WO2015041534A1 - P90rsk in combination with raf/erk/mek - Google Patents

Abstract

The current disclosure relates to pharmaceutical combinations and compositions useful in the treatment of certain types of cancer. The disclosure also relates to method of treatment these certain types of cancer. In particular, the disclosure relates to the combined use of an inhibitor of p90RSK and an inhibitor of another protein of the MAPK/ERK-pathway in the treatment of KRAS-, NRAS-or BRAF-mutated cancer, in particular in NRAS mutated melanoma.

Description

P90RSK in combination with RAF/ERK/MEK Field of the Invention

[001] The current disclosure relates to pharmaceutical combinations and compositions useful in the treatment of certain types of cancer. The disclosure also relates to method of treatment these certain types of cancer. In particular, the disclosure relates to the combined use of an inhibitor of p90RSK and an inhibitor of another protein of the MAPK/ERK-pathway in the treatment of KRAS-, NRAS- or BRAF-mutated cancer, in particular in NRAS mutated melanoma. Prior Art

[002] Cancer is one of the leading causes of death in the Europe and the United

States. 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 as first line therapy, and as second line therapies in treatment of resistant tumors.

[003] Cancer cells are by definition heterogeneous. For example, multiple mutational mechanisms may lead to the development of cancer and mutational mechanisms associated with some cancers may differ between one tissue type and another; it is therefore often difficult to predict whether a specific cancer will respond to a specific chemotherapeutic (Cancer Medicine, 5th edition, Bast et al, B. C. Decker Inc., Hamilton, Ontario).

[004] The treatment of cancer is gradually changing from an organ-centered to a

pathway-centered approach. Cancer cells often have an addiction to the signals that are generated by the cancer-causing genes. Consequently, targeted cancer drugs that selectively inhibit the products of activated oncogenes can have dramatic effects on cancer cell viability. This approach has yielded significant clinical results for Non Small Cell Lung Cancer (NSCLC) having activating mutations in EGFR or translocations of the ALK kinase and for melanoma patients having a BRAF mutant tumor. However, this approach has not been successful in all type of cancers, in particular in cancers characterized by oncogenic mutations in one of the members of the RAS gene family, or in the BRAF. In particular treatment options of NRAS-mutated cancer, for example NRAS-mutated melanoma are limited.

[005] It is therefore goal of the current invention to provide for new and improved

methods of treatment of KRAS, BRAF and NRAS-mutated cancers, as well as to provide for products and therapeutically pharmaceutical combinations for use in such mutant cancers.

Description of the Drawings

[006]

Figure 1 : Combination treatment of p90RSK inhibitor with MEK inhibitor kills or inhibits NRAS mutant melanoma cell proliferation. Cells were seeded equal densities and treated with GSK1120212 (MEKi) and BI-1870 (p90RSK inhibitor) as indicated and stained with crystal violet.

Figure 2: Combination treatment of p90RSK inhibitor with ERK inhibitor kills or inhibits NRAS mutant melanoma cell proliferation. Cells were seeded at equal densities and treated with SCH772984 (ERKi) and BI-1870 (p90RSK inhibitor) as indicated and stained with crystal violet.

Figure 3: Combination treatment of p90RSK inhibitor with ERK inhibitor kills or inhibits BRAF mutant melanoma cell proliferation. Cells were seeded at equal densities and treated with SCH772984 (ERKi) and BI-1870 (p90RSK inhibitor) as indicated and stained with crystal violet.

Figure 4: Combination treatment of p90RSK inhibitor with ERK inhibitor kills or inhibits ERK inhibitor resistant NRAS mutant melanoma cell proliferation. Cells were seeded at equal densities and treated with SCH772984 (ERKi) and BI-1870 (p90RSK inhibitor) as indicated and stained with crystal violet.

Figure 5: Combination treatment of p90RSK inhibitor with MEK inhibitor kills or inhibits proliferation of ERK inhibitor resistant NRAS mutant melanoma cells. Cells were seeded at equal densities and treated with GSK1120212 (MEKi) and BI-1870 (p90RSK inhibitor) as indicated and stained with crystal violet.

Figure 6: ERK inhibitor resistant NRAS mutant melanoma cells reactivate MAPK/ERK signaling in the presence of ERK inhibitor. Cells were treated with SCH772984 (ERKi) or vehicle control as indicated, lysed and probed for MAPK/ERK signaling and AKT.

Description

Definitions

[007] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. For example, conventional molecular biology, microbiology, and recombinant DNA techniques including sequencing techniques are well known among those skilled in the art. Such techniques are explained fully in the literature. See, e.g.,

Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein

"Sambrook, et al., 1989"); DNA Cloning: A Practical Approach, Volumes I and II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic Acid

Hybridization (B. D. Hames & S. J. Higgins eds. (1985)); Transcription And Translation

(B. D. Hames & S. J. Higgins, eds. (1984)); Animal Cell Culture (R. I. Freshney, ed. (1986)); Immobilized Cells And Enzymes (IRL Press, (1986)); B. Perbal, A Practical Guide To Molecular Cloning (1984). Indeed, the present invention is in no way limited to the methods and materials described.

