation levels of canonical

JAK1 in the control non transfected and WT JAK1 construct expression compared to

APA-mutant and Codon-optimized construct (Fig. 2 b). These results indicate that change in dicing potential may interfere with JAK-STAT signal cascade. Thus, we analyzed the mRNA metabolic state of JAK1 well define IFNy signaling pathway in macrophages which plays a crucial role via JAK-STAT signaling cascade in activating and modulating their functions. Strikingly, upon differentiation of HL-60 cells to macrophage following induction with IFNy and LPS, we observed a shift from predominantly intact JAK1 mRNA variants (enabling canonical JAK1 translation) to exclusive dicing variants post-differentiation signal (Data not shown Fig-2e). This metabolic state of JAK1 physically adverse canonical translation over favoring truncated JH1 isoform production. These results strongly suggest that mRNA dicing of

JAK1 and independent JH1 expression plays critical role in JAK-STAT signaling cascade.

[136] Lastly, we assessed the biological impact following inhibition of JAK1 dicing.

Proliferation assays in MCF7 cells showed that APA-mutant and Codon-optimized constructs significantly reduced cell proliferation (Data not shown), corroborated by colony formation assays (Fig. 2d) which correspond to lower expression levels of truncated phospho-JH1 as shown in Fig. 2 b and in line with previous reports which link expression of nuclear JAK1 to proliferation. These findings underscore a critical interplay between canonical full-length JAK1 and truncated JH1 domain expressions, mediated by JAK1 dicing, which in turn influences cellular proliferation rates. In addition, our results may imply that canonical JAK1 phosphorylation may be mediated by heterodimerization with JH1 diced isoform, or at least dependent on its expression.

Example 3. Impact of Genomic Alterations in JAK1 on Cancer Progression

Through RNA Dicing and Modular Expression

[137] Genetic mutations play a critical role in cancer, with the location of these mutations often dictating whether a gene exhibits a loss of function (LOF) or gain-of- function (GOF). JAK1 mutations, found in 1.88% of all cancers, are particularly prevalent in specific cancer types such as endometrial, breast, prostate, colon, and lung adenocarcinomas. Notably, JAK1 LOF mutations are frequently observed in endometrial cancer (14% in TCGA cohort, 10% in CPTAC cohort), with a strong association with higher tumor grades in these cases. In addition, proteomics analysis of tumors with JAK1fs mutations shows an increase in cell cycle markers and proliferation (Fig-3b, data not shown).

[138] Mutation mapping indicates that the majority of JAK1fs mutations occur at position 860, situated between the JH1 and JH2 protein domains. Dogmatic view of nonsense mutation will generally conserve as a LOF mutation. Viewing this genomic alteration from RNA dicing and modular JAK1 expression perspective provides a striking possibility that JAK1fs mutation may indeed silence the expression of the canonical tumor suppressor isoform but will have no affect on the oncogenic diced isoform since the physical position upstream to JH1 ORF remains intact and in frame

(Fig. 3a). Hence, RNA dicing and modular gene expression underscores the potential role of mutation location in determining the functional outcome of JAK1.

[139] To explore this further, we analyzed three CRISPR-Cas9 Base editing gRNAs screens designed to introduce stop codons (BE3-NGG; BE3.9max-NGN and BE4max-

YE1-NGN). Overall, the libraries contained 108 gRNA which introduce stop codons across JAK1 CDS. Proliferation score revealed that stop codons inducing outside the kinase domain resulted in increased cellular proliferation, while those within the domain did not affect proliferation rate (Fig. 3 b, ¢). Additionally, inducing stop codons just downstream of the JH1 domain led to a significant increase in proliferation, possibly due to diminishing inhibitory domain motif known for protein-protein interaction with SOCS1 which regulates JAK1 function. These findings provide robust support for our hypothesis of differential JAK1 expression, driven by RNA dicing, that regulates the distinct metabolic roles of its different isoforms.

Example 4. JAK1fs Mutation Altered JAK1 Function and Increased Malignancy in Endometrial Cancer

[140] Categorically, a nonsense mutation is often considered a loss-of-function (LOF) mutation due to the homogenous gene expression model. However, our findings challenge this perspective, revealing complexities due to the expression of multilayer isoforms.

[141] The standard approach in transcriptome and proteome analysis relies on a homogenous gene expression model. This includes normalizing RNAseg/Mass spectrometry reads/peptides to known gene isoforms. Unfortunately, these methods unable to detect the dynamic expression within genes resulting from RNA dicing. To uncover this hidden dynamic, we analyzed JAK1 expression levels in endometrial carcinoma (EC) patients carrying JAK1860fs/ JAK1 WT, with an emphasis on modular gene expression perspective. We anticipated that RNA dicing would lead to dynamic expression of JAK1 domains. Thus, we independently analyzed RNAseq data from EC patients for each JAK1 domain. Patients with WT JAK1 exhibited similar expression across all JAK1 domains when normalized to JAK1 expression in adjacent normal tissue. Intriguingly, JAK1860fs EC patients displayed significant fluctuation in RNA expression for each domain. These samples also showed a marked decrease in RNA coverage at the JH2 domain following the SH2 domain, and a sharp increase in coverage at the JH1 kinase domain.

This dip in read coverage strongly suggests discontinuous RNA molecules between the SH2 and JH1 domains, indicating independent expression of the JH1 domain in these patients (Fig.4a). We then analyzed Mass spectrometry data from the CPTAC cohort for JAK1 expression in EC patients.

