WO2017204626A1

WO2017204626A1 - Combination therapy - combined map2k4/map3k1 and mek/erk inhibition

Info

Publication number: WO2017204626A1
Application number: PCT/NL2017/050319
Authority: WO
Inventors: Rene Bernards; Xue ZHENG
Original assignee: Stichting Het Nederlands Kanker Instituut-Antoni van Leeuwenhoek Ziekenhuis
Priority date: 2016-05-24
Filing date: 2017-05-19
Publication date: 2017-11-30

Abstract

Disclosed is a new method of treatment of cancer. Inhibition of the MAP3K1-MAP2K4-JNK pathway, and/or inhibitions of various RTK,and inhibition of the MEK-ERK pathway was found to be useful in the treatment of various cancers, including cancers harboring a KRAS mutation. The treatment is in particular useful in patients harboring wild-type MAP2K4 and MAP3K1 and not responding to inhibitors of the MEK-ERK pathway, in particular MEK inhibitors.

Description

Combination therapy - combined MAP2K4/MAP3K1 and MEK/ERK inhibition Background of the invention

Cancer is one of the leading causes of death in the developed world, with an estimated 3.45 million new cases of cancer (excluding non-melanoma skin cancer) and 1 .75 million deaths from cancer in Europe in 2012 (Ferlay et al. European Journal of Cancer (2013) 49, 1374- 1403). Overall it is estimated that more than 1 in 3 people will develop some form of cancer during their lifetime.

Cancer cells are by definition heterogeneous. This is, for example, caused by mutational mechanisms that may lead to the development of cancer and that differ between one tissue type and another; it is therefore often difficult to predict whether a specific cancer will respond to a specific treatment (Cancer Medicine, 5th edition, Bast et al , B. C. Decker Inc. , Hamilton, Ontario).

The treatment of cancer is gradually changing from an organ-centered to a pathway-centered approach. In recent years, significant progress has been made in the treatment of both common and rare cancers. This progress is leading to longer patient survival and improved quality of life. Advances range from targeted therapies for disease settings where previously no effective treatments existed to exciting progress in immunotherapy. In addition, the strategy of combining different types of therapy shows powerful results in large-scale studies. Combination of a chemotherapy drug with standard hormone therapy brought the longest survival improvement for patients with advanced prostate cancer, and adding radiotherapy to standard chemotherapy extended the lives of patients with glioma by 5 years. (Clinical Cancer Advances 2015 - ASCO's Annual Report on Progress Against Cancer; Published in the Journal of Clinical Oncology, January 2015). The focus of cancer treatment is shifting toward targeted therapies aimed at genes and pathways involved in human cancer. Targeted therapy is a treatment that targets specific molecules in or on cancer cells, or in the tumor's immediate surroundings. This type of approach is aimed at blocking the growth and spread of cancer cells while limiting damage to healthy cells. Important molecular targets in targeted therapy include components of signaling pathways.

Signaling pathways normally connect extracellular signals to the nucleus leading to expression of genes that directly or indirectly control cell growth, differentiation, survival, and death. For many cancers it has been established that signaling pathways are dysregulated. The dysregulated signaling pathways may be linked to tumor initiation and/or progression. Targeting such dysregulated pathway may thus provide a beneficial treatment option. Despite recent advances in understanding mechanisms involved in cancer, targeted therapy is not always successful.

For example, one signaling pathway implicated in human oncogenesis is the RAS-RAF-MEK- ERK or MAPK pathway ((Peyssonnaux et al., Biol Cell. 93(l-2):53-62 (2001 )). Numerous efforts to develop therapeutic agents that specifically target the mutated BRAF kinase are underway for melanoma treatment. However, the development of resistance to the BRAF inhibitors has proven to be a major challenge (Wagle et al. J Clin Oncol. 29(22) :3085-96 (201 1 )). Furthermore, these agents have little or no effect in patients whose tumors have a wild-type BRAF.

Another example of the challenges faced in the field relate to M EK inhibition in KRAS mutant lung and colon cancer. One of the attractive features of MEK as a target of inhibition is its structure. It contains a pocket structure, conserved only in MEK proteins, that, upon binding by an inhibitor, results in locking unphosphorylated MEK1/2 into a catalytically inactive state. Because this action does not have an inhibitory effect on the highly conserved adenosine triphosphate binding site pocket, it avoids side effects associated with inhibition of other protein kinases.

Several compounds with potent inhibitory activity specific for MEK1/2 have been investigated. The first MEK inhibitor entered clinical trials in 2000, but until 2014 no MEK inhibitor had been approved for clinical use. This is because, for the most part, the agents investigated have not demonstrated robust clinical activity in most tumor types. One of the possible mechanism by which a cancer becomes or is unresponsive to MEK inhibitor is based on acquiring or inherently possessing resistance to the MEK inhibitor. Indeed preclinical studies have identified distinct mechanisms by which cells acquire resistance to MEK inhibition, including amplification of mutant BRAF, STAT3 upregulation, or mutations in the allosteric pocket of MEK that can directly block binding of inhibitors to the MEK kinase or lead to constitutive MEK kinase activity. MEK mutations have also been described in tumor samples from patients treated with MEK or BRAF inhibitors, showing clinical relevance (Hatzivassiliou, .Mol Cancer Ther 1 1 , 1 143 (2012)) Other studies show that MEK inhibition results in MYC- dependent transcriptional upregulation of ERBB3, which is responsible for intrinsic drug resistance. (Sun et al. Cell Reports 7, 86-93 (2014), suggesting dual targeted inhibition treatment. Dual targeted inhibition of MEK and PI3K pathway effectors (mTOR, PI3K, AKT, IGF-1 R or PI3K/mTOR inhibitors) has also been suggested as a potential strategy to overcome resistance to MEK inhibitor therapy in in KRAS and BRAF mutated colorectal cancers and clinical trials are underway to evaluate multiple combinations of these pathway inhibitors in solid tumors (Reviewed by Temraz Int. J. Mol. Sci. 16, 22976-22988 (2015)).

Despite recent advances in understanding mechanisms involved in cancer and in diagnosis and treatment, drug therapies for metastatic disease are often palliative in nature. Drug therapies seldom offer a long-term cure. There is a constant need for new methods of treatment, either in the form of monotherapy or in the form of combination treatment, combining different new or known drugs, for example as first line therapy, or in case resistance is acquired during treatment.

Thus, there is a need for new agents targeting signaling pathways and new combinations of agents that target multiple pathways that could provide therapeutic benefit for cancer patients.

For the same reason there is need for new methods that allow to establish if a patient is likely or unlikely to respond to a particular treatment. In light of this, products, compositions, combinations, methods and uses for the treatment of cancer would be highly desirable. In particular there is a clear need in the art for reliable, efficient, reproducible and in particular targeted products, compositions, methods and uses that allow treatment of specific cancers. 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. The technical problem is solved by the embodiments characterized in the claims and herein below.

Description Drawings

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

Fig. 1 : MAP2K4 mutant ILC cell lines respond to MEK inhibitor selumetinib. Four ILC cell lines of indicated mutation status were cultured in medium containing the indicated concentration of selumetinib for two weeks. After this, cells were fixed and stained. Fig. 2: MAP3K1 and MAP2K4 knockout block selumetinib induced JNK kinase activation and confer sensitivity to selumetinib.

A. I LC cell lines of indicated mutation status were treated with 2μΜ selumetinib for 6 hours, the levels of p-JNK, JNK, p-JUN, JUN, p-ERK and ERK were determined by western blot analysis. HSP90 was served as a loading control.

B. Control and MAP3K1 or MAP2K4 knockout MDA-MB-468 cells were cultured for two weeks in medium containing the indicated concentration of selumetinib. Then cells were fixed and stained.

Fig. 3: HER receptors are activated by selumetinib.

A. MDA-MB-468 cells were treated with selumetinib for 72 hours, then RNA was extracted and qRT-PCR analysis performed for H ER receptor transcripts.

B. Two individual shRNAs targeting JUN were introduced into MDA-MB-468 cells by lentiviral transduction. Ctrl and JUN knockdown cells were treated with 2μΜ selumetinib for 6 hours (left) or 72 hours (right). The levels of phospho-HER1 -4 and HER1 -4 receptors, p-JUN, JUN, p-ERK and ERK were determined by western blot analysis. HSP90 was served as a loading control.

C. Indicated cells were cultured for two weeks in medium containing increasing concentration of selumetinib alone, dacomitinib alone, or combination of selumetinib and dacomitinib. After this, cells were fixed and stained.

Fig. 4: MAP3K1 and MAP2K4 knockout confer sensitivity to selumetinib in KRAS mutant lung and colon cancer. A. Parental and MAP3K1 knockout H358 were treated with 2μΜ selumetinib for 6 hours (left) or 72 hours (right) and lysates were western blotted for p-JUN, JUN, MAP2K4, p-ERK, ERK. HSP90 served as a control.

B. Control and MAP3K1 knockout H358 cells were treated with 2μΜ selumetinib for 6 hours and lysates were western blotted as in A. C. Control and MAP3K1 or MAP2K4 knockout H358 (upper panel) and HCT1 16 cells (lower panel) were cultured for two weeks in medium containing the indicated concentration of selumetinib. Then cells were fixed and stained.. D. H358 (left) and HCT1 16 (right) cells, both Ctrl and MAP2K4 knockout cells, were injected in nude mice. Once tumors reach 100mm3, mice (six per group) were treated with vehicle or selumetinib (20mg/kg/day). The mean percentage change from the initial tumor volume is shown. Error bars represent standard error of the mean (SEM). Fig. 5: MDA-MB-468 cells were cultured for two weeks in medium containing increasing concentration of selumetinib alone, JNK inhibitor sp600125 alone, or combination of selumetinib and sp600125. After this, cells were fixed and stained.

Fig.6: JNK inhibitor sensitizes MAP3K1/MAP2K4 wild-type cells to MEK inhibitor. Panels A-F. depict cells (MDA-MB-468 breast cancer cells, LoVo colon cancer cells, and H358 lung cancer cells), which were cultured for two weeks in medium containing increasing concentration of selumetinib alone, 0.5 μΜ JNK-IN-8 or 0.5 μΜ SR3306 alone, or combination of selumetinib and JNK inhibitor. At the term of the treatment, the cells were fixed and stained.

Definitions

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. 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. "A," "an," and "the": these singular form terms include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a cell" includes a combination of two or more cells, and the like. "About" and "approximately": these terms, when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1 %, and still more preferably ±0.1 % from the specified value, as such variations are appropriate to perform the disclosed methods. "And/or": The term "and/or" refers to a situation wherein 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.

"Comprising": this term 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.

"Exemplary": this terms means "serving as an example, instance, or illustration," and should not be construed as excluding other configurations disclosed herein.

"A method of treating" or its equivalent: When applied to, for example, cancer, this term refers to a procedure or course of action that is designed to reduce or eliminate the number of cancer cells in an animal, or to alleviate the symptoms of a cancer. "A method of treating" cancer or another proliferative disorder does not necessarily mean that the cancer cells or other disorder will, in fact, be eliminated, that the number of cells or disorder will, in fact, be reduced, or that the symptoms of a cancer or other disorder will, in fact, be alleviated. Often, a method of treating cancer will be performed even with a low likelihood of success, but which, given the medical history and estimated survival expectancy of an animal, is nevertheless deemed an overall beneficial course of action.

"Acquired resistance": this term indicates that a cancer has acquired reduced sensitivity or has become resistant to the effects of a drug after being exposed to it, or a drug targeting the same mechanism or pathway, for a certain period of time. Acquired resistance to the therapy with a drug often manifests either a diminished amount of tumor regression for the same dose of a drug or the need for an increased dose for an equal amount of tumor regression. The term also indicates that a cancer may also become resistant to a first drug after being exposed to a second drug targeting the same mechanism of pathway in the cancer cell. For example, resistance may be acquired to a first ERK-inhibitor due to exposure to a second ERK-inhibitor (and to which the cancer will also have developed resistance). Alternatively, resistance may be intrinsic, i.e. not acquired of induced by the anti-cancer therapy. When, the resistance is intrinsic the tumor cells already originally lack sensitivity to one or more anti- cancer drugs. Since the resistance can be intrinsic or acquired the observed reduction in sensitivity is either compared to fully sensitive "normal" cancer cells, which are responsive to the therapeutically effective dosage of the applied anticancer drug and/or compared to the original sensitivity upon therapy onset. "Antagonist" and "inhibitor": These terms are used interchangeably, and they refer to a compound or agent having the ability to reduce or inhibit a biological function of a target protein or polypeptide, such as by reducing or inhibiting the activity or expression of the target protein or polypeptide, for example by down-regulating, decreasing, suppressing or otherwise regulating the amount and/or activity of the (defined) protein. Accordingly, the terms "antagonist" and "inhibitor" are defined in the context of the biological role of the target protein or polypeptide. An inhibitor need not completely abrogate the biological function of a target protein or polypeptide, and in some embodiments reduces the activity by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%. While some inhibitors herein specifically interact with (e.g. , bind to) the target, compounds that inhibit a biological activity of the target protein or polypeptide by interacting with other members of the signal transduction pathway of which the target protein or polypeptide are also specifically included within this definition. The inhibitors to be used in accordance with the present invention may be selective inhibitors of said (defined) protein; the term "selective" or "selectivity" expresses the biologic fact that at a given compound concentration enzymes (or proteins) are affected to different degrees. In the case of enzymes (or proteins) selective inhibition can be defined as preferred inhibition by a compound at a given concentration. In other words, an enzyme is selectively inhibited over another enzyme when there is a concentration which results in inhibition of the first enzyme whereas the second enzyme is not affected. Non-limiting examples of biological activity inhibited by an inhibitor include those associated with the development, growth, or spread of a tumor. The inhibitors used herein are inhibitors that may display anti-cancer effects, either alone or in combination.