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

[009] As used herein, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. For example, a method for administrating a drug includes the administrating of a plurality of molecules (e.g. 10's, 100's, 1000's, 10's of thousands, 100's of thousands, millions, or more molecules).

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

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

[012] As used herein "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. The terms "cancer," "neoplasm," and "tumor," are used interchangeably and refer to ceils 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 ceils derived from such primary cancer cell, including metastasized cancer cells, and ceil lines derived from cancer ceils.

[0 3] 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," "tumor" or "cancer" in a patient includes treatment of the primary cancer, and, where appropriate, treatment of metastases. [014] As used herein, "in combination with" is intended to refer to all forms of administration that provide a first drug together with a further (second, third) drug. The drugs may be administered simultaneous, separate or sequential and in any order. Drugs administered in combination have biological activity in the subject to which the drugs are delivered. Within the context of the invention, a combination thus comprises at least two different drugs, and wherein one drug is at least a p90RSK- inhibitor and wherein the other drug is at least an inhibitor of another protein of the APK/ERK pathway, as disclosed herein in detail. In an embodiment, in the combination, the p90RSK-inhibitor is a selective inhibitor, and does preferably does not inhibit the "another protein of the MAPK/ERK pathway", as disclosed herein in detail. In an embodiment, in the combination, the inhibitor of another protein of the APK-'ERK pathway, as disclosed herein in detail, is a selective inhibitor, and within the context of the current invention, does not inhibit p90RSK. In a further embodiment, in the combination, both the p90RSK-inhibitor and the inhibitor of "another protein of the MAPK/ERK pathway", as disclosed herein in detail, are selective inhibitors./V'pct

[015] A used herein "compositions", "products" of "combinations" useful in the methods of the present disclosure include those 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 (alone or in combination) and one or more suitable pharmaceutically acceptable excipients or carriers.

[016] As used herein, "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded, it also encompasses the more limiting "to consist of." [017] As used herein, "an effective amount" is meant the amount of an

agent/pharmaceutical compound required to ameliorate the symptoms of a disease relative to an untreated patient. The effective amount of active agent(s) used to practice the present invention for therapeutic treatment of a cancer varies depending upon the manner of administration, the age, body weight, and general health of the subject.

Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount. Thus, in connection with the administration of a drug which, in the context of the current disclosure, is "effective against" a disease or condition indicates that administration in a clinically appropriate manner results in a beneficial effect for at least a statistically significant fraction of patients, such as an improvement of symptoms, a cure, a reduction in at least one disease sign or symptom, 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.

[018] As used herein, in general the term "inhibitor of a (defined) protein, for example ERK, refers to any compound capable of down-regulating, decreasing, suppressing or otherwise regulating the amount and/or activity of the (defined) protein, for example

ERK. 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. To compare compound effects on different enzymes it is important to employ similar assay formats. For the proteins/enzymes as disclosed herein, such assay formats are readily available in the prior art. Thus, within the context of the current invention the different drugs used in the combination may be drugs that selectively inhibit one of the proteins to be inhibited according to the invention in comparison to the other protein(s), for example when used in a clinical setting.

[019] "Mammal" as used herein, refers to any member of the class Mammalia,

including, without limitation, humans and non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs, and the like. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be included within the scope of this term. Preferably the mammal is human.

[020] "Pharmaceutically acceptable" is employed herein to refer to those combinations of a therapeutic as described herein, other drugs or therapeutics, materials,

compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals, without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

[021] "Pharmaceutically-acceptable carrier", as used herein, means a

pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject chemical from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. [022] The terms "protein" or "polypeptide" are used interchangeably and refer to molecules consisting of a chain of amino acids, without reference to a specific mode of action, size, 3 dimensional structure or origin. A "fragment" or "portion" of a protein may thus still be referred to as a "protein". An "isolated protein" is used to refer to a protein which is no longer in its natural environment, for example in vitro or in a recombinant bacterial or plant host ceil.

[023] As used herein "simultaneous" administration 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.

[024] As used herein, "small molecule" is understood to refer to a chemical compound having a molecular weight below 2,500 Daitons, more preferably between 300 and 1 ,500 Daltons, and still more preferably between 400 and 1000 Daitons. it is preferred that these small molecules are organic molecules. In certain embodiments, "small molecule" does not include peptide or nucleic acid molecules.

[025] The term "subject" is intended to include vertebrates, preferably a mammal, including human and non-human mammals such as non-human primates. Human subjects are can be referred to as patients.

[026] As used herein, the terms "treat," treating", "treatment," and the like refer to

reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.