Consistent with the RNAseq results, we observed a similar trend, a decrease in JH2 MS peptide coverage compared to the SH2 domain and an increased coverage in the JH1 domain (Fig.4b). To account for the potential heterogeneity in tumor JAK1 genomic status, we validated our findings in cell lines with homogeneous JAK1860fs mutations which represent full knock down of canonical JAK1 expression.

MS analysis of breast carcinoma CAL51 cell line (JAK1860fs homozygote) revealed over 50% coverage of peptides in the JH1 domain downstream to the homogeneous fs mutation at position 860, confirming in-frame translation past the frameshift site (Fig.4c). As expected, Western blot analysis of the ISHIKAWA EC cell line (JAK1860fs homozygote, Data not shown) did not detect full-length JAK1 in either phosphorylated or non-phosphorylated states (Fig.4d). However, we observed a 37kd phospho-JAK1 band, but not the upper 39kd band compared to MCF7 cell line expressing WT JAK1 (Fig.4d). To further validate the expression of diced JAK1 in this cell line, we introduced JAK1860fs with a C-terminal HA tag.

Western blot analysis revealed a 37kd band in the JAK1860fsHA sample; however, a similar band was also observed in the control, preventing a definitive conclusion about its specificity.

Nonetheless, we detected a specific 27kd band of the truncated isoform, supporting in-frame translation of the truncated product downstream of the fs mutation site (Data not shown). To examine the potential bias in the balance toward diced JAK1isoform expression and its effect on cell cycle progression, we restore WT JAK1 expression in ISHIKAWA cell line using constructs expressing either JAK1 WT or JAK1 Codon-opt.

Contrary to the negligible effect of WT JAK1 expression on MCF7 cell proliferation, ISHIKAWA cells exhibited decreased growth with both WT and Codon-opt constructs (Fig.4e). A colony formation assay corroborated these findings, showing stronger decrease in colony formations in Codon-opt expressing cells (Fig.4f). These results further endorse the independent tumor suppressor function of canonical JAK1 when induced in either modified or unmodified ORFs in canonical JAK1-deficient cells.

Lastly, we performed a single-sample gene set enrichment analysis comparing JAK1 with damaging mutations to WT JAK1 in endometrial carcinoma cell lines (23 wild type vs. 10 mutated

JAK1). Notably, the most significant decrease in gene set clusters was observed in the negative regulation of macrophage activation, a process that reduces the frequency, rate, or extent of macrophage activation (Data not shown). The enforced advantage expression of JH1 compared to canonical JAK1 due to nonsense mutations underscores the biological importance of JAK1 dicing metabolism and further support our hypothesis that JAK1 dicing and JH1 isoform expression are critical for cell cycle and macrophage activation.

Example 5. JAK1 Inhibitor CYT387 Enhanced Efficacy in Cells with JAK1fs

Mutations Compared to WT JAK1

[142] Since JAK1 plays as a key component in the JAK-STAT signaling pathway which upon activation contributes to acquisition of properties required for tumor invasion and metastasis, there has been and currently ongoing a major effort for inhibitors design in cancer drug development. Interestingly, all current JAK1 inhibitors that has been approved by FDA where design to target the kinase JH1 domain. We next focus on the differential impact of JAK1 RNA metabolism and dicing on the efficacy of JAK1 inhibitors, particularly CYT387 (Momelotinib), an FDA-approved drug used for the treatment of myelofibrosis targeting the JH1 kinase domain of JAK1.

Firstly, we examined CYT387's efficacy in relation to JAK1 expression in various cell lines. Omics analysis across a range of concentrations (600pM to 38nM) in 221 and 434 cell lines for transcriptomic and proteomic (correspond) showed no correlation between JAK1 RNA or protein levels and the efficiency of drug administration (Fig.5a), emphasize the obscure connection between current canonical homogenous model for gene expression approach and the efficiency of targeted therapy.

[143] Next, we sub selected all Endometrial Carcinoma cell lines expressing wild type

JAK1 or homozygote frameshift mutation in JAK1 (10 and 5 respectively). Strikingly,

JAK1fs cell lines has shown a significant decrease in cell growth compared to the WT expressed lines in 2.4nM concentration (Fig.5b). Furthermore, treatment efficiency of

CYT387 in EC cell lines shows one of the best outcomes in cells carrying JAK1fs compared to other mutations statuses. Further analysis highlighted CYT387's effectiveness in EC cell lines with JAK1fs mutations, surpassing its impact on other mutation statuses (Fig.5c). Gene set enrichment analysis following CYT387 treatment showed significant downregulation in JAK-STAT pathway-related functions, including macrophage proliferation, cytokine production, and differentiation in JAK1fs mutated lines, contrasting with moderate changes observed in WT JAK1 lines further support the its robust effect on cells with JAK1fs mutation status (Data not shown).

[144] Lastly, we extend this analysis to other cancer types with JAK1fs mutations.

For 22RV1 (Prostate Adenocarcinoma) and CAL51 (Invasive Breast Carcinoma),

CYT387 shown the strongest effect in 600pM/2.4nM concentration compared to WT expressing JAK1 compared to other cell lines from the same tumor types (3 and 19 in total respectively) (Fig.6). These findings suggest that JAK1 inhibitors like CYT387 may be particularly effective in treating cancers with JAK1 nonsense mutations, specifically targeting the oncogenic JH1 JAK1 kinase domain variant resulting from

RNA dicing. This offers a promising avenue for tailored cancer treatment strategies.