"Anti-cancer effect": This refers to the effect a therapeutic agent has on cancer, e.g., a decrease in growth, viability, or both of a cancer cell. The IC50 of cancer cells can be used as a measure the anti-cancer effect. IC50 refers to a measure of the effectiveness of a therapeutic agent in inhibiting cancer cells by 50%. "Alleviating or treating cancer": These terms may be used interchangeably. These terms refer to an approach for obtaining beneficial or desired results including, but not limited to, therapeutic benefit. By therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the patient, notwithstanding that the patient can still be afflicted with the underlying disorder. The term, in the context of specific cancers and/or their pathologies, refers to degrading a tumor, for example, breaking down the structural integrity or connective tissue of a tumor, such that the tumor size is reduced when compared to the tumor size before treatment. "Alleviating" metastasis of cancer includes reducing the rate at which the cancer spreads to other organs.

"Cancer": This term refers to the physiological condition in mammals that is typically characterized by unregulated cell growth. The terms "cancer," "neoplasm," and "tumor," are often used interchangeably to describe cells that have undergone a malignant transformation that makes them pathological to the host organism. Primary cancer cells can be distinguished from non-cancerous cells by techniques known to the skilled person. A cancer cell, as used herein, includes not only primary cancer cells, but also cancer cells derived from such primary cancer cell, including metastasized cancer cells, and cell lines derived from cancer cells. Examples include solid tumors and non-solid tumors or blood tumors. Examples of cancers include, without limitation, leukemia, lymphoma, sarcomas and carcinomas (e.g. colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, lung cancer, melanoma, lymphoma, non-Hodgkin lymphoma, colon cancer, (malignant) melanoma, thyroid cancer, papillary thyroid carcinoma, lung cancer, non-small cell lung carcinoma, and adenocarcinoma of lung.). As is well known, tumors may metastasize from a first locus to one or more other body tissues or sites. Reference to treatment for a "neoplasm, "tumors" or "cancer" in a patient includes treatment of the primary cancer, and, where appropriate, treatment of metastases. "Compositions", "products" or "combinations": These encompass those compositions suitable for various routes of administration, including, but not limited to, intravenous, subcutaneous, intradermal, subdermal, intranodal, intratumoral, intramuscular, intraperitoneal, oral, nasal, topical (including buccal and sublingual), rectal, vaginal, aerosol and/or parenteral or mucosal application. The compositions, formulations, and products according to the disclosure invention normally comprise the drugs/compound/inhibitor (alone or in combination) and one or more suitable pharmaceutically acceptable excipients or carriers. "Combinations" refer to the use of more than one compound or agent to treat a particular disorder or condition. For example, Compound 1 may be administered in combination with at least one additional therapeutic agent, Compound 2. By "in combination with," it is not intended to imply that the combination of Compound 1 and Compound 2 must be administered at the same time and/or formulated for delivery together, although these methods of delivery are within the scope of this disclosure. Compound 1 can be administered concurrently with, prior to (e.g. , 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, 12 weeks, or 16 weeks before), or subsequent to (e.g. , 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, 12 weeks, or 16 weeks after) Compound 2. In general, each therapeutic agent of the combination may be administered at a dose and/or on a time schedule determined for that particular agent. Compound 2 can be administered with Compound 1 herein in a single composition or separately in a different composition. Higher combinations, e.g. , triple therapy, are also contemplated herein.

"Effective amount": this means the amount of a drug which is effective for at least a statistically significant fraction of subjects to treat any symptom or aspect of the cancer. Effective amounts can be determined routinely. The term includes both pharmacological effectiveness and physiological safety. Pharmacological effectiveness refers to the ability of the treatment to result in a desired biological effect in the subject such as improvement of symptoms, a cure, a reduction in disease load, reduction in tumor mass or cell numbers, extension of life, improvement in quality of life, or other effect generally recognized as positive by medical doctors familiar with treating the particular type of disease or condition. Physiological safety refers to the level of toxicity, or other adverse physiological effects at the cellular, organ and/or organism level (often referred to as side-effects) resulting from administration of the treatment.

"Monotherapy": This term refers to the use of an agent individually (also referred to herein as alone) (e.g., as a single compound or agent), e.g., without a second active ingredient to treat the same indication, e.g. , cancer.

"Resistant cancer" or "refractive cancer": These terms refer to when a cancer that has a reduced responsiveness to a treatment, e.g., up to the point where the cancer does not respond to treatment. The cancer can be resistant at the beginning of treatment, or it may become resistant during treatment. The cancer subject may have one or more mutations that cause it to become resistant to the treatment, or the subject may have developed such mutations during treatment. The term "refractory" can refer to a cancer for which treatment (e.g. chemotherapy drugs, biological agents, and/or radiation therapy) has proven to be ineffective. A refractory cancer tumor may shrink, but not to the point where the treatment is determined to be effective. Typically however, the tumor stays the same size as it was before treatment (stable disease), or it grows (progressive disease). "Simultaneous administration": This refers to administration of more than one drug at the same time, but not necessarily via the same route of administration or in the form of one combined formulation. For example, one drug may be provided orally whereas the other drug may be provided intravenously during a patients visit to a hospital. "Separate administration" includes the administration of the drugs in separate form and/or at separate moments in time, but again, not necessarily via the same route of administration. "Sequentially" of "sequential administration" indicates that the administration of a first drug if followed, immediately or in time, by the administration of the second drug, but again, not necessarily via the same route of administration. "Subject" or "patient": this is to indicate the organism to be treated e.g. to which administration is contemplated. The subject may be any subject in accordance with the present invention, including, but not limited to humans (e.g. , a male or female of any age group, e.g. , a pediatric subject (e.g. , infant, child, adolescent) or adult subject (e.g. , young adult, middle-aged adult or senior adult)) and/or other primates (e.g. cynomolgus monkeys, rhesus monkeys); mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, goats, cats, and/or dogs; and/or birds, including commercially relevant birds such as chickens, ducks, geese, quail, and/or turkeys. Preferably the subject is a human patient. Detailed Description

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.

As embodied and broadly described herein, the present invention is directed to the finding by the inventors of the present invention that there is an unexpected relationship between the presence of mutations (e.g. mutations that cause a loss of function of the encoded protein, including deletion or truncation mutations or point mutations leading to loss of function, e.g. that cause loss of enzymatic activity/loss of the ability to phosphorylate substrates) in genes encoding MAP3K1 protein and/or the MAP2K4 protein and response of cancer cells, including KRAS-mutated cancer cells, to treatment with inhibitors of proteins of the MAPK pathway (e.g. said pathway comprising or consisting of the RAS protein, RAF protein, MEK protein (MEK1 and MEK2 proteins) and ERK (MAPK) protein), in particular with inhibitors of the MEK-ERK pathway (e.g. said pathway comprising or consisting of the MEK protein and ERK protein), in particular inhibitors of MEK and/or inhibitors of ERK.

More in particular, it was found by the current inventors that cancer cells carrying inactivating mutations/loss-of-function mutations (e.g. deletion mutations or truncation mutations leading to loss of function of the MAP2K4 protein or MAP3K1 protein, e.g loss of enzymatic activity / loss of the capacity to phosphorylate their respective (natural) substrates) in the gene encoding MAP3K1 protein and/or the gene encoding the MAP2K4 protein are sensitive to inhibition by inhibitors of the MAPK pathway (comprising or consisting of the RAS protein, RAF protein, M EK protein (M EK1 and MEK2 proteins) and ERK (MAPK) protein), in particular with inhibitors of the MEK-ERK pathway (comprising or consisting of the MEK protein and ERK protein), in particular to MEK inhibitors, whereas cancer cells expressing both functional MAP3K1 protein (having enzymatic activity being capable of phosphorylating MAP2K4) and MAP2K4 protein (having enzymatic activity being capable of phosphorylating serine and threonine in its substrates, typically in p38 MAPK and JNK) display reduced sensitivity towards inhibition by such inhibitors. In line therewith, it was found that inhibiting one or more of the proteins of the MAP3K1 -MAP2K4-JNK pathway (e.g. the MAP3K1 protein, the MAP2K4 protein, the JNK1 protein, and/or the JNK2 protein) conferred sensitivity to inhibition with the MAPK pathway inhibitor, in particular with inhibitors of the M EK-ERK pathway, in the cancers with functional MAP3K1 and MAP2K4 proteins.

Likewise, it was found that in cancer cells with a functional MAP3K1 -MAP2K4-JNK pathway (comprising or consisting of MAP3K1 protein, the MAP2K4 protein, the JNK1 protein, and the JNK2 protein), several receptor tyrosine kinases where activated upon treatment with MAPK pathway inhibitors, in particular with inhibitors of the M EK-ERK pathway, (likely via activation of JUN by JNK), whereas reduced or no activation was seen in cancer cells with nonfunctional MAP3K1 protein and/or non-functional MAP2K4 protein (note: mutations are mostly mutually exclusive in cancer, as they act in the same pathway). It is understood that with the term "a functional MAP3K1 -MAP2K4-JNK pathway", it is meant that the proteins of said pathway (said pathway comprising or consisting of MAP3K1 protein, the MAP2K4 protein, the JNK1 protein, and the JNK2 protein) are functional, i.e. they have their expected enzymatic activity, e.g. phosphorylating their respective substrates. The term "a functional MAP3K1 - MAP2K4-JNK pathway" is also understood to include the situation that there are no loss of function mutations (e.g. deletion or truncation mutations causing loss of enzymatic activity or loss of the ability to phosphorylate substrates) in the genes encoding for the proteins of the MAP3K1 -MAP2K4-JNK pathway (MAP3K1 protein, the MAP2K4 protein, the JNK1 protein, and the JNK2 protein). In line therewith, it was found that combining an inhibitor of such receptor tyrosine kinase and an inhibitor of one or more of the proteins of the MAPK pathway (e.g. the RAS protein, RAF protein, MEK protein (MEK1 and MEK2 proteins) and/or ERK (MAPK) protein), in particular with inhibitors of the MEK-ERK pathway, in particular a MEK inhibitor, restored response to treatment in cancer cells that have functional MAP3K1 protein and/or MAP2K4 protein (e.g. wherein the MAP3K1 protein is capable of phosphorylating MAP2K4 and wherein the MAP2K4 protein is capable of phosphorylating serine and threonine on its substrates, typically p38 MAPK and JNK).

The unexpected relationship between the presence of mutations in genes encoding MAP3K1 protein and/or the MAP2K4 protein and response of cancer cells, including KRAS-mutated cancer cells, to treatment with inhibitors of proteins of the MAPK pathway, in particular the MEK-ERK pathway makes it also possible to predict treatment response of a cancer patient to treatment with a MAPK pathway inhibitor, in particular with inhibitors of the MEK-ERK pathway, in particular with an inhibitor of MEK, or to combination of such inhibitor with inhibitors of proteins of the MAP3K1 -MAP2K4-JNK (said pathway comprising or consisting of the MAP3K1 protein, the MAP2K4 protein, the JNK1 protein, and the JNK2 protein) or inhibitors of receptor tyrosine kinases, including, for example, EGFR, HER2, HER3 and HER4. Therefore, there is provided for a combination of an inhibitor of one or more of the proteins of the MAP3K1 -MAP2K4-JNK pathway (e.g. the MAP3K1 protein, the MAP2K4 protein, the JNK1 protein, and/or the JNK2 protein) and an inhibitor of one or more of the proteins of the MAPK pathway (e.g. the RAS protein, RAF protein, MEK protein (M EK1 and MEK2 proteins) and/or ERK (MAPK) protein), in particular an inhibitor of the M EK-ERK pathway for use as a medicament in a subject.

In a preference, there is provided for a combination of an inhibitor of one or more of the proteins of the MAP3K1 -MAP2K4-JNK pathway and an inhibitor of one or more of the proteins of the MAPK pathway, in particular an inhibitor of the M EK-ERK pathway, for use in the treatment of cancer in a subject.

In other words, such combination is useful for use in medicine, in particular in the treatment of a human subject, in particular in the treatment of cancer. Indeed the subject considered herein is typically a human. However, the subject can be any mammal for which cancer treatment is desired. Thus, the invention described herein can be applied to both human and veterinary applications. MAP3K1 -MAP2K4-JNK pathway and inhibitors

The skilled person is well acquainted with the proteins that form the MAP3K1 -MAP2K4-JNK pathway and means to inhibit the activity of these proteins. The MAP3K1 -MAP2K4-JNK pathway comprises proteins encoded by, respectively the MAP3K1 gene, the MAP2K4 gene and the JNK1 and JNK2 genes. In other words, the MAP3K1 -MAP2K4-JNK pathway comprises or consists of the MAP3K1 protein, the MAP2K4 protein, the JNK1 protein, and the JNK2 protein.