[027] The term "wild type" as is understood in the art refers to a polypeptide or

polynucleotide sequence that occurs in a native population without genetic modification. As is also understood in the art, a "mutant" includes a polypeptide or polynucleotide sequence having at least one modification to an amino acid or nucleic acid compared to the corresponding amino acid or nucleic acid found in a wild type polypeptide or polynucleotide, respectively. Cancers that are either wild type or mutant for NRAS, KRAS or BRAF are identified by known methods. For example, wild type or mutant NRAS/BRAF/KRAS cancer cells can be identified by DNA amplification and sequencing techniques, DNA and RNA detection techniques, including, but not limited to Northern and Southern blot, respectively, and/or various biochip and array technologies. Wild type and mutant polypeptides can be detected by a variety of techniques including, but not limited to immunodiagnostic techniques such as ELISA, or Western blot. Mutation tests are, for example, available via fttp://therapy. collabrx.com/melanoma/molecular_analysis/ (as per 27 July 2013).

Detailed description

[028] The current disclosure is based on the surprising finding that a combination of an inhibitor of the protein (enzyme) p90RSK and at least one inhibitor of another protein of the MAPK ERK pathway is synergistic, i.e. produces an effect greater than the effect of the individual drugs, or even greater than the sum of the their individual effects, in inhibiting proliferation of or inducing apoptosis in a cancer in a mammal, preferably a human, wherein the cancer is selected from the group consisting of NRAS-, KRAS- and BRAF-mutated cancer, preferably NRAS-mutated cancer, for example, but not limited to NRAS-, KRAS- and BRAF-mutated melanoma, for example NRAS-mutated melanoma or lung cancer. The inhibitors in the combination may, in one embodiment be selective inhibitors (or a selective inhibitor). In addition, the claimed combination works also in those cells that are relatively insensitive to inhibition by the other inhibitor of a protein of the MAPK/ERK pathway alone (e.g. a RAF-inhibitor alone, an ERK-inhibitor alone, a MEK-inhibitor alone, or a p90RSK-inhibitor alone). Such cells are also referred to as resistant cancer cells and do not normally respond to treatment. The cancer may be resistant at the beginning of treatment (often called intrinsic resistance), or it may become resistant during treatment (also called refractory cancer) (often called acquired resistance. In other words, in one embodiment the cancer is a NRAS-, KRAS- and BRAF-mutated cancer, preferably melanoma, that is or has become relatively insensitive or resistant to the other inhibitor (i.e. not a p90RSK inhibitor) of a protein of the

MAPK/ERK-pathway, preferably, that has become relatively insensitive or resistant to an ERK-inhibitor, a MEK-inhibitor, and/or a RAF-inhibitor, i.e. has or acquired resistance. The term "acquired resistance" indicates that the cancer becomes resistant to the effects of the drug after being exposed to it for a certain period of time.

[029] The inventors of the present invention have demonstrated, via experiments, that the combination of a p90RSK-inhibitor and at least one inhibitor of another protein of the MAPK/ERK pathway, for example a MEK-inhibitor, a ERK-inhibitor, and/or a RAF- inhibitor manifests an unexpected and strong synergistic, therapeutic effect on the treatment of NRAS-, KRAS- and BRAF-mutated cancer, preferably NRAS-mutated cancer, for example, NRAS-mutated melanoma. The invention thus provides for improved treatment strategies by employing the combination at least two different drugs or compounds, directed to inhibiting the combination of proteins/enzymes as disclosed herein. This for the first time allows to optimize the drug treatment by specifically optimizing treatment so as to inhibit the combination of proteins/enzymes in the best possible way, for example by applying selective inhibitors. For example, by the combination, the dose of each of the drugs in the combination may be optimized in order to achieve optimal treatment effect. For example the individual dose of a first individual drug in the combination may be optimized to achieve optimal inhibition of a first protein, and a second, third or further drug in the combination may be optimized to achieve optimal inhibition of the other protein/enzyme to be inhibited, and as detailed herein. In addition, the invention allows for the treatment with various and different combinations of inhibitors of the proteins/enzymes to be inhibited, as detailed herein. This is very useful in case, for example, for an individual patient, certain drugs or drug combinations are not well tolerated or lead to undesired further complications. The current invention allows for the replacement of a drug in such combination, or of the combination by another drug combination, in accordance with the invention and in order to overcome undesired effects or, again optimize treatment of the patient. In addition, when using the

combination, the dose of the individual drugs may be lowered compared to when the drugs are used individually, which may be beneficial in view of toxicity.

[030] The combination disclosed herein exhibits (therapeutic) co-operation and/or synergy when used to treat a subject or patient. Such effect may be demonstrated by the showing that the combination is superior to one or other of the constituents used as at a given, for example, optimum dose.

[031] In a first aspect of the current disclosure, there is provided for a combination of a p90RSK- inhibitor and an inhibitor of another protein of the MAPK/ERK pathway in the treatment of a cancer selected from the group consisting of NRAS-, KRAS- and BRAF- mutated cancer, preferably a NRAS-mutated cancer.

[032] The combination therapy disclosed herein is particular suitable for use in patients that carry mutations in the genes encoding a RAS protein and/or BRAF protein, leading to proteins with aberrant function.