[145] Next, we extended our experiment to include an additional JAK inhibitor, CEP- 33779. Similar to our previous results, treatment with CEP-337798 showed stronger inhibition of cell growth in EC cell lines carrying the JAK1fs mutation at a concentration of 2.4nM. In the breast cancer cell line, we observed the same trend as seen with

CYT387, where CAL51 exhibited one of the strongest responses to treatment (Fig.6).

For the 22RV1 cell line, we also detected strong inhibition at 2.4nM, a trend that was similarly observed in the other two prostate adenocarcinoma cell lines with wild-type

JAK1 expression (data not shown).

Methods

[148] Cell culture

[147] HeLa, MCF7, ISHIKAWA and HL-60 cell lines were grown in Dulbecco’s modified Eagle’s medium, supplemented with 10% (HL-60 with 20%) fetal calf serum, 100 units/ml penicillin, and 100 pg/mL streptomycin at 37°C. Cell lines were regularly tested for Mycoplasma contamination. Cell lines were authenticated by expression analysis based on RNA-seq.

[148] Terminator phosphate-dependent TEX treatment

[149] For RNA-seq analysis, DNase I-treated and poly(A)-selected RNA was subjected to treatment with Terminator 5'-Phosphate-Dependent Exonuclease (Epicentre; TER51020) following the manufacturer’s instructions. The reaction was then deactivated, and RNA was subsequently purified using the RNA Clean-Up and

Concentration MICRO-Elute Kit (Norgen; 61000).

[150] Colony Formation

[151] In a 6-well plate, 3250 cells were seeded per well. Media was refreshed after 4 to 5 days, and the experiment was concluded on day 10/11 after seeding. The cells were washed with PBS and fixed using 3.7% formaldehyde (Sigma). Subsequently, the cells were stained with 0.1% Crystal Violet.

[152] Western blotting

[153] Cell lysates, protein quantification and SDS-PAGE were performed as previously described . Fractionations were run on 4-15% gradient gels (TGX, Bio-Rad) using the Laemmli buffer system and blotted on nitrocellulose (pore size 0.2 um; Pall).

Blots were routinely blocked with 5% nonfat dried milk diluted in TBST for 1 h and then, depending on the manufacturer, incubated with primary antibodies for 1 h or overnight.

[154] Subsequent staining was performed with the appropriate LI-COR secondary antibodies. Visualization was performed by use of an Odyssey infrared scanning device (LI-COR).

[155] Immunoprecipitation

[156] CDS of JAK1860fs- HA was cloned in pLV-Puro-CMV (Vector Builder) and expressed in ISHIKAWA cell line. Pull-down assays were performed as previously described: cleared cell lysates (500 ug) overexpressing the HA-tag proteins were incubated for 2 h at 4°C with immobilized GST-fusion proteins (62.5 pmol). Beads were extensively washed and bound proteins eluted with Laemmli buffer. Correct loading in all pull-down experiments was confirmed by Ponceau staining. Co- immunoprecipitation experiments were performed as previously described. Total cell lysates were obtained as previously described

[157]

[158] and 30 ug were employed in all immunoblotting experiments.

[159] Nuclear/Cytoplasmic RNA Extraction

[160] For nuclear and cytoplasmic protein extraction, two million MCF7 cells were utilized. The NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Fisher

Scientific, Catalogue Number 78833) was employed for this procedure.

[161] Lentiviral Production and Transduction

[162] To produce lentivirus, 4 x 1046 HEK293T cells were seeded per 100-mm dish one day prior to transfection. For each transfection, 10 ug of the pCDH reporter, 5 ug of pMDL RRE, 3.5 pg pVSV-G, and 2.5 ug of pRSV-REV plasmids were mixed in 500

ML of serum-free DMEM. Next, 500 UL of serum-free DMEM containing 63 pL of a 1 mg/mL PEI solution was added. The entire mix was vortexed and left for 15 minutes at room temperature, after which it was added to the HEK293T cells for transfection.

The following day, the medium was replaced with RPMI. Lentivirus-containing supernatants were collected 48 and 72 hours after transfection and snap-frozen in liquid nitrogen. Target cells were transduced on two consecutive days by supplementing the lentiviral supernatant with 8 pg/mL polybrene (Sigma). One day after the final transduction, transduced cells were selected by adding 2 pg/mL puromycin to the medium.

[163] Real-time PCR

[164] Forreverse transcription, 1 ug of total RNA was utilized and reverse transcribed using the Tetro cDNA synthesis kit in accordance with the manufacturer’s instructions.

Subsequently, real-time PCR was conducted using the SensiFAST SYBR real-time

PCR kit (Bioline). The obtained data were normalized to the human endogenous control (GAPDH) and analyzed using the AACt model, unless stated otherwise.

Primers

[165] GAPDH:

F: ACAACTTTGGTATCGTGGAAGG. (SEQ ID NO: 2)

R: GCCATCACGCCACAGTTTC (SEQ ID NO: 3)

[166] JAK1:

UP:

F: TGACGAGAACACCAAGCTCT (SEQ ID NO: 4)

R: GAGAATGACGCCACACTGAC (SEQ ID NO: 5)

DOWN:

F: TGCACAGAAGACGGAGGAAA (SEQ ID NO: 8)

R: GAACGTATTGCCGAGAACCC (SEQ ID NO: 7)

[167] JAK1 (Constructs specific to WT and APA-mut JAK1):

UP:

F: CAAAAAAGCAGGCTGCCAC (SEQ ID NO: 8)

R: GTGTACTCTCCACTGCCCAG (SEQ ID NO: 9)

DOWN:

F: GCCCACCTAACTGTCCAGAT (SEQ ID NO: 10)