Within the context of the current invention, a MAP3K1 -MAP2K4-JNK pathway inhibitor is a compound that specifically or selectively (i.e. selectively reduce the target's activity as compared to off- target signaling activity, via direct or indirect interaction with the target) inhibits signaling through the MAP3K1 -MAP2K4-JNK pathway (e.g. through affecting signaling mediated by the MAP3K1 protein, the MAP2K4 protein, the JNK1 protein, and/or the JNK2 protein and combination thereof). The MAP3K1 -MAP2K4-JNK pathway inhibitor may do so by, for example, reducing the biological activity (e.g. enzymatic activity or ability to phosphorylate substrates) of one or more of the proteins of the pathway (e.g. the MAP3K1 protein, the MAP2K4 protein, the JNK1 protein and/or the JNK2 protein), or by reducing expression of an mRNA encoding one or more of the proteins of the pathway (e.g. the MAP3K1 protein, the MAP2K4 protein, the JNK1 protein and/or the JNK2 protein), or by reducing the expression of one or more of the proteins of the pathway (e.g. the MAP3K1 protein, the MAP2K4 protein, the JNK1 protein and/or the JNK2 protein). For example, with the term "a MAP2K4 inhibitor", it is meant a compound that may reduce the biological activity of MAP2K4 protein (e.g. reduced its ability to phosphorylate serine and threonine in its substrates, typically p38 MAPK and JNK); or that may reduce the expression of an mRNA encoding a MAP2K4 polypeptide or protein; or that may reduce the expression of a MAP2K4 polypeptide or protein. As will be understood by the skilled person, the above is likewise applicable with respect to inhibitors of the other proteins of the MAP3K1 -MAP2K4-JNK pathway (e.g. the MAP3K1 protein, the JNK1 protein and/or the JNK2 protein).

MAP2K4 (mitogen-activated protein kinase kinase 4; also known as MKK4, MEK4, or SEK1 ) is a dual-specificity protein kinase that phosphorylates serine and threonine, as well as tyrosine residues. This 399 amino acid protein typically activates two downstream targets, p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun N-terminal kinase (JNK) (see, for example, Pavese et al. (2014) PloS ONE 9(7): e102289. doi: 10.1371/journal. pone.0102289.). Genetic alterations in the MAP2K4 gene have been recently found in comprehensive genomic analyses of cancers (Michaut et al. (2016) Scientific Reports | 6: 18517 | DOI: 10.1038/srep18517). The MAP2K4 has been identified as an important therapeutic target for inhibiting. One known inhibitor is genistein (Xu et al. (2009) J Natl Cancer Inst 101 : 1 141-1 155. doi: 10.1093/jnci/djp227). However, genistein exerts a wide range of biological effects, and alternative inhibitors have been identified (including various chalcones and cayanidin; Krishna et al. , (2013) PloS ONE 8(12) : doi: 10.1371/annotation/5edeb1 de-b76c-4fd9-a3f6- 0f1 cf45e3905). Other examples, including KBU-2046, are disclosed in WO2009/049214. One other example is the use of miRNA to inhibit expression of MAP2K4. For example, miR27-a has been suggested to target MAP2K4 (Pan et al. Cell Physiol Biochem. 2014; 33(2):402-12).

MAP3K1 or MEKK1 (Mitogen-activated protein kinase kinase kinase 1 ; MEK kinase 1 ) is a 196-kDa serine-threonine kinase that belongs to the MAP3K family and the STE superfamily. MAP3K1 was originally identified as the mammalian homolog of the yeast MAP3Ks Ste1 1 and Byr2. In addition to the conserved kinase domain, MAP3K1 has several unique structural characteristics that mediate its specific activities compared with other MAP3Ks. The kinase domain of MAP3K1 is located at the C-terminus. MAP3K1 selectively phosphorylates and activates MAP2K4, which in turn phosphorylates and activates JNK. MAP3K1 is activated by a variety of stimuli such as growth factors, pro-inflammatory cytokines, microtubule disruption, cell shape disturbance, cold temperature, mild hyperosmolarity, and other cell stresses and activation of full length MAP3K1 stimulates the MAP2K4/7-JNK pathway (Pham et al. Genes Cancer. 2013; 4(1 1 -12): 419^126.) Studies have demonstrated that MAP3K1 functions in cell survival, apoptosis, and cell motility/migration in multiple normal and tumor cell types. Genetic alterations in the MAP3K1 gene have been recently found in comprehensive genomic analyses of cancers (Michaut et al. (2016) Scientific Reports | 6: 18517 | DOI: 10.1038/srep18517). Nonsense mutations were observed (19% of observed mutations) but the majority of mutations result in a frame-shift deletion or insertion (59%) and are predicted to be inactivating, creating non-functional MAP3K1 protein. As discussed above, down- regulation, loss of function mutations, and homozygous deletion of the MAP3K1 downstream target MAP2K4 have also been reported in clinical samples from several types of cancer such as prostate, pancreatic, and ovarian cancer (reviewed in (Pham et al. Genes Cancer. 2013; 4(1 1 -12): 419-426; Huijts et al. Breast Cancer Res 2007; 9: R78; Easton et al. Nature 2007; 447: 1087-93.)

Inhibitors of MAP3K1 include, for example, (5Z)-7-Oxozeaenol (Enzo Life Sciences, Farmingdale, USA). JUN N-terminal kinase (JNK), or c-JUN N-terminal kinase (JNK) is a serine threonine protein kinase that phosphorylates and activates c-JUN, a component of the transcription factor activator protein- 1 (AP-I) (Leppa and Bohmann, Oncogene, 1999, 258: 6158-6162). Three distinct genes (JNK1 , JNK2 and JNK3) encoding 10 splice variants have been identified. JNK1 and JNK2 are expressed in a wide variety of tissues, whereas JNK3 is mainly expressed in neurons, and to a lesser extent in heart and testes. Members of JN K family are activated by pro-inflammatory cytokines such as tumor necrosis factor a (TNF-a) and interleukin-ΐ β (IL-1 β), as well as environmental stresses. The activation of JNKs is mediated by phosphorylation of Thr-183 and Tyr-185.

JN K has been identified as an important target for inhibition and various inhibitors are known. Examples includes CC-401 (Cellgene), SP600125, BI78D3 and BI87G9, several of which are discussed by Cicencas (MAP Kinase (2015) 4:5700 (pp 32). Other examples are disclosed in, for example, US9180159 (describing a peptide based inhibitor), EP2283009, WO2008/028860, WO2006/038001 and in Alam et al. (2007) Bioorganic & Medicinal Chemistry Letters 17 (12): 3463-3467. JNK inhibitors are well-known and various may be obtained from, for example, Celgene, Roche Takeda AZ, Abbott and Merck.

As mentioned herein elsewhere, in addition to the above described and exemplified inhibitors of the various proteins of the MAP3K1 -MAP2K4-JNK pathway, the used inhibitors may also be inhibitors that inhibit (gene) expression of one or more of the proteins of the MAP3K1 - MAP2K4-JNK pathway, for example by interfering with mRNA stability or translation. In one embodiment the MAP3K1 -MAP2K4-JNK pathway inhibitor is selected from small interfering RNA (siRNA), which is sometimes referred to as short interfering RNA or silencing RNA, or short hairpin RNA (shRNA), which is sometimes referred to as small hairpin RNA. The skilled person knows how to design such small interfering nucleotide sequence, for example as described in handbooks such as Doran and Helliwell RNA interference: methods for plants and animals Volume 10 CABI 2009. MAPK pathway and inhibitors

The skilled person is well acquainted with the proteins that form the MAPK pathway and means to inhibit the activity of these proteins. The MAPK pathway comprises four proteins, RAS, RAF, MEK (MEK1 and MEK2) and ERK (MAPK). The MEK-ERK pathway consists of MEK and ERK. The MEK-ERK pathway thus forms a part of the MAPK pathway, and is herein the preferred part of the MAPK pathway. Wherein herein reference is made to the MAPK pathway, it is thus contemplated to also, and preferably, to refer to the MEK-ERK pathway. Activated RAS activates the protein kinase activity of RAF kinase. RAF kinase phosphorylates and activates MEK (MEK1 and MEK2). MEK phosphorylates and activates a mitogen-activated protein kinase (MAPK).

Within the context of the current invention, a MAPK pathway inhibitor or MEK-ERK pathway inhibitor is a compound that specifically or selectively (i.e. selectively reduce the target's activity as compared to off- target signaling activity, via direct or indirect interaction with the target) inhibits signaling through the MAPK pathway (e.g. through affecting signaling mediated by the RAS protein, the RAF protein, the MEK protein (MEK1 and MEK2 proteins) and/or the ERK (MAPK) protein) or MEK-ERK pathway through affecting signaling mediated by the MEK protein and/or the ERK protein). The MAPK or MEK-ERK pathway inhibitor may do so by, for example, reducing the biological activity (e.g. enzymatic activity or ability to phosphorylate substrates) of one or more of the proteins of the pathway (e.g. reducing the enzymatic activity of the RAS protein, RAF protein, MEK protein (MEK1 and MEK2 proteins) and/or ERK (MAPK) protein), or my reducing expression of an mRNA encoding one or more of the proteins of the pathway, or my reducing the expression of one or more of the proteins of the pathway. For example, with the term "a MEK inhibitor", it is meant a compound that may reduce the biological activity of MEK; or that may reduce the expression of an mRNA encoding a MEK polypeptide or protein; or that may reduce the expression of a MEK polypeptide or protein. As will be understood by the skilled person, the above is likewise applicable with respect to inhibitors of the other proteins of the MAPK pathway and the MEK- ERK pathway.

A RAS protein is a polypeptide belonging to the RAS family, more in particular to polypeptides as encoded by HRAS, KRAS, and NRAS in humans. The RAS protein is a GTP-binding protein having the function to transduce signals to e.g. RAF protein in the MAPK signaling pathway.

RAS inhibitors are known to the skilled person. Non-limitative examples include farnesyltransferase inhibitors including SCH66336 (Lonafarnib), R1 15777 (Zarnesta), BMS- 15 214662 and FTI-277, the geranylgeranyltransferase I inhibitor (GGTI)-2166 and transfarnesylthiosalicylic acid (FTS, Salirasib).

A RAF protein is a polypeptide belonging to the RAF kinase family. RAF kinases are a family of three serine/threonine-specific protein kinases that are related to retroviral oncogenes. The three RAF kinase family members are ARAF (A-RAF; for example Genbank Accession NO: NP001243125 ), BRAF (B-RAF; (for example, Genbank Accession NO: NP004324)) and CRAF (C-RAF; (e.g. Gene accession number 5894; Refseq RNA Accessions NM_002880.3 ; protein NP_002871 .1 ), and are well-known to the skilled person. RAF kinase inhibitors are known to the skilled person. Non-limitative examples include the compounds GW5074, BAY 43-9006, CHI R-265 (Novartis), Vemurafenib, PLX4720 (Tsai et al. 2008 PNAS 105(8):3041 ), PLX4032 (RG7204), GDC-0879 (Klaus P. Hoeflich et al. Cancer Res.2009 April 1 ;69:3042-3051 ), sorafenib tosylate (e.g. from Bayer and Onyx Pharmaceuticals as Nexavar), dasatinib (also known as BMS-354825, e.g. as produced by Bristol-Myers Squibb and sold under the trade name Sprycel), AZ628 from Genentech, LGX818 from Novartis, dabrafenib (e.g. Tafinlar™ capsule, made by GlaxoSmithKline, LLC), GSK21 18436, BGB659 (Yao et al. 2015 Cancer Cell 28(3): 370-383), TAK-632 (Takeda), LY3009120 (Peng et al. 2015 Cancer Cell 28(3): 384-398), MLN2480 (Takeda/Millennium), and PLX4032-4720 (Plexxikon).

A MEK polypeptide is a polypeptide having serine/threonine protein kinase activity. For example MEK1 and MEK2 phosphorylates and activates MAPK (ERK). Another example is MEK3. MEK comprises both MEK1 and MEK2: MAP/ERK kinase 1 , MEK1 , PRKMK1 , MAPKK1 , MAP2K1 , MKK1 are the same enzyme, known as MEK1 , MAP/ERK kinase 2, MEK2, PRKMK2, MAPKK2, MAP2K2, M KK2 are the same enzyme, known as M EK2. /pet

Examples of MEK inhibitors, include but are not limited to the MEK inhibitors PD184352 and PD98059, inhibitors of MEKI and MEK2 U0126 (see Favata, M. , et al. , J. Biol. Chem. 273, 18623, 1998) and SL327 (Carr et al Psychopharmacology (Berl). 2009 Jan;201 (4):495-506), and those MEK inhibitors discussed in Davies et al (2000) (Davies et al Biochem J. 351 , 95- 105). Another example is PDI 84352 (Allen, Lee et al Seminars in Oncology, Oct. 2003, pp. 105-106, vol. 30) or trametinib, which has been approved for treatment of BRAF mutant melanoma under the name Mekinist. MEK162 (Novartis) is another example. Other known MEK inhibitors may be selected from PD-325901 (Pfizer), GDC-0973 (cobimetinib)(Genentech), PD-184352 (Allen and Meyer Semin Oncol. 2003 Oct;30(5 Suppl 16): 105-16.), PD- 318088 (Tecle et al Medicinal Chemistry Letters Volume 19, Issue 1 , 1 January 2009, Pages 226-229), AZD6244 (Phase II , Dana Farber, AstraZeneca; WO2007/076245.) and CI-1040 (Lorusso et al Journal of clinical oncology 2005, vol. 23, no23, pp. 5281 -5293), selumetinib (AZD6244), TAK-733, or Honokiol. Preferred examples of MEK inhibitors include Trametinib (GSK), Cobimetinib (GDC-0973) (Genentech/Exelixis), MEK162 (Novartis/Array BioPharma), AZD6244 (AstraZeneca/Array BioPharma), R05126766 (Roche/Chugai), GDC-0623 (Genentech/Chugai), and PD0325901 (Pfizer). An ERK protein is a polypeptide having serine/threonine protein kinase activity, e.g. ERK phosphorylates and activates MAP (microtubule-associated proteins), and having at least 85% amino acid identity to the amino acid sequence of a human ERK, e.g. to ERK1 (e.g. Gene accession number 5595; Refseq RNA Accessions NM_001040056.2; protein NP_001035145.1 ) or ERK2 (e.g. Gene accession number 5594; Refseq RNA Accessions NM_002745.4 ; protein NP_002736.3).