[033] The term "RAS protein" as used herein means any protein which is a member of the ras-subfamily, a subfamily of GTPases involved in cellular signaling. As is known in the art, activation of RAS causes cell growth, differentiation and survival. RAS proteins include, but are not limited to, HRAS, KRAS and NRAS. The proteins differ significantly only in the C-terminal 40 amino acids.

[034] These proteins are GTPases that function as molecular switches regulating

pathways responsible for proliferation and cell survival. RAS proteins are normally tightly regulated by guanine nucleotide exchange factors (GEFs) promoting GDP dissociation and GTP binding and GTPase-activating proteins (GAPs) that stimulate the intrinsic

GTPase activity of RAS to switch off signaling. Aberrant RAS function is associated with hyper-proliferative developmental disorders and cancer and in tumors is associated with a single mutation typically at codons 12, 13 or 61. A comprehensive overview of RAS mutations in cancer was reported by Prior et al (2012) Cancer Res; 2457 - 67.

[035] The combination therapy disclosed herein is suitable for use in patients with

KRAS-mutated (also referred to as or KRAS-mutant) cancer, and in a preferred embodiment particular useful in patients that are characterized by having a KRAS- mutant melanoma. The term "KRAS-mutated cancer", and thus KRAS-mutated melanoma are 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 mutationaliy 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.

[036] The GTPase KRAS, also known as V-Ki-ras2 Kirsten rat sarcoma viral oncogene homo!og or KRAS, is a protein that in humans is encoded by the KRAS gene (e.g. Gene accession number 3845; Refseq RNA Accessions NM_004985.3; protein NP_004976.2). Like other members of the Ras family, the KRAS protein is a GTPase and is an early player in many signal transduction pathways. KRAS acts as a molecular on/off switch. Once it is turned on it recruits and activates proteins necessary for the propagation of growth factor and other receptors' signal, such as c-Raf and PI 3-kinase.

[037] In a preferred embodiment, the combination therapy disclosed herein is for use in patients with NRAS-mutated (also referred to as or NRAS-mutant) cancer, and in a preferred embodiment particular useful in patients that are characterized by having a NRAS-mutated melanoma. The term "NRAS-mutated cancer" and therefore NRAS- mutated melanoma are well known to the skilled person. A comprehensive overview of RAS mutations, including NRAS-mutations, in cancer was reported by Prior et al (2012) Cancer Res; 2457 - 67. NRAS-mutant cells promote ontogenesis due to being mutationaliy activated, in most cases, again at codon 12, 13 and 61.

[038] The NRAS protein is a GTPase enzyme that in humans is encoded by NRAS (neuroblastoma RAS viral (v-ras) oncogene homolog) gene (e.g. Gene accession number 4893; Refseq RNA Accessions N J302524.4; protein NPJ3Q2515.1). The N-ras gene specifies two main transcripts of 2Kb and 4.3Kb, both transcripts appear to encode identical proteins as they differ only in the 3' untranslated region.

[039] The combination therapy disclosed herein is suitable for use in patients with

BRAF-mutated (also referred to as or BRAF-mutant) cancer, and in a preferred embodiment particular useful in patients that are characterized by having a BRAF- mutant melanoma. The term "BRAF-mutated cancer" and therefore BRAF-mutated melanoma are well known to the skilled person. BRAF (e.g. Gene accession number 673; Refseq RNA Accessions NM_004333.4; protein NP_004324.2) is a member of the RAF family, which includes ARAF and CRAF in humans (Ikawa, Mol Cell Biol. 8(6):2651- 4 (1988)). BRAF is a serine/threonine protein kinase and participates in the

RAS/RAF/MEK/ERK mitogen activated protein kinase pathway (MAPK pathway, see

Williams & Roberts, Cancer Metastasis Rev. 13(1): 105-16 (1994); Fecher et al 2008 Curr Opin Oncol 20, 183-189, or Cargnello M, Roux PP. Microbiol Mol Biol Rev. 2011 Mar; 75(1 ):50-83). Activation and function of the MAPKs and their substrates, the MAPK- activated protein kinases.). Approximately 40-60% of (cutaneous) melanomas carry a mutation in the BRAF protein. Approximately 90% of these mutations result in the substitution of glutamic acid for valine at codon 600 (BRAF V600E, although other mutations are also known (e.g. BRAF V600K and BRAF V600R). Such mutation in BRAF typically leads to proliferation and survival of melanoma cells (Davies et al Nature 2002; 417:949-54; Curtin et al N Engl J Med 2005;353:2135-47), through activation of the MAPK/ERK pathway. As is well-known to the skilled person, this pathway plays a significant role in modulating cellular responses to extracellular stimuli, particularly in response to growth factors, and the pathway controls cellular events including cell proliferation, cell-cycle arrest, terminal differentiation and apoptosis (Peyssonnaux et al., Biol Cell. 93(l-2):53-62 (2001)).