R: CTTTGTACAAGAAAGCTGGGTT (SEQ ID NO: 11)

[168] JAK1 (Constructs specific to Codon optimized JAK1):

UP:

F: TCCGAGACCCTAAAACCGAG (SEQ ID NO: 12)

R: CAGGTTCCGTTGTCTGATGC (SEQ ID NO: 13)

DOWN:

F: TGGGAGATCTGCTACAACGG (SEQ ID NO: 14)

R: GCATAATGGCCCGGAAGAAG (SEQ ID NO: 15)

[169] 5'end sequencing

[170] 20 ug of total RNA was polyA selected and then was treated with E.coli purified

AlkB for demethylation of the RNA as previously describe (Zheng et al., 2015). Next, we construction uncapped 5'-specific sequencing libraries as previously describe (Pelechano et al., 2016). The experiment was performed in two biological replicates.

[171] Ribosome profiling (Ribo-seq)

[172] Libraries from cultured cells were prepared as described previously (Loayza-

Puch et al., 2013), with an addition of harringtonine treatment for 5 min that added to the cell culture medium (final concentration of 2 pg/mL -Santa Cruz sc-204771A) prior to CHX treatment. The experiment was performed in two biological replicates.

[173] Hlumina RNA-sequencing

[174] RNA was extracted from Hela cells using QIAzol Reagent (15,596-018, Ambion life technologies) according to the manufactures protocol followed by DNase treatment. PolyA selected RNA was isolated using Oligotex kit (QIAGEN) and further processed with SMARTer Stranded RNA-Seq Kit (Takara; 634,839) and illumina

Truseq Stranded mRNA Library Prep kit. or 3-mRNA-Seq Library Prep Kit (lexogen; 015UG009V0211).

[175] RNA-Seg analysis

[176] Trimming and filtering of raw reads

[177] NextSeq basecall files were converted to FASTQ files using the bcl2fastq (v.2.15.0.4) program with default parameters.

[178] QC preprocessing

[179] Raw reads were inspected for quality issues with FastQC (v.0.11.2) and were quality trimmed at both ends to a quality threshold of 32. Adapter sequences were then removed using cutadapt (version 1.7.1) through the Trim Galore! interface (version 0.3.7), leaving only reads of length above 15 nt. The remaining reads were further filtered to remove very low-quality reads, using the fastq_quality_filter (FASTX package, version 0.0.14), with a quality threshold of 20 at 90% or more of the read positions.

[180] Genomic mapping of RNA-seq data

[181] The processed FASTQ files were mapped (using TopHat, v.2.0.13)22 to the human genome and transcriptome (hg19). Reads that, after processing, were left as a pair, as well as reads for which only one of the pair mates remained, were used for further analyses. Mapping allowed up to 2 mismatches per read, a maximum gap of 5 bases, and a total edit distance of 7.

[182] 3'-End RNA-Seq Analysis

[183] Trimming and Filtering of Raw Reads

[184] NextSeq basecall files were converted to FASTQ files using bcl2fastq (v.2.17.1.14). Reads were screened and preprocessed similarly to the described process above. An additional step involved removing polyA sequences from the 3’ ends of reads, which was carried out with cutadapt. A 75-mer oligo-A sequence was used as the “adapter,” and a minimal overlap of 2 was required for the removal process.

[185] Nanopore RNA Sequencing

[186] Poly(A) selected RNA (500 ng) from control or TEX treated samples was prepared for nanopore direct RNA sequencing, generally following the ONT SQK-

RNA002 kit protocol, which includes the optional reverse transcription step recommended by ONT. RNA sequencing on the Min! ON was performed using ONT R9

Flow Cells. The experiment was conducted with two biological replicates.

[187] Ribosome Profiling (Ribo-seq)

[188] Libraries from cultured cells were prepared following the procedure described previously (Loayza-Puch et al., 2013), with the addition of harringtonine treatment for 5 minutes in the cell culture medium (final concentration of 2 pg/mL - Santa Cruz sc- 204771A) prior to CHX treatment. The experiment was conducted with two biological replicates.

[189] Mass Spectrometry

[190] Data Sets

[191] Two publicly available TMT-based proteomic spectrum files were downloaded and used for the peptide quantitation analysis of JAK1. The uterine corpus endometrial carcinoma (UCEC) dataset with the CPTAC study identifier of PDC000125[pmid: 32059778], and The Cancer Cell Line Encyclopedia (CCLE) dataset with MassIVE identifier of MSV000085836[pmid: 31978347].

[192] Databases

[193] The concatenated protein database of human reference proteome database from UniProt (Release 2023 01, 20,603 entries) and universal contaminant database[pmid:35793413]. To false discovery rate control purpose, the protein database was attached decoy sequences via FragPipe proteome search program (v19.1).

[194] Protein Identification and Quantification

[195] The built-in workflow “TMT10” was used with the adjustments described next.

For CCLE dataset, the downloaded raw files were converted to mzML using the

ProteoWizard MSConvert tool (v3.0.20287) and for UCEC dataset, we used the mzML files without conversion. For MSFragger (v3.7) [pmid:28394336] settings, digestion enzyme, trypsin; number of tolerable termini (Cleavage) was set 1 (SEMI) with clipping

N-terminal methionine; variable modification, M: 15.9949 (Oxidation), protein N-term.: 42.0106 (Acetyl), peptide N-term. and S: 229.16293 (TMT); fixed modification,

C:57.02146 (Carbamidomethyl), K:229.16293 (TMT). For validation settings,

MSbooster (v1.1.11) was used for RT and spectra prediction; PSM validation:

Percolator (v3.05) [pmid: 27572102] with tdc option with minimum probability of 0.5.