ERK inhibitors are known to the skilled person, and includes such inhibitors as disclosed in WO2002058687, for example SL-327 (Carr et al Psychopharmacology (Berl). 2009 Jan;201 (4):495-5060). Further ERK inhibitors may be found in WO2002058687, AU2002248381 , US20050159385, US2004102506, US2005090536, US2004048861 , US20100004234, HR201 10892, WO201 1 163330, TW200934775, EP2332922, WO201 1041 152, US201 1038876, WO2009146034, HK1 1 17159, WO2009026487, WO20081 15890, US2009186379, WO2008055236, US2007232610, WO2007025090, and US2007049591. Further non-limiting examples or ERK-inhibitors include BVD-523, FR180204 (CAS No. 865362-74-9), Hypothemycin (CAS no. 76958-67-3), MK-8353, SCH9003531 , Pluripotin (CAS no. 839707-37-8), SCH772984 (CAS no. 942183-80-4), and VX-1 1 e (Cas no. 896720-20-0). Preferred examples of ERK inhibitors include SCH772984 (Merck/Schering- Plough), VTX1 1 e (Vertex) and GDC-0994 (Roche/Genentech).

As mentioned herein elsewhere, in addition to the above described and exemplified inhibitors of the various proteins of the MAPK pathway (e.g. comprising or consisting of the RAS protein, RAF protein, M EK protein (MEK1 and MEK2 proteins) and ERK (MAPK) protein) or of the MEK-ERK pathway (e.g. comprising or consisting of the MEK protein and the ERK protein), the used inhibitors may also be inhibitors that inhibit (gene) expression of a protein of the pathway, for example by interfering with mRNA stability or translation. In one embodiment the MAPK pathway inhibitor or MEK-ERK pathway inhibitor is selected from small interfering RNA (siRNA), which is sometimes referred to as short interfering RNA or silencing RNA, or short hairpin RNA (shRNA), which is sometimes referred to as small hairpin RNA. The skilled person knows how to design such small interfering nucleotide sequence, for example as described in handbooks such as Doran and Helliwell RNA interference: methods for plants and animals Volume 10 CABI 2009. The skilled person understands that the amount or dosage as well as the dosage regimen will depend on the (combination of inhibitors) employed (and as described herein). Determining suitable combinations within the context of the present disclosure is within the ordinary skill of the skilled person. For example, in one set of embodiments, one or all inhibitors are administered in a therapeutically effective (i.e. , therapeutic) amount or dosage. A "therapeutically effective amount" or "effective amount" is an amount of the inhibitor that, when administered to a patient by itself, effectively treats the cancer (for example, inhibits tumor growth, stops tumor growth, or causes tumor regression), although such amount may not be effective for 100% of subjects similarly treated for the disease or condition under consideration, even though such dosage is deemed an "effective amount" by the skilled person. In another set of embodiments, one or all inhibitors are administered in a sub- therapeutically effective amount or dosage. A sub-therapeutically effective amount is an amount of the inhibitor that, when administered to a patient by itself, does not completely inhibit over time the biological activity of the intended target to an extent to be considered "therapeutically effective" on its own. Whether administered in therapeutic or sub-therapeutic amounts, the inhibitors or combination of inhibitors, as disclosed herein, should be effective in treating the cancer. A sub-therapeutic amount of first inhibitor can be an effective amount if, when combined with the second inhibitor, the combination is effective in the treatment of a cancer. In some embodiments, the combination of compounds exhibits a synergistic effect (i.e. , greater than additive effect) in treating the cancer. In different embodiments, depending on the combination and the effective amounts used, the combination of compounds can inhibit tumor growth, achieve tumor stasis, or even achieve substantial or complete tumor regression.

As shown in the Examples, it has surprisingly be found that inhibiting signaling through both the MAP3K1 -MAP2K4-JNK pathway (e.g. through affecting signaling mediated by the MAP3K1 protein, the MAP2K4 protein, the JNK1 protein, and/or the JNK2 protein) and through the MAPK pathway (e.g. through affecting signaling mediated by the RAS protein, the RAF protein, the MEK proteins (M EK1 and MEK2 proteins ) and the ERK (MAPK) protein), in particular the MEK-ERK pathway (e.g. through affecting signaling mediated by the MEK protein and/or the ERK protein), i.e. by an, or at least one, inhibitor of one or more of the proteins of the MAP3K1 -MAP2K4-JNK pathway (e.g. the MAP3K1 protein, the MAP2K4 protein, the JNK1 protein, and/or the JNK2 protein) and an, or at least one, inhibitor of one or more of the proteins of the MEK-ERK pathway (e.g. the MEK protein and/or the ERK protein), shows surprising desirable response of cancer cells, or tumors, in particular in cases wherein such cancer cells, or tumor, does not, or only modestly respond to treatment with a MEK-ERK pathway inhibitor alone (as detailed herein). Combined inhibition of the MAP3K1 -MAP2K4- JNK pathway (said pathways comprising or consisting of the MAP3K1 protein, the MAP2K4 protein, the JNK1 protein, and/or the JNK2 protein) and the MEK-ERK pathway (e.g. said pathway comprising or consisting of the MEK protein and/or the ERK protein) thus allows for the treatment of cancer, in particular of cancers that do not, or only modestly respond to treatment with a MEK-ERK pathway inhibitor alone. Another consequence of the current invention is that it explains previous disappointing effects with treatment with MEK-ERK pathway inhibitors alone in various cancers and re-opens the possibility of using MEK-ERK pathway inhibitors, for example MEK inhibitors, in such cases, by combining with MAP3K1 -MAP2K4-JNK pathway inhibition. In particular it was found that the combination of an inhibitor of one or more proteins of the MAP3K1 -MAP2K4-JNK pathway and an inhibitor of one or more of the proteins of the MAPK pathway, in particular the MEK-ERK pathway allows the treatment of cancers or tumors that are characterized by the presence, in its genetic material, of a gene that encodes a functional MAP2K4 protein (wherein the MAP2K4 protein has enzymatic activity/is capable of phosphorylating serine and threonine in its substrates, typically in p38 MAPK and JNK), preferably wherein the gene encodes a wild-type MAP2K4 protein (e.g. wherein the wild-type MAP2K4 protein has enzymatic activity/is capable of phosphorylating serine and threonine in its substrates, typically in p38 MAPK and JNK) and a gene that encodes a functional MAP3K1 protein (wherein the MAP3K1 protein has enzymatic activity or is capable of phosphorylating MAP2K4), preferably wherein the gene encodes a wild-type MAP3K1 protein (wherein the wild-type MAP3K1 protein has enzymatic activity/is capable of phosphorylating MAP2K4).

As detailed elsewhere herein, genetic alterations in the MAP3K1 and MAP2K4 genes have been recently found in comprehensive genomic analyses of invasive lobular breast cancers (Michaut et al. (2016) Scientific Reports | 6: 18517 | DOI: 10.1038/srep18517). Such genetic alterations include deletion, nonsense mutations and mutations that result in a frame-shift deletion or insertion, and are predicted to be inactivating (e.g. causing loss of function), creating non-functional MAP3K1 protein (e.g. wherein the MAP3K1 protein has lost its enzymatic activity or has lost its ability to phosphorylate MAP2K4) or MAP2K4 protein (e.g. wherein the MAP2K4 protein has lost its enzymatic activity or has lost its ability to phosphorylate serine and threonine in its substrates, typically in p38 MAPK and JN K), thereby causing reduced or absent functional signaling through the MAP3K1 -MAP2K4-JNK pathway. Down-regulation, loss of function mutations, and homozygous deletion of MAP3K1 and MAP2K4 has also been described by others in other cancers.

As shown in the Examples, it was surprisingly found that cancers characterized by reduced (e.g. less than 40%, 30%, 20%, 10%, 5% of normal activity) or absent signaling activity through the MAP3K1 -MAP2K4-JNK pathway respond well to treatment with a MEK-ERK pathway protein inhibitor, whereas cancers that express functional MAP2K4 proteins and functional MAP3K1 proteins, and thus display a functional MAP3K1 -MAP2K4-JNK pathway do not, or hardly, respond to treatment with a MEK-ERK pathway protein inhibitor. Cancers that comprise a gene that encodes a functional MAP2K4 protein and MAP3K1 protein, and thus express functional MAP2K4 and functional MAP3K1 proteins (as shown by normal or significant signaling activity through the MAP3K1 -MAP2K4-JNK pathway) do, surprisingly, respond well to the MEK-ERK pathway inhibitor when at the same time the MAP3K1 - MAP2K4-JNK pathway is inhibited. Therefore there is also provided for the combination of an inhibitor of one or more of the proteins of the MAP3K1 -MAP2K4-JNK pathway and an inhibitor of one or more of the proteins of the MEK-ERK pathway for use as a medicament, in particular in the treatment of cancer, wherein the subject or cancer in the subject is characterized by a gene that encodes a functional MAP2K4 protein, preferably wherein the gene encodes a wild-type MAP2K4 protein and a gene that encodes a functional MAP3K1 protein, preferably wherein the gene encodes a wild-type MAP3K1 protein (e.g. wherein the wild-type MAP3K1 protein has enzymatic activity or is capable of phosphorylating MAP2K4). Likewise there is provided for the combination of an inhibitor of a one or more of the proteins of the MAP3K1 -MAP2K4-JNK pathway and an inhibitor of one or more of the proteins of the MEK-ERK pathway for use as a medicament, in particular in the treatment of cancer, wherein the subject or cancer in the subject is characterized by the expression of functional MAP2K4 protein (e.g. wherein the MAP2K4 protein has enzymatic activity or is capable of phosphorylating serine and threonine in its substrates, typically in p38 MAPK and JNK, wherein said MAP2K4 protein is preferably a wild-type MAP2K4 protein having these characteristics, and wherein the gene encoding said MAP2K4 protein does not comprise or contain loss of function mutation(s) causing loss of function, e.g. lost of enzymatic activity or phosphorylation activity) and the expression of functional MAP3K1 protein (e.g. wherein the MAP3K1 protein has enzymatic activity or is capable of phosphorylating MAP2K4, wherein said MAP3K1 protein is preferably a wild-type MAP3K1 protein having these characteristics, and wherein the gene encoding said MAP3K1 protein does not comprise or contain loss of function mutation(s) causing loss of function, e.g. lost of enzymatic activity or phosphorylation activity).

The skilled person knows how to determine if a subject or patient, or the cancer in the patient comprises a gene that encodes for a functional MAP2K4 protein (as defined herein) and/or if a gene encodes for a functional MAP3K1 protein (as defined herein), or not. Likewise, the skilled person knows how to determine if a subject or patient, or the cancer in the patient expresses a functional MAP2K4 protein and/or a functional MAP3K1 protein (as defined herein). The skilled person may, for example, determine the sequence, or part of the sequence of the genes encoding the MAP2K4 protein and/or the MAP3K1 protein and analyze the sequence for the presence of loss-of-function mutations (e.g. mutations such as deletion or truncation causing a loss of function such as lost of enzymatic activity or lost of the ability to phosphorylate substrates). Alternatively the skilled person may, using an enzyme assay, determine the activity of the MAP2K4 protein (e.g. determining the enzymatic activity of the MAP2K4 protein in terms of its ability to phosphorylate serine and threonine in its substrates, typically in p38 MAPK and JNK) and/or the MAP3K1 protein (e.g. determining its enzymatic activity of the MAP3K1 protein in terms of its ability to phosphorylate MAP2K4) in material obtained from the subject or cancer in the subject and compare the activity determined with activity obtained from one or more control samples.

After establishing that the subject or cancer in the subject comprises a gene encoding for a functional MAP2K4 protein and MAP3K1 protein, the patient may be treated with the combination of an inhibitor of one or more of the proteins of the MAP3K1 -MAP2K4-JNK pathway and an inhibitor of one or more of the proteins of the MEK-ERK pathway. It is noted that treatment with the combination of an inhibitor of one or more of the proteins of the MAP3K1 -MAP2K4-JNK pathway and an inhibitor of one or more of the proteins of the MAPK pathway, in particular the MEK-ERK pathway, is not limited to subjects or cancers in subjects that display functional MAP2K4 and MAP3K1 proteins of genes , but, as will be understood by the skilled person, is highly preferred.

Although not limited thereto, in an embodiment the cancer in the subject is breast cancer, including invasive lobular breast cancer, colon cancer, pancreatic cancer or lung cancer.. In particular such cancer, and in particular such cancers, characterized by functional MAP3K1 - MAP2K4-JNK pathway (e.g. functional MAP3K1 and MAP2K4 protein, as discussed herein elsewhere), can advantageously be treated with the combination of an inhibitor of one or more of the proteins of the MAP3K1 -MAP2K4-JNK pathway and an inhibitor of one or more of the proteins of the MEK-ERK pathway, in particular a M EK inhibitor.

Although not limited thereto, in an embodiment the cancer in the subject is a KRAS mutated cancer, in particular a KRAS-mutated lung cancer, pancreatic cancer or a KRAS-mutated colon cancer. As described herein elsewhere, in both pre-clinical models of cancer and in clinical trials, the results obtained with MAPK pathway inhibitors, in particular MEK and ERK inhibitors, have been disappointing, in particular in KRAS mutated cancers. The current disclosure surprisingly shows that such inhibitors, in particular inhibitors of the MEK-ERK pathway, may be effective in KRAS-mutated cancers, and in particular such cancers, characterized by functional MAP3K1 -MAP2K4-JNK pathway (e.g. functional MAP3K1 and MAP2K4 protein, as discussed herein elsewhere), when combined with inhibition of the MAP3K1 -MAP2K4-JNK pathway, e.g. with an inhibitor of one or more of the proteins of the MAP3K1 -MAP2K4-JNK pathway.