[040] The amino acid sequence of BRAF, NRAS or KRAS protein and any other

protein mentioned herein, and variations thereof are available in GenBank, accessible via http://www.ncbi.nlm.nih.gov/genbank/.

[041] The disclosed combination comprises a p90RSK-inhibitor and at least one

inhibitor of another protein of the MAPK/ERK pathway. The skilled person is well aware of such p90RSK-inhibitors and such inhibitors of other proteins of the MAPK/ERK pathway, as these are readily available in the scientific literature or in various patent documents.

[042] A p90RSK polypeptide (e.g. EC 2.7.1 1.1 ; e.g. Gene accession numbers 6195, 6197, 6196, or 27330; Refseq RNA Accessions NM_0Q 1006665, NM_0Q2953,

NM_004586, NM_001006932, NM_021135, or NM_014496; protein NPJ301006666.1 ,

NP_004577.1 , N P_001006933.1 , or NP_05531 1.1), protein or peptide is to indicate a protein of the ribosomal s6 kinase (rsk) a family of protein kinases. There are two subfamilies of rsk, p90RSK, also known as MAPK-activated protein kinase-1 (MAPKAP- K1), and p70RSK, also known as S6-H1 Kinase or simply S6 Kinase. There are at least four variants of p90RSK in humans, RSK 1- 4. RSKs are serine/threonine kinases and are activated by the MAPK/ERK pathway. The RSK protein is a MAP kinase activated protein kinase (MAPKAP kinase) and described in, e.g., Leukemia, 17: 1263-1293 (2003). p90RSK is phosphorylated and activated by Erk1 and -2 in response to many growth factors, polypeptide hormones and neurotransmitters. RSK has been shown to directly promote cell survival by regulating the expression and activation of pro-survival proteins such as CREB (cyclic adenosine monophosphate response element binding protein). The combination of promoting cell survival and prevention of apoptosis by p90RSK causes excessive cell survival, eventually leading to diseases such as cancer and autoimmune disorders.

[043] It has been found that p90RSK2 is overexpressed in more than 50% of human breast cancers, validating the family as a potential target for drug design.

[044] The amino acid sequence of p90RSK enzyme, or other proteins mentioned

herein, and variations thereof are available in GenBank, accessible via

http://www.ncbi.nlm.nih.gov/genbank/ by entering either the numbers mentioned above or entering the relevant protein name.

[045] By p90RSK biological activity is meant any function of p90RSK, such as herein.

By p90RSK inhibitor is meant a compound that reduces the biological activity of p90RSK; or that reduces the expression of an mRNA encoding ap90RSK polypeptide; or that reduces the expression of a p90RSK polypeptide. As used herein, an "effective amount of an inhibitor ", e.g. an "effective amount of a p90RSK inhibitor" is the amount of inhibitor required to inhibit expression of, in the case of p90RSK, p90RSK or inhibit activity of p90RSK.

[046] The inhibitor may, in one embodiment, be a selective inhibitor. Non-limitative examples of p90RSK inhibitors include, for example, Kaempherol-3-0-(4'-0-acetyl-a-L- rhamnopyranoside), or those disclosed in EP1845778. Further examples are provided in the Table 1 below. The activity of RSK protein or inhibitory activity of a p90RSK inhibitor can be determined by the method described in, e.g., EMBO J., 14: 674-684 (1995) or in EP1845778.

[047] Table 1 : p90RSK inhibitors compound name CAS number formula

BI-D1870 501437-28-1 C19H23F2N502

BRD 7389 376382-11-5 C24H18N202

FMK 821794-92-7 C18H19FN402

HH-5709 185313-17-1

IDDBCP287099

IDDBCP292177

SL 0101-1 , SL- 010, SL-0101-1 77307-50-7 C25H24012 [048] In another embodiment the p90RSK inhibitor may inhibit (gene) expression of p90RSK, for example by interfering with mRNA stability or translation. In one

embodiment the p90RSK inhibitor is selected from small interfering RNA (siRNA), which is sometimes known as short interfering RNA or silencing RNA, or short hairpin RNA (shRNA), which is sometimes known 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.

[049] The p90RSK inhibitor according to the present invention may be a binding agent such as an antibody which specifically binds p90RSK, thereby inhibiting its function.

[050] The inhibitor of the another protein of the MAPK/ERK pathway may be any

inhibitor that reduces the activity of one or more proteins that belong to the MAPK/ERK pathway.

[051] The MAPK/ERK pathway is well-known to the skilled person and is one of the four parallel mitogen activated protein kinase (MAPK) signaling pathways identified:

ERK1/ERK2, JNK, p38 and ERK5.