Forisobaric quantification, TMT-10 label type was selected and reference channel was set as TMT-126 channel for both UCEC and CCLE dataset, normalization was done at peptide level of median centering, minimal accepted purity was 0.75.

[196] Data Interpretation

[197] For UCEC, we selected 7 patients with JAK1 mutation of 860 frame-shift: C3N- 00850-02, C3L-01744-01, C3N-00389-04, C3N-01212-03, C3L-01257-01, C3N- 00321-01, C3N-01219-03. For CCLE, we selected CAL51, IGROG1, HEC265,

HEC108, ISHIKAWAHERAKLIOO2ER, 22RV1, MFE319, SNU1 and MFE296. In CCLE dataset, CAL51 has 3 replicates and we used all. The median normalized peptide level ratio acquired via FragPipe were used to generate JAK1 peptide level relative quantitation data.

[198] JAK1 inhibitors screen

[199] CYT387 and CEP-33779 JAK1 inhibitors were analyzed using the Prism

Repurposing Secondary Screen. This public resource contains data on the growth inhibitory activity of 4,518 drugs tested across 578 human cancer cell lines (Corsello

SM, et al. 2020. Discovering the anti-cancer potential of non-oncology drugs by systematic viability profiling. Nat Cancer. 2020 Feb;1(2):235-248. doi: 10.1038/s43018-019-0018-6. Epub 2020 Jan 20. PMID: 32613204; PMCID:

PMC7328899.PMID: 32613204). The secondary PRISM Repurposing dataset includes results from pooled-cell line chemical-perturbation viability screens for 1,448 compounds, screened against 489 cell lines using an 8-step, 4-fold dilution starting from 10 uM.

[200] Having now fully described this invention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation.

[201] Reference to known method steps, conventional methods steps, known methods or conventional methods is not in any way an admission that any aspect, description, or embodiment of the present invention is disclosed, taught, or suggested in the relevant art.

PO99199NL STICHTING HET NEDERLANDSKANKERINSTITUUT-ANTONI VAN

LEEUWENHOEK ZIEKENHUIS Janus kinase (JAK) inhibitors for treating cancer 16 279 AA PAT source 1..279 mol_type protein organism Homo sapiens

KRIRDLGEGHFGKVELCRYDPEGDNTGEQVAVKSLKPESGGNHIADLKKEIEILRNLYHENIVK

YKGICTEDGGNGIKLIMEFLPSGSLKEYLPKNKNKINLKQQLKYAVQICKGMDYLGSRQYVHR

DLAARNVLVESEHQVKIGDFGLTKAIETDKEYYTVKDDRDSPVFWYAPECLMQSKFYIASDV

WSFGVTLHELLTYCDSDSSPMALFLKMIGPTHGQMTVTRLVNTLKEGKRLPCPPNCPDEVYQL

MRKCWEFQPSNRTSFQNLIEGFEALLK 22 DNA PAT source 1..22 mol_type other DNA organism synthetic construct acaactttggtatcgtggaagg 19 DNA PAT source 1..19 mol_type other DNA organism synthetic construct gccatcacgccacagtttc 20 DNA PAT source 1..20 mol_type other DNA organism synthetic construct tgacgagaacaccaagctct 20 DNA PAT source 1..20 mol_type other DNA organism synthetic construct gagaatgacgccacactgac 20 DNA PAT source 1..20 mol_type other

DNA organism synthetic construct tgcacagaagacggaggaaa 20 DNA PAT source 1..20 mol_type other DNA organism synthetic construct gaacgtattgccgagaaccc 19 DNA

PAT source 1..19 mol_type other DNA organism synthetic construct caaaaaagcaggctgccac 20 DNA PAT source 1..20 mol_type other DNA organism synthetic construct gtgtactctccactgcccag 20 DNA PAT source 1..20 mol_type other

DNA organism synthetic construct gcccacctaactgtccagat 22 DNA PAT source 1..22 mol_type other DNA organism synthetic construct ctttgtacaagaaagctgggtt 20 DNA

PAT source 1..20 mol_type other DNA organism synthetic construct tccgagaccctaaaaccgag 20 DNA PAT source 1..20 mol_type other DNA organism synthetic construct caggttccgttgtctgatgc 20 DNA PAT source 1..20 mol_type other

DNA organism synthetic construct tgggagatctgctacaacgg 20 DNA PAT source 1..20 mol_type other DNA organism synthetic construct gcataatggcccggaagaag 840 DNA

PAT source 1..840 mol_type genomic DNA organism Homo sapiens aagaggatccgtgacttgggagagggccactttgggaaggttgagctctgcaggtatgaccccgaaggggacaata caggggagcaggtggctgttaaatctctgaagcctgagagtggaggtaaccacatagctgatctgaaaaaggaaat cgagatcttaaggaacctctatcatgagaacattgtgaagtacaaaggaatctgcacagaagacggaggaaatggt attaagctcatcatggaatttctgccttcgggaagccttaaggaatatcttccaaagaataagaacaaaataaacctca aacagcagctaaaatatgccgttcagatttgtaaggggatggactatttgggttctcggcaatacgttcaccgggactt ggcagcaagaaatgtccttgttgagagtgaacaccaagtgaaaattggagacttcggtttaaccaaagcaattgaaa ccgataaggagtattacaccgtcaaggatgaccgggacagccctgtgttttggtatgctccagaatgtttaatgcaatc taaattttatattgcctctgacgtctggtcttttggagtcactctgcatgagctgctgacttactgtgattcagattctagtc ccatggctttgttcctgaaaatgataggcccaacccatggccagatgacagtcacaagacttgtgaatacgttaaaag aaggaaaacgcctgccgtgcccacctaactgtccagatgaggtttatcaacttatgaggaaatgctgggaattccaac catccaatcggacaagctttcagaaccttattgaaggatttgaagcacttttaaaataa