The term "KRAS-mutated cancer" is well known to the skilled person. A comprehensive overview of RAS mutations, including KRAS-mutations, in cancer was reported by Prior et al (2012) Cancer Res; 2457 - 67. KRAS-mutant cells promote oncogenesis due to being mutationally activated, in most cases, at codon 12, 13 and 61 . In total forty-four separate point mutations have been characterized in RAS isoforms, with 99.2% in codons 12, 13 and 61. The protein product of the normal KRAS gene performs an essential function in normal tissue signaling, and the mutation of a KRAS gene is an essential step in the development of many cancers. It is preferred the inhibitor of the protein of the MAP3K1 -MAP2K4-JNK pathway is an inhibitor of MAP2K4 and/or an inhibitor of MAP3K1 . It was found that, in particular inhibiting activity of these proteins is effective in the combination with the MEK-ERK pathway inhibitor.

In a preference, the inhibitor of the protein of the MEK-ERK pathway is an inhibitor of MEK and/or an inhibitor of ERK, preferably wherein the inhibitor is trametinib, selumetinib, cobimetinib, binimetinib or pimasertib. More preferably, the inhibitor is a MEK inhibitor.

As defined herein elsewhere, inhibitors are not limited to compounds that directly inhibit the activity of the targeted protein, but also include compounds that specifically or selectively inhibit the protein by inhibiting expression of the protein and/or transcription of the gene encoding the protein and/or translation of the transcript of the gene encoding the protein. Therefore, there is also provided for the combination of an inhibitor of one or more of the proteins of the MAP3K1 -MAP2K4-JNK pathway and an inhibitor of one or more of the proteins of the MEK-ERK pathway wherein the inhibitor of the protein of the MAP3K1 - MAP2K4-JNK pathway and/or the inhibitor of the MEK-ERK pathway inhibits expression of the protein and/or transcription of the gene encoding the protein and/or translation of the transcript of the gene encoding the protein, or wherein the inhibitor of the protein inhibits the enzymatic activity of said protein. The skilled person knows how to determine whether a compound (selectively of specifically) inhibits expression of the protein and/or transcription of the gene encoding the protein and/or translation of the transcript of the gene encoding the protein. In addition, and as mentioned herein, the skilled person understand how to provide for a compound that inhibits by inhibiting expression of the protein and/or transcription of the gene encoding the protein and/or translation of the transcript of the gene encoding the protein, for example by providing a small interfering RNA (siRNA), which is sometimes referred to as short interfering RNA or silencing RNA, or short hairpin RNA.

Although not limited thereto, in some embodiment the cancer in the subject that is to be treated with the combination of an inhibitor of one or more of the proteins of the MAP3K1 - MAP2K4-JNK pathway and an inhibitor of one or more of the proteins of the MEK-ERK pathway is a cancer that is or has acquired resistance to a MEK inhibitor and/or an ERK inhibitor. In particular the cancer that is or has acquired resistance to a MEK inhibitor and/or an ERK inhibitor is further characterized by the presence of a gene that encodes a functional MAP2K4 protein, preferably wherein the gene encodes a wild-type MAP2K4 protein and a gene that encodes a functional MAP3K1 protein, preferably wherein the gene encodes a wild- type MAP3K1 protein, or is characterized by the expression of functional MAP2K4 protein and the expression of functional MAP3K1 protein, for the reasons already discussed herein elsewhere.

The MAPK pathway is activated in more than 30% of human cancers, most commonly via mutation in the KRAS oncogene. In particular MEK inhibitors and ERK inhibitors are considered for use in the treatment of these cancers. However, (acquired) resistance to these inhibitors has been documented both in preclinical and clinical samples, and the current invention provides a new strategy to overcome this resistance, i.e. by providing a combination of an inhibitor of one or more of the proteins of the MAP3K1 -MAP2K4-JNK pathway and an inhibitor of one or more of the proteins of the MEK-ERK pathway.

Also provided is an inhibitor of one or more of the proteins of the MAP3K1 -MAP2K4-JNK pathway (e.g. the MAP3K1 protein, the MAP2K4 protein, the JNK1 protein, and/or the JNK2 protein) for use in the treatment of cancer in a subject, wherein the inhibitor is administrated simultaneously, separately or sequentially with an inhibitor of one or more of the proteins of the MEK-ERK pathway (e.g. the MEK protein and/or the ERK protein). Likewise there is provided for an inhibitor of one or more of the proteins of the MEK-ERK pathway (e.g. the MEK protein and/or the ERK protein) for use in the treatment of cancer in a subject, wherein the inhibitor is administrated simultaneously, separately or sequentially with an inhibitor of one or more of the proteins of the MAP3K1 -MAP2K4-JNK pathway (e.g. the MAP3K1 protein, the MAP2K4 protein, the JN K1 protein, and/or the JN K2 protein).

Depending on the inhibitors employed, the skilled person understands the inhibitors may be provided to the subject either in the same formulation or in different formulations. The inhibitors may be provided via the same route of administration or via different routes of administration. The inhibitors may be provided at the same time or at different moments during the treatment with the combination.

As can be witnessed from the Examples, it was surprisingly found that inhibiting the MAP3K1 - MAP2K4-JNK pathway (e.g. said pathway comprising or consisting of the MAP3K1 protein, the MAP2K4 protein, the JNK1 protein, and the JNK2 protein), for example in cancers characterized by functional MAP2K4 and MAP3K1 proteins, causes the cells to become sensitized to a MEK-ERK pathway inhibitor, specifically a MEK inhibitor or an inhibitor of ERK. In contrast these cells do not, or hardly respond to the MEK or ERK inhibitor under conditions the MAP3K1 -MAP2K4-JNK pathway is functional or not inhibited. Therefore, there is provided for an inhibitor of one or more of the proteins of the MAP3K1 -MAP2K4-JNK pathway (e.g. the MAP3K1 protein, the MAP2K4 protein, the JNK1 protein, and the JNK2 protein) for use in sensitizing a cell in vivo, preferably a cancer cell (e.g. present in a subject, e.g. human), even more preferably a KRAS-mutated cancer cell in vivo, e.g. present in a subject such as a human, to a MEK inhibitor and/or an ERK inhibitor.

In line therewith, there is provided for an in vitro method, the method being performed outside the subject (for example, in vitro) when the subject is a human, for sensitizing a cancer, e.g. cancer cells cultured or maintained in vitro, to a MEK or ERK inhibitor, the method comprising inhibiting the MAP3K1 -MAP2K4-JNK pathway (e.g. said pathway comprising or consisting of the MAP3K1 protein, the MAP2K4 protein, the JNK1 protein, and the JNK2 protein).

Therefore, there is provided for the use of an inhibitor of one or more of the proteins of the MAP3K1 -MAP2K4-JNK pathway (e.g. the MAP3K1 protein, the MAP2K4 protein, the JNK1 protein, and the JNK2 protein) for sensitizing a cell in vitro, preferably a cancer cell (e.g. cultured in vitro), even more preferably a KRAS-mutated cancer cell in vitro, e.g. cultured in vitro, to a MEK inhibitor and/or an ERK inhibitor. Also provided is a product, preferably a pharmaceutical product, which comprises an inhibitor of a protein of the MAP3K1 -MAP2K4-JNK pathway and an inhibitor of a protein of the MEK- ERK pathway, as a combined preparation for simultaneous, separate or sequential use in the treatment of cancer in a subject. The product thus comprises the combination of inhibitors, although the inhibitors may be present in the product separate from each other, for example in separate compartments (e.g. in separate blisters or sachets or other forms).

As can be witnessed from the Examples, it was found that, depending whether a cancer cell carries a functional MAP3K1 -MAP2K4-JNK pathway, for example as exemplified by the presence of functional MAP2K4 protein and MAP3K1 protein, the cells are sensitive or not to treatment with an inhibitor of (a protein of) the MAPK pathway, in particular with a MEK or ERK inhibitor.

In particular it was found that cancer or cancers cells characterized by a gene that encodes a non-functional MAP2K4 protein and/or a gene that encodes a non-functional MAP3K1 protein, or characterized by the expression of non-functional MAP2K4 protein and/or the expression of non-functional MAP3K1 protein, or characterized by reduced expression of functional MAP2K4 protein and/or reduced expression of functional MAP3K1 protein, form a defined subgroup that respond well to treatment (e.g. monotherapy) with an inhibitor of one or more of the proteins of the MAPK pathway, or MEK-ERK pathway, in particular an inhibitor of MEK or ERK.

Consequently, it was now for the first time surprisingly found that this particular and defined group of cancer patients may benefit form therapy, e.g. monotherapy, with an inhibitor of a protein of the MEK-ERK pathway. This group of patients has not been identified before as a specific group of patients within the population, as a group that would show better response to the treatment with an inhibitor of one or more of the proteins of the MEK-ERK pathway, in particular an ERK inhibitor or a MEK inhibitor. Thus with the current invention overall treatment of cancer can be improved by treating this group of patients with an inhibitor of a protein of the MEK-ERK pathway, in particular a MEK inhibitor or an ERK inhibitor.

Therefore, according to another aspect of the invention, there is provided for an inhibitor of one or more of the proteins of the MEK-ERK pathway for use in the treatment of cancer in a subject wherein the subject or cancer in the subject is characterized by a gene that encodes a non-functional MAP2K4 protein and/or a gene that encodes a non-functional MAP3K1 protein. Likewise there is provided for an inhibitor of one or more of the proteins of the MEK- ERK pathway for use in the treatment of cancer in a subject, wherein the subject or cancer in the subject is characterized by the expression of non-functional MAP2K4 protein and/or the expression of non-functional MAP3K1 protein, or wherein the subject or cancer in the subject is characterized by reduced expression of functional MAP2K4 protein and/or reduced expression of functional MAP3K1 protein. Preferably the inhibitor is a MEK inhibitor or an ERK inhibitor. Preferably the cancer is a KRAS-mutated cancer. Preferably the cancer is a colon cancer, lung cancer or breast cancer. The cancer may be a cancer that has or has acquired resistance to treatment with a MEK inhibitor or an ERK inhibitor. Indeed the skilled person will understand that preferences and explanations described within the context of other aspect or embodiments described herein likewise apply to the above aspect and embodiments of the current invention.

As discussed herein elsewhere, the results obtained by the current inventors brought forward the unexpected relationship between the presence of mutations (e.g. mutations causing loss of function as deletion truncation mutations causing loss of enzymatic activity or loss ability to phosphorylate substrates) in genes encoding MAP3K1 protein and/or the MAP2K4 protein and response of cancer cells, including KRAS-mutated cancer cells, to treatment with inhibitors of proteins of the MEK-ERK pathway, in particular inhibitors of ERK and/or MEK. Directly connected thereto, and within the same inventive concept, it was found that in cancer cells with a functional MAP3K1 -MAP2K4-JNK pathway, several receptor tyrosine kinases where activated upon treatment with MEK-ERK pathway inhibitors (likely via activation of JUN by JNK), whereas reduced of no activation was seen in cancer cells with non-functional MAP3K1 protein and/or non-functional MAP2K4 protein. In line therewith it was found that combining an inhibitor of such receptor tyrosine kinase and an inhibitor of one or more of the proteins of the MEK-ERK pathway, in particular a MEK inhibitor restored response to treatment in cancer cells that have functional MAP3K1 and MAP2K4 protein.

Thus according this aspect of the invention, there is provided for a combination of an inhibitor of a receptor tyrosine kinase, preferably wherein the inhibitor is an inhibitor of epidermal growth factor receptor, platelet-derived growth factor receptor (beta), and/or human epidermal growth factor receptor (for example EGFR (epidermal growth factor receptor), HER (human epidermal growth factor receptor) 2, HER3 or HER 4) and an inhibitor of one or more of the proteins of the MEK-ERK pathway, in particular an inhibitor of MEK and/or ERK, for use in the treatment of cancer in a subject, wherein the subject or cancer in the subject is characterized by a gene that encodes a functional MAP2K4 protein, preferably wherein the gene encodes a wild-type MAP2K4 protein and a gene that encodes a functional MAP3K1 protein, preferably wherein the gene encodes a wild-type MAP3K1 protein. In a preferred embodiment, the subject or cancer in the subject is characterized by the expression of functional MAP2K4 protein and the expression of functional MAP3K1 protein.

Preferably the inhibitor is a MEK inhibitor or an ERK inhibitor. The skilled person is well aware of the role different receptor tyrosine kinase play in cancer, and of inhibitors of such kinases.

Within the context of the current invention, an inhibitor of a receptor tyrosine kinase, preferably an inhibitor of epidermal growth factor receptor, platelet-derived growth factor receptor (beta), and/or human epidermal growth factor receptor (for example EGFR, HER2, HER3 or HER 4) is a compound that specifically or selectively (i.e. selectively reduce the target's activity as compared to off- target signaling activity, via direct or indirect interaction with the target) inhibits activity of such kinase. The inhibitor may do so by, for example, reducing the biological activity of the receptor tyrosine kinase, epidermal growth factor receptor, platelet-derived growth factor receptor (beta), and/or human epidermal growth factor receptor (for example EGFR, HER2, HER3 or HER 4), or my reducing expression of an mRNA encoding such kinase, or my reducing the expression of such kinase. For example, with a HER2 inhibitor is meant a compound that may reduce the biological activity of HER2; or that may reduce the expression of an mRNA encoding a HER2 polypeptide; or that may reduce the expression of a HER2 polypeptide. As will be understood by the skilled person, the above is likewise applicable with respect to inhibitors of other receptor tyrosine kinases, preferably epidermal growth factor receptor, platelet-derived growth factor receptor (beta), and/or human epidermal growth factor receptor (for example EGFR, HER2, HER3 or HER 4).