[052] The pathways are involved in cellular events such as growth, differentiation and stress responses (J. Biol. Chem. (1993) 268, 14553-14556). These four pathways are linear kinase cascades in that MAPKKK phosphorylates and activates MAPKK, and MAPKK phosphorylates and activates MAPK. To date, seven MAPKK homologs (MEK1 ,

MEK2, MKK3, MKK4/SEK, MEK5, MKK6, and MKK7) and four MAPK families (ERK1/2, JNK, p38, and ERK5) have been identified. Activation of these pathways regulates the activity of a number of substrates through phosphorylation. These substrates include: transcription factors such as TCF, c-myc, ATF2 and the AP-1 components, fos and Jun; cell surface components EGF-R; cytosolic components including PHAS-I, p90rsk, cPLA2 and c-Raf-1 ; and cytoskeleton components such as tau and MAP2. MAPK signaling cascades are involved in controlling cellular processes including proliferation, differentiation, apoptosis, and stress responses.

[053] Of the known MAPK signaling pathways, the MAPK/ERK pathway (also referred to as RAF-MEK-ERK pathway or Ras-Raf-MEK-ERK pathway) mediates proliferative and anti-apoptotic signaling from growth factors and oncogenic factors such as Ras and Raf mutant phenotypes that promote tumor growth, progression, and metastasis. By virtue of its central role in mediating the transmission of growth-promoting signals from multiple growth factor receptors, the MAPK/ERK pathway provides molecular targets with potentially broad therapeutic applications in, for example, cancerous and noon- cancerous hyperproliferative disorders, immunomodulation and inflammation.

[054] Within the context of the current invention a protein of the MAPK/ERK pathway includes ERK, MEK, RAF, and RSK proteins, as discussed below. [055] In a preferred embodiment, the protein of the MAPK/ERK pathway is selected from the group consisting of RAF, MEK, and ERK, and combination of two or three thereof. Thus in a preferred embodiment, the inhibitor of the another protein of the MAPK/ERK pathway is selected from the group consisting of a RAF-inhibitor, an ERK- inhibitor, and a MEK-inhibitor.

[056] In a preferred embodiment more than one inhibitor of a protein of the MAPK/ERK pathway is used. For example, two, three or four inhibitors of one or more proteins of the MAPK/ERK pathway are used in the combination therapy disclosed herein, i.e. in combination with a p90RSK inhibitor or one, two, three or more p90RSK inhibitors. For example, at least one p90RSK-inhibitor may be combined with at least one MEK- inhibitor and/or at least one ERK-inhibitor, and/or at least one RAF inhibitor.

[057] A RAF protein, polypeptide or peptide is to indicate a polypeptide having

serine/threonine protein kinase activity. 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) and CRAF (C-RAF; for example Genbank

Accession NO: NP002871).

[058] For example, BRAF (for example, Genbank Accession NO: NP004324)

phosphorylates and activates MEK (MEK1 and MEK2). BRAF (or BRAF) is a member of the RAF family, which includes ARAF and CRAF in humans (Ikawa, Mol Cell Biol.

8(6):2651-4 (1988)). BRAF is a serine/threonine protein kinase and participates in the RAS/RAF/MEK/ERK mitogen activated protein kinase pathway (MAPK pathway, see Williams & Roberts, Cancer Metastasis Rev. 13(1): 105-16 (1994); Fecher et al 2008 Curr Opin Oncol 20, 183-189).

[059] CRAF (e.g. Gene accession number 5894; Refseq RNA Accessions

NM_002880.3; protein NPJ3G2871.1) acts as a MAP3 kinase, initiating the entire kinase cascade of the MAPK/ERK pathway.

[060] These amino acid sequence of BRAF, CRAF and ARAF enzymes, other proteins mentioned herein, and variations thereof are available in GenBank, accessible via http://www.ncbi.nlm.nih.gov/genbank/ by entering either the numbers mentioned above or entering the relevant protein name.

[061] By RAF biological activity is meant any function of RAF, such as enzymatic

activity, kinase activity, or signaling the MAPK/ERK pathway.

[062] By RAF inhibitor, for example a BRAF inhibitor, is meant a compound that

reduces the biological activity of RAF, for example BRAF; or that reduces the expression of an mRNA encoding a RAF polypeptide, for example BRAF; or that reduces the expression of a RAF polypeptide, for example BRAF. RAF kinase inhibitors as used herein include efficient inhibitors of RAF kinase, particularly CRAF kinase inhibitors and wild and mutated BRAF kinase inhibitors, e.g. including inhibitors of mutant BRAF kinase.

[063] RAF kinase inhibitors, e.g. low molecular compounds, are known to the skilled person. Any RAF inhibitor, including any pharmaceutical agent having RAF inhibitory activity or selective RAF inhibitors may be utilized in the present invention. Examples of

RAF kinase inhibitors include the compounds GW5074, BAY 43-9006, CHIR-265, and compounds as defined in US 6,987,1 19, WO98022103, WO99032436,

WO2006/084015, WO2006/125101 , WO2007/027855, WO2005/004864,

WO2005/028444, WO03082272, WO2005/032548, and WO2007030377,

WO2005028444, WO03082272, WO2005032548, and WO2007030377. BRAF inhibitors are described, for instance, in WO 2010/114928, WO 2005/123696, WO2007/002325, WO 2006/003378, WO 2006/024834, WO 2006/024836, WO 2006/040568, WO

2006/067446 and WO 2006/079791 , WO02/24680 and WO03/022840, WO

2005/047542, AU 2003/286447, US 2004/0096855, AU2002/356323, WO2005089443, WO2006/053201 , US20050267060, WO2008120004, US20090181371 ,

WO2008120004 which patent applications can be referenced to the extent of their disclosure of BRAF inhibitors and methods of making and using the same.