Claims (20)

Translated fromEnglish

CONCLUSIESCONCLUSIONS1. Een Janus Kinase (JAK)-remmer voor gebruik bij de behandeling van kanker in een subject, waarbij de subject een tumor heeft, waarbij de tumor gekenmerkt wordt door expressie van een isovorm van een Janus Kinase 1 (JAK1) eiwit, waarbij de isovorm: - ten minste een JH1-kinasedomein van het JAK1-eiwit bevat; en - geen JH2 (pseudo)kinase domein van het JAK1 eiwit bevat en/of geen JH2 (pseudo)kinase domein, of een deel daarvan, bevat dat in staat is tot (auto)remmende interactie met het JH1 kinase domein van het JAK1 eiwit.1. A Janus Kinase (JAK) inhibitor for use in the treatment of cancer in a subject, wherein the subject has a tumor, the tumor being characterized by expression of an isoform of a Janus Kinase 1 (JAK1) protein, wherein the isoform: - contains at least a JH1 kinase domain of the JAK1 protein; and - does not contain a JH2 (pseudo)kinase domain of the JAK1 protein and/or does not contain a JH2 (pseudo)kinase domain, or part thereof, capable of (auto)inhibitory interaction with the JH1 kinase domain of the JAK1 protein.2. De JAK-remmer voor gebruik volgens de vorige conclusie, waarbij de isovorm van een Janus Kinase 1 (JAK1) geen JH2 (pseudo)kinasedomein van het JAK1-eiwit bevat.2. The JAK inhibitor for use according to the preceding claim, wherein the isoform of a Janus Kinase 1 (JAK1) does not contain a JH2 (pseudo)kinase domain of the JAK1 protein.3. De JAK-remmer voor gebruik volgens een van de voorgaande conclusies, waarbij de tumor wordt gekenmerkt door de aanwezigheid van een JAK1-gen met een of meer mutaties, waarbij de mutatie wordt gekozen uit een frameshiftmutatie, een splicevariant mutatie, een puntmutatie, een nonsense mutatie of een combinatie daarvan.The JAK inhibitor for use according to any preceding claim, wherein the tumor is characterized by the presence of a JAK1 gene with one or more mutations, wherein the mutation is selected from a frameshift mutation, a splice variant mutation, a point mutation, a nonsense mutation or a combination thereof.4. De JAK-remmer voor gebruik volgens een van de voorgaande conclusies, waarbij de tumor wordt gekenmerkt door de aanwezigheid van een JAK1-gen met een of meer mutaties, waarbij ten minste één mutatie een frameshiftmutatie is.The JAK inhibitor for use according to any preceding claim, wherein the tumor is characterized by the presence of a JAK1 gene with one or more mutations, wherein at least one mutation is a frameshift mutation.5. De JAK-remmer voor gebruik volgens een van de voorgaande conclusies, waarbij de tumor wordt gekenmerkt door de aanwezigheid van een JAK1-gen met een of meer mutaties, waarbij ten minste één mutatie wordt gekozen uit de groep bestaande uit een K860Nfs frameshiftmutatie, een P430R frameshiftmutatie, een L431V frameshiftmutatie, een K142R frameshiftmutatie en een 1143D frameshiftmutatie.5. The JAK inhibitor for use according to any preceding claim, wherein the tumor is characterized by the presence of a JAK1 gene with one or more mutations, wherein at least one mutation is selected from the group consisting of a K860Nfs frameshift mutation, a P430R frameshift mutation, an L431V frameshift mutation, a K142R frameshift mutation and an 1143D frameshift mutation.6. De JAK-remmer voor gebruik volgens een van de voorgaande conclusies, waarbij de tumor wordt gekenmerkt door de aanwezigheid van een JAK 1-gen met een of meer mutaties, waarbij ten minste één mutatie een nonsense mutatie is, waarbij de genoemde nonsense mutatie bij voorkeur optreedt in een positie stroomopwaarts van een nucleïnezuursequentie die codeert voor het JH1-domein, en waarbij de genoemde nonsense mutatie zorgt voor de expressie van een JAK1-isovorm zoals gedefinieerd in een van de voorgaande conclusies.The JAK inhibitor for use according to any one of the preceding claims, wherein the tumor is characterized by the presence of a JAK1 gene with one or more mutations, wherein at least one mutation is a nonsense mutation, said nonsense mutation preferably occurring in a position upstream of a nucleic acid sequence encoding the JH1 domain, and wherein said nonsense mutation provides for the expression of a JAK1 isoform as defined in any one of the preceding claims.7. De JAK-remmer voor gebruik volgens een van de voorgaande conclusies, waarbij de isovorm van een Janus Kinase 1 (JAK1)-eiwit bestaat uit een aminozuursequentie die ten minste 85 % identiek is, bij voorkeur 90 % - 100 % identiek aan de aminozuursequentie SEQ ID NO: 1.7. The JAK inhibitor for use according to any preceding claim, wherein the isoform of a Janus Kinase 1 (JAK1) protein consists of an amino acid sequence that is at least 85% identical, preferably 90% - 100% identical to the amino acid sequence SEQ ID NO: 1.8. De JAK-remmer voor gebruik volgens een van de voorgaande conclusies, waarbij de JAK-remmer een remmer is van ten minste JAK1 of een remmer die de kinaseactiviteit van het JH1-kinasedomein kan remmen.8. The JAK inhibitor for use according to any one