Preferably the inhibitor of a receptor tyrosine kinase, preferably an inhibitor of epidermal growth factor receptor family, (for example EGFR, HER2, HER3 or HER 4) is dacomitinib, lapatinib, neratinib, afatinib.

As mentioned herein elsewhere, in addition to the above described and exemplified inhibitors of receptor tyrosine kinase, preferably of epidermal growth factor receptor, platelet-derived growth factor receptor (beta), and/or human epidermal growth factor receptor (for example EGFR, HER2, HER3 or HER 4), the used inhibitors may also be inhibitors that inhibit (gene) expression of such kinase, for example by interfering with mRNA stability or translation. In one embodiment such inhibitor is selected from small interfering RNA (siRNA), which is sometimes referred to as short interfering RNA or silencing RNA, or short hairpin RNA (shRNA), which is sometimes referred to as small hairpin RNA. The skilled person knows how to design such small interfering nucleotide sequence, for example as described in handbooks such as Doran and Helliwell RNA interference: methods for plants and animals Volume 10 CABI 2009.

Preferably the cancer is a KRAS-mutated cancer. Preferably the cancer is a colon cancer, lung cancer or breast cancer. The cancer may be a cancer that has or has acquired resistance to treatment with a M EK inhibitor or an ERK inhibitor. Indeed the skilled person will understand that preferences and explanations described within the context of other aspects or embodiments described herein likewise apply to the above aspect and embodiments of the current invention. Thus, for example, also provided is an inhibitor of a receptor tyrosine kinase, preferably wherein the inhibitor is an inhibitor of epidermal growth factor receptor (for example EGFR, HER2, HER3 or HER 4) for use in the treatment of cancer in a subject, wherein the inhibitor is administrated simultaneously, separately or sequentially with an inhibitor of one or more of the proteins of the MEK-ERK pathway, and wherein the subject or cancer in the subject is characterized by a gene that encodes a functional MAP2K4 protein, preferably wherein the gene encodes a wild-type MAP2K4 protein and a gene that encodes a functional MAP3K1 protein, preferably wherein the gene encodes a wild-type MAP3K1 protein, or wherein the subject or cancer in the subject is characterized by the expression of functional MAP2K4 protein and the expression of functional MAP3K1 protein.

Thus, for example, also provided is an inhibitor of one or more of the proteins of the MEK- ERK pathway for use in the treatment of cancer in a subject, wherein the inhibitor is administrated simultaneously, separately or sequentially with an inhibitor of a receptor tyrosine kinase, preferably wherein the inhibitor is an inhibitor of epidermal growth factor receptor family (for example EGFR, HER2, HER3 or HER 4), and wherein the subject or cancer in the subject is characterized by a gene that encodes a functional MAP2K4 protein, preferably wherein the gene encodes a wild-type MAP2K4 protein and a gene that encodes a functional MAP3K1 protein, preferably wherein the gene encodes a wild-type MAP3K1 protein, or wherein the subject or cancer in the subject is characterized by the expression of functional MAP2K4 protein and the expression of functional MAP3K1 protein. According to another aspect of the current invention, there is provided for a method for predicting treatment response of a cancer in a subject, preferably a KRAS-mutated cancer.

In particular the method of the current invention provides for a marker that is predictive of response to treatment, preferably monotherapy treatment with an inhibitor of one or more of the proteins of the MEK-ERK pathway, in particular a MEK inhibitor and/or a ERK inhibitor.

As witnessed from the Examples, it was surprisingly found that cancers in subjects, wherein the cancer is characterized by the presence of a gene that encodes a functional MAP2K4 protein, preferably wherein the gene encodes a wild-type MAP2K4 protein and a gene that encodes a functional MAP3K1 protein, preferably wherein the gene encodes a wild-type MAP3K1 protein or wherein the cancer in the subject is characterized by the expression of functional MAP2K4 protein and the expression of functional MAP3K1 protein do not respond well to treatment with a MEK or ERK inhibitor alone but do respond to the combination of such MEK or ERK inhibitors in the combination as disclosed herein.

In contrast, cancers in subjects, wherein the cancer is characterized by the presence of a gene that encodes a non-functional MAP2K4 protein and/or a gene that encodes a nonfunctional MAP3K1 protein or wherein the cancer in the subject is characterized by the expression of non-functional MAP2K4 protein and/or the expression of non-functional MAP3K1 protein do respond well to treatment with a MEK or ERK inhibitor alone, i.e. to monotherapy. In other words, analysis of the gene(s) encoding the MAP2K4 protein and/or the MAP3K1 protein, or the protein as expressed by the subject, or the cancer in the subject may be used to predict responsiveness of the patient to a given treatment.

Therefor the is provided for the use of MAP2K4 protein and/or MAP3K1 protein, or genes encoding such proteins, or mRNA transcripts thereof, for predicting response to treatment with a MEK inhibitor or an ERK inhibitor.

Therefore there is provided for a method for predicting treatment response of a cancer in a subject, preferably a KRAS-mutated cancer and wherein the treatment comprises a) monotherapy treatment with a M EK inhibitor or a ERK inhibitor; or

b) combination treatment with an inhibitor of one or more of the proteins of the MAP3K1 -MAP2K4-JNK pathway (e.g. the MAP3K1 protein, the MAP2K4 protein, the JNK1 protein, and/or the JNK2 protein) and an inhibitor of one or more of the proteins of the MEK- ERK pathway (e.g. the MEK protein and/or the ERK protein); or

c) combination treatment with an inhibitor of one or more of the proteins of the MAP3K1 -MAP2K4-JNK pathway (e.g. the MAP3K1 protein, the MAP2K4 protein, the JNK1 protein, and/or the JNK2 protein) and an inhibitor of a receptor tyrosine kinase, preferably wherein the inhibitor is an inhibitor of epidermal growth factor receptor family proteins, such as EGFR, HER2, H ER 3 or HER 4, wherein the method comprises the step of determining in tissue or cells obtained from said subject, in particular in cancer cells from said subject, the presence of -functional MAP2K4 protein and/or non-functional MAP2K4 protein;

-functional MAP3K1 protein and/or non-functional MAP3K1 protein; and/or

-genes encoding such functional protein or non-functional protein, or mRNA transcripts thereof wherein

-absence or reduced presence of a functional protein, or a gene encoding such functional protein, or mRNA transcript thereof, is predictive for good response to monotherapy treatment with a MEK inhibitor or an ERK inhibitor

-presence of a functional protein, or a gene encoding such functional protein, or mRNA transcript thereof, is predictive for poor response to monotherapy treatment with a MEK inhibitor or an ERK inhibitor; -presence of a functional protein, or a gene encoding such functional protein, or mRNA transcript thereof is predictive for good response to combination treatment with an inhibitor of one or more of the proteins of the MAP3K1 -MAP2K4-JNK pathway and an inhibitor of one or more of the proteins of the MEK-ERK pathway;

-presence of a functional protein, or a gene encoding such functional protein, or mRNA transcript thereof is predictive for good response to combination treatment with an inhibitor of one or more of the proteins of the MAP3K1 -MAP2K4-JNK pathway and an inhibitor of a receptor tyrosine kinase, preferably wherein the inhibitor is an inhibitor of epidermal growth factor receptors, (e.g. EGFR, HER2, HER3 and/or HER4); and/or

-presence of non-functional protein, or a gene encoding such non-functional protein, or mRNA transcript thereof, is predictive for good response to monotherapy treatment with a MEK inhibitor or an ERK inhibitor.

The skilled person is well aware of the method to determine the presence of absence of such gene, mRNA, or functional or non-functional protein (e.g. having less than 40%, 30%, 20%, 10%, 5% of activity of wild-type functional protein in the cell).

Thus, MAP2K4 and/or MAP3K1 genes, mRNA and proteins serve as prognostic markers for efficacy of MEK inhibitors or ERK inhibitors in patients suffering from cancer. Efficacy of M EK inhibitors and ERK inhibitors can be prognosticated in a patient suffering from cancer by obtaining a biological sample from the patient and then analyzing the sample for the presence or absence of functional or non-functional MAP2K4 and/or MAP3K1 protein, or genes encoding such protein, and mRNA transcripts thereof. In a preferred embodiment, the sample is analyzed for the presence of mutant (for instance a deletion or truncation mutation causing a loss of function), i.e. non-functional MAP2K4 and/or MAP3K1 (protein, genes or mRNA) as defined herein.

In another preferred embodiment, the sample is analyzed for the presence of functional MAP2K4 and MAP3K1 (protein, genes or mRNA). The presence of non-functional MAP2K4 and/or MAP3K1 , in the biological sample of the patient is indicative of good response to treatment with a MEK inhibitor or a ERK inhibitor. The presence of functional MAP2K4 and/or MAP3K1 , in the biological sample of the patient is indicative of non or reduced response to treatment with a MEK inhibitor or a ERK inhibitor and warrant treatment with the combinations as disclosed herein. Accordingly, treatment regimens should be selected for the patients. Biological samples which can be screened are samples containing DNA, mRNA and/or protein. Examples include, but are not limited to, tumor biopsy samples and blood or serum samples obtained from the patient. Preferably the cancer is a KRAS-mutated cancer. Preferably the cancer is a colon cancer, lung cancer or breast cancer. The cancer may be a cancer that has or has acquired resistance to treatment with a M EK inhibitor or an ERK inhibitor. Indeed the skilled person will understand that preferences and explanations described within the context of other aspects or embodiments described herein likewise apply to the above aspect and embodiments of the current invention.

Also provided is a method of treating cancer in a subject, preferably KRAS-mutated cancer, wherein the method comprises the simultaneous, separate or sequential administering to the subject of an inhibitor of one or more of the proteins of the MAP3K1 -MAP2K4-JNK pathway and an inhibitor of one or more of the proteins of the MEK-ERK pathway or the simultaneous, separate or sequential administering to the subject of an inhibitor of one or more of the proteins of the MEK-ERK pathway and an inhibitor of a receptor tyrosine kinase, preferably wherein the inhibitor is an inhibitor of epidermal growth factor receptor, platelet-derived growth factor receptor, and/or human epidermal growth factor receptor.

Preferably the subject or cancer in the subject is characterized by a gene that encodes a functional MAP2K4 protein, preferably wherein the gene encodes a wild-type MAP2K4 protein and a gene that encodes a functional MAP3K1 protein, preferably wherein the gene encodes a wild-type MAP3K1 protein. Preferably the subject or cancer in the subject is characterized by the expression of functional MAP2K4 protein and/or the expression of functional MAP3K1 protein.

Also provided is a method of treating cancer in a subject, preferably KRAS-mutated cancer, wherein the method comprises the simultaneous, separate or sequential administering to the subject of an inhibitor of one or more of the proteins of the MAP3K1 -MAP2K4-JNK pathway (e.g. the MAP3K1 protein, the MAP2K4 protein, the JNK1 protein, and/or the JNK2 protein) and an inhibitor of one or more of the proteins of the MEK-ERK pathway (e.g. (e.g. the MEK protein and/or the ERK protein) or the simultaneous, separate or sequential administering to the subject of an inhibitor of one or more of the proteins of the MEK-ERK pathway (e.g. the MEK protein and/or the ERK protein) and an inhibitor of a receptor tyrosine kinase, preferably wherein the inhibitor is an inhibitor of epidermal growth factor receptor, platelet-derived growth factor receptor, and/or human epidermal growth factor receptor, and wherein the subject or cancer in the subject is characterized by a gene that encodes a functional MAP2K4 protein, preferably wherein the gene encodes a wild-type MAP2K4 protein (as defined herein) and a gene that encodes a functional MAP3K1 protein, preferably wherein the gene encodes a wild-type MAP3K1 protein (as defined herein). Preferably the subject or cancer in the subject is characterized by the expression of functional MAP2K4 protein and/or the expression of functional MAP3K1 protein (as defined herein).

Preferably the cancer is a KRAS-mutated cancer. Preferably the cancer is a colon cancer, lung cancer or breast cancer. The cancer may be a cancer that has or has acquired resistance to treatment with a MEK inhibitor or an ERK inhibitor. Indeed the skilled person will understand that preferences and explanations described within the context of other aspects or embodiments described herein likewise apply to the above aspect and embodiments of the current invention.

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. Examples

(References cited in the example are listed at the end of the example)

Example 1

Introduction.

The genetic aberrations that lie at the heart of the cancerous process create a dependency on these aberrations, a situation referred to as "oncogene addiction" (1 ). Inhibition of these signals using drugs that selectively inhibit these so called "driver" pathways often leads to massive clinical responses. It is estimated that over 30% of all human cancers are driven by mutations in RAS genes. For example, some 95% of pancreatic cancers, 45% of colorectal cancers and 35% of lung cancers have mutations in the KRAS gene (2). In spite of massive efforts, RAS proteins have resisted drug development efforts (3). RAS proteins connect growth factor signaling to multiple downstream pathways, including the RAF-MEK-ERK pathway (also known as the mitogen activated protein kinase (MAPK) pathway) and the Pl- 3Kinase (PI-3K) pathway. These pathways contribute to oncogenesis through stimulation of cell proliferation and escape from apoptosis.

Given the "undruggable" nature of RAS proteins, drug development efforts have focused on the kinases in the pathways downstream of RAS, including the MEK kinases. However, in both pre- clinical models of cancer and in clinical trials, the results obtained with these MEK inhibitors have been disappointing. The only notable exception is the use of M EK inhibitors in BRAF and NRAS mutant melanomas (4-6). Thus, identifying predictive biomarkers for MEK inhibitor response and potential combination therapies that enhance MEK inhibitor effectiveness are essential for the future clinical use of inhibitors of the MAPK pathway, including MEK and/or ERK inhibitors.