[064] Preferred RAF inhibitors include 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), erlotinib (e.g. as marketed by Genentech and OSI pharmaceuticals as Tarceva, LGX818 from Novartis, Dabrafenib (Tafinlar™ capsule, made by GlaxoSmithKline, LLC), or derivatives thereof.

[065] Preferably, the derivative of the BRAF inhibitor is a salt. Thus, according to the invention the RAF inhibitor may be selected from the group consisting of dasatinib, erlotinib hydrochloride, dabrafenib, gefitinib, imatinib mesilate, lapatinib, sorafenib tosylate and sunitinib malate. Preferably the RAF inhibitor is sorafenib tosylate.

Particularly preferred is Vemurafenib (also known as PLX4032, RG7204 or R05185426, e.g. marketed as Zelboraf, from Plexxikon (Daiichi Sankyo group) and Hoffmann-La

Roche).

[066] In one embodiment the RAF inhibitor is selected from any one of the BRAF

inhibitors disclosed in WO2006/024834, W02006/067446, PCT/GB2006/004756, or is selected from any one of CHIR-265 (Novartis), XL281 (Exelixis) or PLX4032 (Plexxikon, or Roche). In one embodiment the RAF inhibitor is selected from any one of the BRAF inhibitors disclosed in WO2008120004. Other BRAF inhibitors include GSK21 18436, benzenesulfonamide, N-[3-[5-(2-amino-4-pyrimidinyl)-2-(1 , 1-dimethylethyl)-4-thiazolyl]- 2-fluorophenyl]-2,6-difluoro-, methanesulfonate (1 : 1), N-{3-[5-(2-aminopyrimidin-4-yl)-2- (1 , 1-dimethylethyl)thiazol-4-yl]-2-fluorophenyl}-2,6- difluorobenzenesulfonamide monomethanesulfonate (Clin Cancer Res. 201 1 ; doi: 10.1158/1078-0432;

http://www.ama-assn.org/resources/doc/usan/dabrafenib.pdf). A non-limitative overview of well-known RAF inhibitors is provided in Table 2.

[067] Table 2 Examples or RAF-inhibitors:

CAS

compound name cellular target(s) number formula

wild-type c-Raf, B-Raf

AZ 628 V600E 878739-06-1 C27H25N502

B-Raf V600E, wild type

B-Raf, c-Raf, Abl-1 , 1188910-76-

CEP-32496 CSF-1 R 0 C24H22F3N505

GDC 0879 B-Raf (B-Raf V600E) 905281-76-7 C19H18N402

GSK2118436, 1195765-45- Dabrafenib 7 C23H20F3N5O2S2

GW 5074 c-Raf 220904-83-6 C15H8Br2IN02

c-Raf (maybe also A-

L-779,450 Raf?) 303727-31-3 C20H 14CIN3O

MLN-2480, BIIB- 1096708-71- 024 2 C17H12CI2F3N702S

c-Raf, c-Src, c-Abl,

NVP-BHG712 EphB4, VEGFR2 940310-85-0 C26H20F3N7O

11 11636-35-

PF-04880594 B-Raf, c-Raf 1 C19H16F2N8

B-Raf V600E, c-Raf-1

PLX-4720 Y340D/Y341 D 918505-84-7 C17H14CIF2N303S

PLX4032,

Vemurafenib,

Zelboraf, B-Raf V600E, C-Raf,

RG7204, ACK1 , FGR, KHS1 ,

R05185426 SRMS 918504-65-1 C23H18CIF2N303S

RAF265, CHIR- 265 B-Raf, C-Raf, VEGFR 927880-90-8 C24H16F6N60

Regorafenib, BAY c-Raf, VEGFR1 ,

73-4506, Fluoro- VEGFR2, VEGFR3,

Sorafenib PDGFRβ, Kit, RET 755037-03-7 C21 H15CIF4N403

R05126766 Raf/MEK1/2 B-Raf (10fold more

SB 590885 potent than for C-Raf) 405554-55-4 C27H27N502

Sorafenib

Tosylate,

Nexavar, Bay 43- c-Raf, B-Raf, VEGFR- 9006 2, PDGFR-beta 475207-59-1 C21 H16CIF3N403.C7H803S

c-Raf, (B-Raf 10fold

ZM 336372 less) 208260-29-1 C23H23N303

[068] In particular examples, the RAF inhibitor is a small interfering nucleotide

sequence capable of inhibiting RAF activity, such as siRNA using one or more small double stranded RNA molecules. For example, RAF activity in a cell can be decreased or knocked down by exposing (once or repeatedly) the cell to an effective amount of the appropriate small interfering nucleotide sequence. 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. A variety of techniques can be used to assess interference with RAF activity of such small interfering nucleotide sequence, such as described in WO 2005/047542, for example by determining whether the candidate small interfering nucleotide sequence decreases BRAF activity. Candidate small interfering nucleotide sequences that are capable of interference may be selected to further analysis to determine whether they also inhibit proliferation of melanoma cells, for example by assessing whether changes associated with inhibition of proliferation of melanoma cells occurs in melanoma cells.