of the preceding claims, wherein the JAK inhibitor is an inhibitor of at least JAK1 or an inhibitor capable of inhibiting the kinase activity of the JH1 kinase domain.9. De JAK-remmer voor gebruik volgens een van de voorgaande conclusies, waarin de JAK-remmer wordt gekozen uit de groep bestaande uit Momelotinib (CYT387), CEP- 33779 (CAS-nr. 1257704-57-6), Ruxolitinib, Itacitinib, Tofacitinib, Oclacitinib, Baricitinib, Peficitinib, Upadacitinib, Fedratinib, Delgocitinib, Filgotinib, Abrocitinib, Pacritinib, Deucravacitinib, Ritlecitinib, AZD1480 (CAS-nr. 935666-88-9), Gandotinib, Upadacitinib, Gusacitinib, Cerdulatinib en combinaties daarvan.9. The JAK inhibitor for use according to any one of the preceding claims, wherein the JAK inhibitor is selected from the group consisting of Momelotinib (CYT387), CEP- 33779 (CAS No. 1257704-57-6), Ruxolitinib, Itacitinib, Tofacitinib, Oclacitinib, Baricitinib, Peficitinib, Upadacitinib, Fedratinib, Delgocitinib, Filgotinib, Abrocitinib, Pacritinib, Deucravacitinib, Ritlecitinib, AZD1480 (CAS No. 935666-88-9), Gandotinib, Upadacitinib, Gusacitinib, Cerdulatinib and combinations thereof.10. De JAK-remmer voor gebruik volgens een van de voorgaande conclusies, waarbij de remmer Momelatinib is.10. The JAK inhibitor for use according to any one of the preceding claims, wherein the inhibitor is Momelatinib.11. De JAK1-remmer voor gebruik volgens een van de voorgaande conclusies, waarbij de tumor een tumor van epitheelweefsel is.11. The JAK1 inhibitor for use according to any preceding claim, wherein the tumor is an epithelial tissue tumor.12. De JAK1-remmer voor gebruik volgens een van de voorgaande conclusies, waarbij de tumor een niet-epitheliale weefseltumor is.12. The JAK1 inhibitor for use according to any preceding claim, wherein the tumor is a non-epithelial tissue tumor.13. Een in vitro methode voor de selectie van een subject die aan kanker lijdt als kandidaat voor een kankertherapie, waarbij de methode omvat het bepalen in een monster dat tumormateriaal bevat dat van de subject is verkregen: - de aanwezigheid van een isovorm van een Janus Kinase 1 (JAK1) eiwit zoals gedefinieerd in een van de conclusies 1 - 12;13. An in vitro method for selecting a subject suffering from cancer as a candidate for a cancer therapy, the method comprising determining in a sample containing tumor material obtained from the subject: - the presence of an isoform of a Janus Kinase 1 (JAK1) protein as defined in any one of claims 1 to 12;- de aanwezigheid van een JAK1-gen met een of meer mutaties zoals gedefinieerd in een van de bovenstaande conclusies 1-12; en/of - de aanwezigheid van een RNA-molecuul dat, wanneer vertaald, zorgt voor een isovorm van een Janus Kinase 1 (JAK1)-eiwit zoals gedefinieerd in een van de conclusies 1 - 12, en waarbij als de isovorm van een Janus Kinase 1 (JAK1) eiwit en/of het JAK1-gen met een of meer mutaties en/of de RNA-molecule aanwezig is in het monster, de subject geselecteerd wordt als kandidaat voor de kankertherapie.- the presence of a JAK1 gene with one or more mutations as defined in any of the above claims 1 to 12; and/or - the presence of an RNA molecule that, when translated, gives rise to an isoform of a Janus Kinase 1 (JAK1) protein as defined in any of claims 1 to 12, and wherein if the isoform of a Janus Kinase 1 (JAK1) protein and/or the JAK1 gene with one or more mutations and/or the RNA molecule is present in the sample, the subject is selected as a candidate for cancer therapy.14. De in-vitro methode volgens claim 13, waarbij de therapie omvat het toedienen van ten minste één JAK-remmer aan de subject.14. The in vitro method of claim 13, wherein the therapy comprises administering at least one JAK inhibitor to the subject.15. De in-vitro methode volgens een van de conclusies 13 - 14, waarbij het monster wordt gekozen uit een tumorbiopt, een vloeistofmonster met tumorcellen en combinaties daarvan.15. The in vitro method according to any one of claims 13 to 14, wherein the sample is selected from a tumor biopsy, a fluid sample containing tumor cells and combinations thereof.16. De in-vitro methode volgens een van de conclusies 13-15, waarbij de methode verder omvat het bepalen of de isovorm van het Janus Kinase 1 (JAK1) het gevolg kan zijn van een endonuclease-splitsing van een mRNA-molecuul dat een 5'Uncapped Polyadenylated Transcript (5'UPT) vormt dat codeert voor de isovorm van het Janus Kinase 1 (JAK1) eiwit.16. The in vitro method of any one of claims 13 to 15, wherein the method further comprises determining whether the Janus Kinase 1 (JAK1) isoform can result from endonuclease cleavage of an mRNA molecule that forms a 5'Uncapped Polyadenylated Transcript (5'UPT) encoding the Janus Kinase 1 (JAK1) protein isoform.17. De in-vitro methode volgens een van de conclusies 13 - 16, waarbij de methode verder omvat het in contact brengen van een monster omvattende tumorcelmateriaal afkomstig van een persoon die geselecteerd is als kandidaat voor de kankertherapie, met een of meer