The current inventors and others have recently described the identification of oncogenic driver mutations in, for example, invasive lobular breast cancers (I LCs). Both studies identified recurring mutations in MAP3K1 and MAP2K4 (7, 8). The MAP3K1 and MAP2K4 mutations are loss of function mutations that tend to be mutually exclusive, including nonsense and frame shift mutations and an inactivating missense MAP2K4 mutation (Ser56Leu), which interferes with MAP2K4 kinase activity (7-9). Other large-scale genomic studies also found that MAP3K1 and MAP2K4 frequently carry inactivating mutations in different types of cancers, being most prominent in invasive ductal breast cancers: MAP3K1 9% and MAP2K4 7% (10), followed by cancers of prostate, stomach and diffuse large B cell lymphoma (10-15) (http://www.cbioportal.org).

The MAP3K1 -MAP2K4-JNK cascade activates JUN, which in combination with FOS, forms the Activator Protein-1 (AP-1 ) transactivator complex that controls a number of cellular processes including differentiation, proliferation, and apoptosis (16). The significant number of MAP3K1 and MAP2K4 mutations in different types of cancers is still understood poorly due to their dual roles in cell survival and apoptosis. MAP3K1 can promote cell survival through activation of MAP2K4/7-JNK-JUN, MAP2K1/2-ERK1/2 and NF-κΒ, while a MAP3K1 kinase domain generated by caspase-3 cleavage can induce apoptosis (1 1). Consequently, both activating and inactivating mutations in these genes are seen in cancer (2) (http://www.cbioportal.org). In addition it is unknown whether mutations in MAP3K1 or MAP2K4 cause a vulnerability that can be targeted with specific drugs. We show here an unexpected relationship between MAP3K1 and MAP2K4 mutations and response to MEK inhibitors.

Methods

Cell lines and cell culture, inhibitors and antibodies

I LC cell lines CAMA-1 , EVSA-T, HCC1 187, HCC2218, MDA-MB-134VI , MDA-MB-330, MDA- MB-453, M DA-MB-468, M PE600, OCUB-F, OCUB-M, SK-BR-3, SK-BR-5, SUM44PE, ZR-75- 30 were kind gifts from Dr. Mieke Schutte (Josephine Nefkens Institute, Erasmus University Medical Center, Rotterdam, The Netherlands). H358, HCT1 16, LoVo and DLD1 cell lines were purchased from American Type Culture Collection (ATCC). All the cell lines were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, glutamine and Penicillin (Gibco) at 37 °C in 5% C0₂.

Selumetinib (S1008) and dacomitinib (S2727) were purchased from Selleck Chemicals. Antibodies against p-JNK (T183/Y185) (4668), JNK (9252), p-c-JUN (S63) (2361), c-JUN (2315), MAP2K4 (9152), p-ErbB2 (Y1221/1222) (2243), ErbB2 (4290), p-ErbB3 (Y1222) (4784), ErbB3 (4754), p-ErbB4 (Y1284) (4757) and ErbB4 (4795) were purchased from Cell Signaling Technology. Antibodies against p-ERK (E-4) and HSP90 (H-1 14) were purchased from Santa Cruz Biotechnology. Antibody against p-EGFR (Y1068) (ab5644) and MAP3K1 (ab55653) was purchased from Abeam. Antibody against EGFR (06-847) was purchased from Millipore.

Short-term growth inhibition assays

I LC cell lines were cultured and seeded into 384-well plates (1000-3000 cells per well, depending on growth rate). After 24 hours incubation, three-fold serial dilutions of drugs were added to final drug concentrations ranging from 0.0005-10 microM. Cell viability was measured with the CellTiter-Blue assay (Roche) after treatment with drug for 72 hours. The relative survival of different I LC cell lines in the presence of drug was normalized against control conditions (untreated cells) after subtraction of background signal. H358, HCT1 16, LoVo and DLD1 Ctrl and MAP3K1 or MAP2K4 knockout cells were cultured and seeded into 96-well plates (500 cells per well). After 24 hours incubation, three-fold serial dilutions of drugs were added to final drug concentrations ranging from 0.0005-10 microM (medium was changed after 3 days). Cell viability was measured with the CellTiter-Blue assay (Roche) after treatment with drug for 7 days. The relative survival of different I LC cell lines in the presence of drug was normalized against control conditions (untreated cells) after subtraction of background signal.

Long-term cell proliferation assays

Cells were cultured and seeded into 6-well plates at density of 1 -2 * 10⁴ cells per well, depending on growth rate and were cultured in the medium containing the indicated drugs for two weeks (medium was changed twice a week). After this, cells were fixed with 4% formaldehyde in PBS and stained with 0.1 % crystal violet in water. Protein lysate preparation and western blot

Cells were plated in completed medium. After 24 hours incubation, cells were treated under indicated conditions. Then the cells were washed twice with PBS and lysed in RIPA buffer supplemented with Complete Protease Inhibitors (Roche), Phosphatase Inhibitor Cocktails I I and II I (Sigma). The lysates were then resolved by electrophoresis in Novex NuPAGE gels and followed by western blotting. shRNA and lentiviral transduction The lentiviral based RNAi Consortium (TRC) human genome-wide shRNA collection (TRCHslO) was used in making gene knockdown cell lines. Individual lentiviral plasmids containing shRNAs against JUN were collected from TRC library and lentiviruses were produced as described at http:// www.broadinstitute.org/rnai/public/resources/protocols. In brief, HEK293T cells were transfected with lentiviral vectors using calcium phosphate method. Lentiviral supernatants were collected and transduced into target cells with polybrene (1 mg/ml). Stable gene knockdown cell lines were selected with puromycin (2 microg/ml).

CRISPR/Cas9 mediated gene knockout

The lentiviral based CRISPR/Cas9 mediated gene knockout cell lines were produced as described at http://genome-engineering.org/gecko. In brief, sequence of individual sgRNAs against MAP3K1 and MAP2K4 were collected from genome-scale CRISPR knock-out (GeCKO) libraries, and then cloned to LentiCRISPRv2 vector. To make lentivirus, HEK293T cells were co-transfected by lentiCRISPRv2 plasmids contacting individual sgRNAs and packaging plasmids. Lentiviruses were collected and transduced into target cells with polybrene (1 mg/ml). After puromycin (2 microg/ml) selection, single clones were cultured and knockout clones were identified.

RNA isolation and analysis

Cells were harvested and total RNA was isolated using Trizol (Invitrogen). For real-time PCR analysis, cDNA was synthesized from total RNA using Maxima Universal First Strand cDNA Synthesis Kit (Thermo scientific). The resulting cDNA was subjected to PCR analysis with gene-specific primers using Biosystems 7500 Real-Time PCR Systems (life technologies). The housekeeping gene GAPDH was used as the internal control. The PCR products were detected by measurement of the SYBR Green (Roche).

Cell proliferation assay

Indicated cells were cultured and seeded into 96-well plates at a density of 1000 cells per well. 24 hours later, drugs were added using HP D300 Digital Dispenser (HP) at indicated concentrations. Cells were imaged every 4 hours in IncuCyte ZOOM (Essen Bioscience). Phase-contrast images were collected and analyzed to detect cell proliferation based on cell confluence. phospho-RTK activation analysis

The phospho-RTK activation analysis was done following the manufacturers' instruction of Human Phospho-Receptor Tyrosine Kinase Array Kit (R&D). Briefly, cells were lysed and incubated with blocked array membranes overnight. Then the array membranes were washed and incubated with Anti-Phospho-Tyrosine-HRP Detection Antibody. The arrays were then washed and processed using a luminol based chemical reagent, and followed by X-ray films exposure.

In vivo mice xenograft studies

Dacomitinib and selumetinib was dissolved in Cremophor EL/DMSO (Sigma). All animals were performed according to protocols approved by the Animal Ethics Committee of the Netherlands Cancer Institute in accordance with the Dutch Act on Animal Experimentation. MDA-MB-468 cells (3.5 ^χ 10⁶ cells per mouse) were injected subcutaneously in the right posterior flank of 7-week-old immunodeficient Balb/C female nude mice. Tumor formation was monitored twice a week. When the tumor volume reached approximately 100 mm³, mice were randomly (6 mice per group) either treated orally 5 days on and 2 days off with vehicle, selumetinib (20mg/kg of body weight by daily gavage), dacomitinib (3.75mg/kg of body weight by daily gavage) or their combination at the same dose as monotherapy. H358 Ctrl and MAP2K4 knockout cells (5 ^χ 10⁶ cells per mouse) or HCT1 16 Ctrl and MAP2K4 knockout cells (1 * 10⁶ cells per mouse) were injected subcutaneously in the right posterior flank of 7- week-old immunodeficient Balb/C female nude mice. Tumor formation was monitored once a week. When the tumor volume reached approximately 100 mm³, mice were randomly (6 mice per group) either treated orally with vehicle or selumetinib (20mg/kg of body weight by daily gavage).

Results

Recurrent MAP3K1 and MAP2K4 mutations sensitize to MEK inhibitors

The MAP3K1 and MAP2K4 genes act in the in the JNK pathway downstream of receptor tyrosine kinases and mutations in these genes are also seen lobular breast and other cancers. To study whether the MAP3K1 and MAP2K4 mutations identified in ILCs give rise to vulnerability that can we exploited therapeutically, we used a panel of 15 ILC cells lines that we sequenced previously (7). Among the 15 ILC cell lines, we found that MDA-MB-134VI and MPE600 had inactivating mutations in MAP2K4 (Ser56Leu and c.219-1G<C).

To find genotype-drug response relationships in ILCs that can be exploited therapeutically, we examined drug sensitivity of the ILC cell line panel in relation to their genotypes. Given the frequent mutations in the MAPK pathway in ILC patients, we focused initially on drugs that act on this pathway. The drugs that are most advanced clinically are the MEK inhibitors, as exemplified by trametinib and selumetinib (5, 6). The data showed that only two cell lines in the panel were sensitive to selumetinib (AZD6244): the MAP2K4 mutant cell lines MDA-MB- 134VI and MPE600 . To confirm these findings in long-term colony formation assays, we tested the effect of selumetinib on proliferation in two MAP2K4 wild-type and two MAP2K4 mutant I LC cell lines. Figure 1 shows that the two MAP2K4 mutant I LC cell lines are more sensitive to selumetinib as compared to wild-type ILC cells in the long-term proliferation assay. We also quantified cell proliferation in short-term assays using the Incucyte system that detects cell confluence over time. The data of the four I LC cell lines indicate that selumetinib treatment reduces cell proliferation in MAP2K4 mutant cells, but not in the wild-type cells.

MEK inhibitor activates JNK kinase dependent on a functional MAP3K1-MAP2K4 pathway. To explore the mechanism of sensitivity of MAP2K4 mutant I LC cells to MEK inhibition, biochemical analyses of the ERK and JNK signaling pathways were performed in both MAP2K4 mutant and wild-type cells. In MAP2K4 wild-type cells, selumetinib treatment resulted in activation of JNK kinase, as evidenced by an increase in JNK phosphorylation (p- JNK) and its downstream target JUN (p-JUN, Figure 2A). In contrast, JNK activation was not evident in the two MAP2K4 mutant cells (M DA- MB- 134V I and M PE600), as evidenced by low phosphorylation of JNK and JUN.

To ask if the activation of JNK by selumetinib is dependent on MAP2K4, we generated isogenic derivatives of MDA-MB-468 lacking MAP3K1 or MAP2K4 using CRISPR/Cas9 mediated gene knockout. The MEK inhibitor induced phosphorylation of JUN was blocked by MAP3K1 and by MAP2K4 knockout, indicating that JUN activation by MEK inhibitor was dependent on the presence of functional MAP3K1 -MAP2K4 pathway. Importantly, we observed that MAP3K1 or MAP2K4 knockout cells gained sensitivity to selumetinib, both in long-term colony formation assays (Figure 2B) and in short-term proliferation assays (data not shown). Together, these data suggest that activation of JN K pathway is a common response to MEK inhibitor in MAP3K1;MAP2K4 wild-type ILC cells, whereas blockade of the MAP3K1 - MAP2K4 pathway by gene knockout or loss of function mutation, or by inhibition of activity of the pathway, confers sensitivity to M EK inhibitor. MEK inhibition activates HER receptors in MAP3K1MAP2K4 wild-type cells.

The transcription factor JUN is known to activate several RTKs, including the epidermal growth factor receptor (EGFR) (17-20), HER2 (21 ) and platelet-derived growth factor receptor-beta (PDGFRB) (22). In view of the surprising finding above, we therefore tested whether MEK inhibitor treatment would lead to activation of any of these RTKs through an effect on JUN in MAP3K1;MAP2K4 wild-type I LCs. We performed Receptor Tyrosine Kinase (RTK) blots on both wild-type and MAP2K4 mutant cells treated both with or without selumetinib. We found that before MEK inhibitor treatment, phosphorylation levels of HER RTKs were lower in MAP2K4 mutant cells than in wild-type cells. In wild-type cells all four HER RTKs (EGFR, HER2, HER3, HER4) were activated upon MEK inhibition, which was not seen in the two I LC cells mutated for MAP2K4.