anti-tumormiddelen, bij voorkeur een of meer JAK-remmers, en het bepalen van een verandering in de proliferatie van de tumorcellen vergeleken met een controle in afwezigheid van genoemde anti-tumormiddelen, en waarbij als de proliferatie afneemt in aanwezigheid van genoemde anti-tumormiddel, bij voorkeur JAK-remmer, de subject wordt geselecteerd als kandidaat voor een kankertherapie waarbij de therapie bestaat uit het toedienen van ten minste één anti-tumormiddel, bij voorkeur JAK-remmer, aan de subject.17. The in vitro method of any one of claims 13 to 16, wherein the method further comprises contacting a sample comprising tumor cell material from a subject selected as a candidate for cancer therapy with one or more anti-tumor agents, preferably one or more JAK inhibitors, and determining a change in proliferation of the tumor cells compared to a control in the absence of said anti-tumor agents, and wherein if proliferation decreases in the presence of said anti-tumor agent, preferably JAK inhibitor, the subject is selected as a candidate for cancer therapy, the therapy comprising administering at least one anti-tumor agent, preferably JAK inhibitor, to the subject.18. Een in vitro methode voor het screenen van kandidaat-agentia die:18. An in vitro method for screening candidate agents that:- binden aan een isovorm van een Janus Kinase 1 (JAK1) eiwit zoals gedefinieerd in een van de conclusies 1 - 12; en/of - de activiteit van een isovorm van een Janus Kinase 1 (JAK1) eiwit zoals gedefinieerd in een van de conclusies 1 - 12 moduleren; Waarbij de methode omvat de stappen van - het in contact brengen van de isovorm van een Janus Kinase 1 (JAK1) eiwit zoals gedefinieerd in een van de conclusies 1 - 12 met een kandidaat-agent; en - het detecteren van binding van de kandidaat-agent met en/of het detecteren van modulatie van de activiteit van de isovorm van het Janus Kinase 1 (JAK1)-eiwit; - naar keuze, het detecteren van binding van de kandidaat-agent met en/of het detecteren van modulatie van de activiteit van een Janus Kinase 1 (JAK1) eiwit.- binding to an isoform of a Janus Kinase 1 (JAK1) protein as defined in any of claims 1 to 12; and/or - modulating the activity of a Janus Kinase 1 (JAK1) protein isoform as defined in any of claims 1 to 12; wherein the method comprises the steps of - contacting the Janus Kinase 1 (JAK1) protein isoform as defined in any of claims 1 to 12 with a candidate agent; and - detecting binding of the candidate agent with and/or detecting modulation of the activity of the Janus Kinase 1 (JAK1) protein isoform; - optionally, detecting binding of the candidate agent with and/or detecting modulation of the activity of a Janus Kinase 1 (JAK1) protein.19. De in-vitro methode voor screening volgens conclusie 18, waarbij het detecteren van modulatie van de activiteit van de isovorm van het Janus Kinase 1 (JAK1)-eiwit omvat het detecteren van modulatie van de tumorigene activiteit van de isovorm van het Janus Kinase 1 (JAK1)-eiwit in vergelijking met de tumorigene activiteit in afwezigheid van de kandidaat-agent, en/of waarin het detecteren van modulatie van de activiteit van de isovorm van het Janus Kinase 1 (JAK1)-eiwit het detecteren van kinaseactiviteit van de isovorm van het Janus Kinase 1 (JAK1)-eiwit omvat in vergelijking met de kinaseactiviteit van de isovorm van het Janus Kinase 1 (JAK1)-eiwit in afwezigheid van de kandidaat agent.19. The in vitro method of screening according to claim 18, wherein detecting modulation of the activity of the Janus Kinase 1 (JAK1) protein isoform comprises detecting modulation of the tumorigenic activity of the Janus Kinase 1 (JAK1) protein isoform compared to the tumorigenic activity in the absence of the candidate agent, and/or wherein detecting modulation of the activity of the Janus Kinase 1 (JAK1) protein isoform comprises detecting kinase activity of the Janus Kinase 1 (JAK1) protein isoform compared to the kinase activity of the Janus Kinase 1 (JAK1) protein isoform in the absence of the candidate agent.20. De in-vitro methode voor screening volgens een van de conclusies 18 - 19, waarbij de methode de stappen omvat van: - het detecteren van binding van de kandidaat-agent met en/of het detecteren van modulatie van de activiteit van een Janus Kinase 1 (JAK1) eiwit; en - vergelijken van een gedetecteerde binding en/of modulatie van de activiteit van een Janus Kinase 1 (JAK1)-eiwit met de binding en/of modulatie van de activiteit van het Janus Kinase 1 (JAK1)-eiwit zoals gedefinieerd in een van de conclusies 1 - 12.20. The in vitro method for screening according to any one of claims 18 to 19, the method comprising the steps of: - detecting binding of the candidate agent with and/or detecting modulation of the activity of a Janus Kinase 1 (JAK1) protein; and - comparing a detected binding and/or modulation of the activity of a Janus Kinase 1 (JAK1) protein with the binding and/or modulation of the activity of the Janus Kinase 1 (JAK1) protein as defined in any one of claims 1 to 12.