A qRT-PCR analysis showed increased HER receptors transcripts after 72 hours selumetinib treatment in wild-type cells (Figure 3A). The activation of HER receptors was also observed in HCC1 187, another MAP3K1;MAP2K4 wild-type cell line (data not shown). This activation of HER RTKs was JUN dependent, as JUN knockdown decreased the phosphorylation and total levels of four HER receptors (Figure 3B). Moreover, JUN suppression inhibited activation of the HER receptors resulting from MEK inhibition (Figure 3B). We also observed that in wild- type cells p-ERK level came back after 72 hours MEK inhibitor treatment, which is likely explained by the slow kinetics of transcriptional activation of the HER receptors. The above findings suggest that inhibition of MEK in MAP3K1;MAP2K4 wild-type cells results to a feedback activation of multiple RTKs through activated JUN that limits the response to MEK inhibitor treatment. This implicates that cancers cells, such as I LCs, or KRAS-mutated cancers, that are wild-type for both MAP2K4 and MAP3K1 require co-treatment with a small molecule pan-HER inhibitor, like dacomitinib for MEK inhibitors to be effective. To test this directly, we treated two wild-type cell lines (MDA-MB-468 and HCC1 187) with a combination of selumetinib and dacomitinib. Figure 3C shows that MEK inhibitor selumetinib displays strong synergy with pan-HER inhibitor dacomitinib. To test this synergy in vivo, we injected MBA-MB-468 breast cancer cells into immuno-deficient nude mice. When tumors reached a volume of 100 mm³, we treated with vehicle, selumetinib, dacomitinib or the combination of the two drugs. The data shows that again a striking synergy was observed between selumetinib and dacomitinib, consistent with the notion that blocking the feedback loop at the level of HER receptors reinstates sensitivity to MEK inhibitors.

KRAS mutant cancer cells are also sensitized to MEK inhibitors by MAP3K1 or MAP2K4 mutation.

We have shown previously that KRAS mutant colon and lung cancer cells are sensitized to MEK inhibitors by pan-HER inhibitors (23). To ask whether in these cells the MAP3K1 and MAP2K4 kinases are involved in intrinsic resistance to MEK inhibitors, we generated a number of KRAS mutant colon and lung cancer cells lacking MAP3K1 or MAP2K4 through CRISPR/Cas9 mediated knockout.

Figure 4A shows that KRAS mutant H358 NSCLC cells activate JUN in response to MEK inhibition, but this activation is absent in MAP2K4 knockout derivatives. Note the lower levels of JUN in the MAP2K4 knockout cells, which is explained by the feed forward loop in which active JUN induces JUN expression (24). Similar results were seen in MAP3K1 knockout H358 cells (Figure 4B). Importantly, H358 lacking MAP3K1 or MAP2K4 became sensitive to selumetinib monotherapy, both in long-term colony formation and in short-term proliferation assays (Figure 4C). Essentially identical results were obtained in KRAS mutant HCT1 16, LoVo and DLD-1 colon cancer cells in which MAP2K4 had been ablated by CRISPR knockout.

Next, we tested the response of MAP2K4 knockout H358 and HCT1 16 cells to MEK inhibition in vivo. We again observed that MAP2K4 knockout conferred sensitivity to selumetinib monotherapy (Figure 4D). Together, these data indicate that mutation of the MAP3K1 - MAP2K4-JNK-JUN pathway prevents a feedback activation of HER family RTK receptors upon MEK inhibition, making such cells responsive to MEK monotherapy inhibition. Finally, to test whether inhibition of JNK kinases shows synergy with MEK inhibitors, RAS wild type MDA-MB-468 breast cancer cells were cultured for two weeks in media containing increasing concentration of the MEK inhibitor selumetinib, the JNK inhibitor SP600125 or the combination of selumetinib and SP600125. After this, the cells were fixed and stained. Figure 5 shows that concentrations of selumetinib up to 4 uM did not affect proliferation rates of the MDA-MB-468 cells. Similarly, culturing of MDA-MB-468 breast cancer cells with 4 uM of SP600125 had little effect on proliferation. However, combining selumetinib and SP600125 resulted in a significant inhibition of proliferation, indicating that inhibition of MEK and JNK kinases is synergistic in these cells

JNK inhibitor sensitize MAP3K1/MAP2K4 wild-type cells to MEK inhibitor

In order to test whether inhibition of the MAP3K1-MAP2K4-JNK pathway in cancer cells that do not have inactivating mutations in the genes of the MAP3K1 -MAP2K4-JNK pathway (e.g. MAP3K1 gene, the MAP2K4 gene, the JNK1 gene, and the JN K2 gene) synergizes with M EK inhibition, we treated cells with inhibitors of the MAP3K1 -MAP2K4-JNK pathway, which consist of two different JNK kinase inhibitors. Specifically, we used both JNK-IN-8 and SR3306 in three cell types that are non-responsive to MEK inhibition, namely MDA-MB-468 breast cancer cells, LoVo colon cancer cells, and H358 lung cancer cells. The cells were cultured with increasing concentration of selumetinib in the presence and absence of JNK inhibitors for two weeks and stained thereafter (see Figure 6).

The results are shown in Figure 6. Specifically, Figure 6 shows that both the JNK inhibitor JNK-IN-8 and the JNK inhibitor SR3306 synergized in growth inhibition with selumetinib in MAP3K1 ;MAP2K4 wild-type cells (see Figure 6 panels A-F). In other words, it was found that combining the JNK inhibitor JNK-IN-8 with selumetinib resulted into a synergistic effect on growth inhibition of cancer cells which express functional MAP3K1/MAP2K4 (wild-type MAP3K1/MAP2K4). The same effect was observed when combining the JNK inhibitor SR3306 with selumetinib.

Discussion.

The data presented here provide a potential explanation for the disappointing performance of MEK inhibitors in a variety of clinical studies (5, 6). We find here that inhibition of MEK kinases in both RAS wild type and RAS mutant tumors results in a feedback activation of the parallel MAK3K1 -MAP2K4-JNK-JUN pathway, resulting in activation of a number of HER family receptor tyrosine kinases that limit the efficacy of M EK monotherapy.

We show that cancer cells that have inactivating mutations in MAP3K1 or MAP2K4 are sensitive to MEK inhibitor monotherapy. Such mutations are present in some 100,000 patients diagnosed in the US alone annually (2) (http://www.cbioportal.org). As such, our data provide a DNA guided biomarker strategy to identify patients that are most likely to respond to MEK inhibition. Moreover, we find that inhibition of HER RTKs with dacomitinib sensitizes KRAS wild type breast cancer cells to MEK inhibition through blockade of the feedback loop. Our data also predict that inhibitors of the MAP3K1 , MAP2K4 or JNK kinases may show synergy with MEK inhibition in a variety of cancers.

References

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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.

While this invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications. This application is intended to cover any variations, uses, or adaptations of the inventions following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth as follows in the scope of the appended claims. 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.

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.

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. 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.

Claims

1 . A combination of an inhibitor of a protein of the MAP3K1 -MAP2K4-JNK pathway and an inhibitor of a protein of the MEK-ERK pathway for use as a medicament in a subject.

2. A combination of an inhibitor of a protein of the MAP3K1 -MAP2K4-JNK pathway and an inhibitor of a protein of the MEK-ERK pathway for use in the treatment of cancer in a subject.

3. The combination for use of any one of the previous claims wherein the subject or cancer in the subject is characterized by a gene that encodes a functional MAP2K4 protein, preferably wherein the gene encodes a wild-type MAP2K4 protein and a gene that encodes a functional MAP3K1 protein, preferably wherein the gene encodes a wild-type MAP3K1 protein.

4. The combination for use of any one of the previous claims wherein the subject or cancer in the subject is characterized by the expression of functional MAP2K4 protein and/or the expression of functional MAP3K1 protein.

5. The combination for use of any one of the previous claims wherein the cancer is KRAS-mutated cancer, breast cancer, colon cancer, lung cancer, KRAS-mutated lung cancer or KRAS-mutated colon cancer.

6. The combination for use of any one of the previous claims wherein the inhibitor of the protein of the MAP3K1 -MAP2K4-JNK pathway is an inhibitor of MAP2K4 and/or an inhibitor of

MAP3K1 .

7. The combination for use of any one of the previous claims wherein the inhibitor of the protein of the MEK-ERK pathway is an inhibitor of M EK and/or an inhibitor of ERK, preferably wherein the inhibitor is trametinib, selumetinib, cobimetinib, binimetinib or Pimasertib.

8. The combination for use of any one of the previous claims wherein the inhibitor of the protein of the MAP3K1 -MAP2K4-JNK pathway and/or the inhibitor of the MEK-ERK pathway inhibits expression of the protein and/or transcription of the gene encoding the protein and/or translation of the transcript of the gene encoding the protein, or wherein the inhibitor of the protein inhibits the activity of said protein.

9. The combination for use of any one of the previous claims wherein the cancer is a cancer that is or has acquired resistance to a MEK inhibitor and/or an ERK inhibitor.

10. An inhibitor of a protein of the MAP3K1 -MAP2K4-JNK pathway for use in the treatment of cancer in a subject, wherein the inhibitor is administrated simultaneously, separately or sequentially with an inhibitor of a protein of the MEK-ERK pathway.

1 1 . An inhibitor of a protein of the MEK-ERK pathway for use in the treatment of cancer in a subject, wherein the inhibitor is administrated simultaneously, separately or sequentially with an inhibitor of a protein of the MAP3K1 -MAP2K4-JNK pathway.

12. An inhibitor of a protein of the MAP3K1 -MAP2K4-JNK pathway for use in sensitizing a cell, preferably a cancer cell, even more preferably a KRAS-mutated cancer cell, to a MEK inhibitor and/or a ERK inhibitor.

13. A product comprising an inhibitor of a protein of the MAP3K1 -MAP2K4-JNK pathway and an inhibitor of a protein of the MEK-ERK pathway, as a combined preparation for simultaneous, separate or sequential use in the treatment of cancer in a subject.

14. An inhibitor of a protein of the MEK-ERK pathway for use in the treatment of cancer in a subject wherein the subject or cancer in the subject is characterized by a gene that encodes a non-functional MAP2K4 protein and/or a gene that encodes a non-functional MAP3K1 protein.

15. An inhibitor of a protein of the MEK-ERK pathway for use in the treatment of cancer in a subject, wherein the subject or cancer in the subject is characterized by the expression of non-functional MAP2K4 protein and/or the expression of non-functional MAP3K1 protein, or wherein the subject or cancer in the subject is characterized by reduced expression of functional MAP2K4 protein and/or reduced expression of functional MAP3K1 protein.

16. A combination of an inhibitor of a receptor tyrosine kinase, preferably wherein the inhibitor is an inhibitor of epidermal growth factor receptors, EGFR, HER2, EGFR, H ER3 and HER4, and an inhibitor of a protein of the MEK-ERK pathway for use in the treatment of cancer in a subject, wherein the subject or cancer in the subject is characterized by a gene that encodes a functional MAP2K4 protein, preferably wherein the gene encodes a wild-type MAP2K4 protein and a gene that encodes a functional MAP3K1 protein, preferably wherein the gene encodes a wild-type MAP3K1 protein.

17. The combination for use of claim 16 wherein the subject or cancer in the subject is characterized by the expression of functional MAP2K4 protein and the expression of functional MAP3K1 protein.

18. A method for predicting treatment response of a cancer in a subject, preferably a KRAS-mutated cancer and wherein the treatment comprises

a) monotherapy treatment with a MEK inhibitor or a ERK inhibitor; or

b) combination treatment with an inhibitor of a protein of the MAP3K1 -MAP2K4-JNK pathway and an inhibitor of a protein of the MEK-ERK pathway; or

c) combination treatment with an inhibitor of a protein of the MAP3K1 -MAP2K4-JNK pathway and an inhibitor of a receptor tyrosine kinase, preferably wherein the inhibitor is an inhibitor of epidermal growth factor receptor, platelet-derived growth factor receptor, and/or human epidermal growth factor receptor, wherein the method comprises the step of determining in tissue or cells obtained from said subject, preferably from cancer cells from said subject, the presence of

-functional MAP2K4 protein and/or non-functional MAP2K4 protein;

-functional MAP3K1 protein and/or non-functional MAP3K1 protein; and/or

-genes encoding such functional protein or non-functional protein, or mRNA transcripts thereof, wherein

-presence of a functional protein, or a gene encoding such functional protein, or mRNA transcript thereof, is predictive for poor response to monotherapy treatment with a

MEK inhibitor or an ERK inhibitor;

-presence of a functional protein, or a gene encoding such functional protein, or mRNA transcript thereof, is predictive for good response to combination treatment with an inhibitor of a protein of the MAP3K1 -MAP2K4-JNK pathway and an inhibitor of a protein of the

MEK-ERK pathway;

-presence of a functional protein, or a gene encoding such functional protein, or mRNA transcript thereof, is predictive for good response to combination treatment with an inhibitor of a protein of the MAP3K1 -MAP2K4-JNK pathway and an inhibitor of a receptor tyrosine kinase, preferably wherein the inhibitor is an inhibitor of epidermal growth factor receptor, platelet-derived growth factor receptor, and/or human epidermal growth factor receptor; and/or

19. A method of treating cancer in a subject, preferably KRAS-mutated cancer, wherein the method comprises the simultaneous, separate or sequential administering to the subject of an inhibitor of a protein of the MAP3K1 -MAP2K4-JNK pathway and an inhibitor of a protein of the MEK-ERK pathway or the simultaneous, separate or sequential administering to the subject of an inhibitor of a protein of the MEK-ERK pathway and an inhibitor of a receptor tyrosine kinase, preferably wherein the inhibitor is an inhibitor of epidermal growth factor receptors,.

20. The method of claim 19, wherein the subject or cancer in the subject is characterized by a gene that encodes a functional MAP2K4 protein, preferably wherein the gene encodes a wild-type MAP2K4 protein and a gene that encodes a functional MAP3K1 protein, preferably wherein the gene encodes a wild-type MAP3K1 protein.

21. The method of any one of claims 19 - 20 wherein the subject or cancer in the subject is characterized by the expression of functional MAP2K4 protein and the expression of functional MAP3K1 protein.