[069] The RAF inhibitor according to the present invention may be a binding agent such as an antibody which specifically binds activated and/or mutated BRAF such as the ones described in WO 2005/047542, or as described in US 2004/0096855.

[070] A RAF inhibitor has RAF inhibitor activity, or in other words reduces activated (or mutated) RAF activity, which activity may be verified by method known to the skilled person, for example those disclosed in EP0986382B1.

[071] A ERK polypeptide or peptide is to indicate 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 ERK1 (e.g. Gene accession number 5595; Refseq RNA Accessions NM_001040056.2; protein NP_Q0 035145.1) or ERK2 (e.g. Gene accession number 5594; Refseq RNA Accessions NM_002745.4; protein NP_002736.3). The amino acid sequence of ERK enzymes, other proteins mentioned herein, and variations thereof are available in GenBank, accessible via http://www.ncbi.nlm.nih.gov/genbank/ by entering either the numbers mentioned above or entering the relevant protein name.

[072] By ERK biological activity is meant any function of ERK, such as enzymatic activity, kinase activity, the ability to phosphorylate an ERK substrate, or signaling the MAPK/ERK pathway.

[073] By ERK inhibitor is meant a compound that reduces the biological activity of ERK; or that reduces the expression of an mRNA encoding an ERK polypeptide; or that reduces the expression of an ERK polypeptide. An ERK inhibitor can inhibit one member, several members or all members of the family of ERK kinases.

[074] ERK (extracellularly regulated kinase) is a group of MAP kinases which regulate the growth and proliferation of cells (Bokemeyer et al. 1996, Kidney Int. 49, 1187).

[075] Embodiments of the invention include an ERK inhibitor that inhibits or reduces ERK protein expression, amount of ERK protein or level of ERK translation, amount of ERK transcript or level of ERK transcription, stability of ERK protein or ERK transcript, half-life of ERK protein or ERK transcript, prevents the proper localization of an ERK protein or transcript; reduces or inhibits the availability of ERK polypeptide, reduces or inhibits ERK activity; reduces or inhibits ERK, binds ERK protein, or inhibits or reduces the post-translational modification of ERK, including its phosphorylation. In analogy, the above described inhibitory action are also to be construed to apply, in comparable fashion to any inhibitor described herein for its specific target (e.g. a BRAF inhibitor for

BRAF, a p90RSK inhibitor for p90RSK etc.). In some embodiments the inhibitor is a selective inhibitor.

[076] In some embodiments of the present invention, the ERK inhibitor is an ERK inhibitor such 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, HR20110892, WO201 1163330, TW200934775, EP2332922, WO2011041152, US2011038876, WO2009146034, HK11 17159, WO2009026487, WO2008115890, US2009186379, WO2008055236, US2007232610, WO2007025090, and US2007049591. Reference is made to said documents with respect to their content regarding ERK inhibitors, and methods for making the same.

[077] Examples or ERK-inhibitors are also shown in Table 3 below.

[078] Table 3: ERK inhibitors

compound cellular

name target(s) CAS number formula BVD-523 ERK1/2

FR 180204 ERK1/2 865362-74-9 C18H 13N7

Hypothemycin 76958-67-3 C19H2208

MK-8353,

SCH900353 ERK1

Pluripotin ERK1 , Ras-GAP 839707-37-8 C27H25F3N802

SCH772984 ERK1/2 942183-80-4 C34H34N802

VX-11⁶, ERK- 1 1e, TCS ERK

11 e ERK2 896720-20-0 C24H20CI2FN5O2

[079] In particular examples, the ERK inhibitor is a small interfering nucleotide

sequence capable of inhibiting ERK activity, such as siRNA using one or more small double stranded RNA molecules. For example, ERK activity in a cell can be decreased or knocked down by exposing (once or repeatedly) the cell to an effective amount of the appropriate small interfering nucleotide sequence. 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. Candidate small interfering nucleotide sequences that are capable of interference may be selected to further analysis to determine whether they also inhibit proliferation of melanoma cells, for example by assessing whether changes associated with inhibition of proliferation of melanoma cells occurs in melanoma cells.

[080] The skilled person knows that analogues, derivatives or modified versions of the above-documented ERK inhibitors may be used in the context of the present invention, as long as such analogues, derivatives or modified versions have ERK inhibitor activity.

[081] The ERK inhibitor according to the present invention may be a binding agent such as an antibody which specifically binds ERK, thereby inhibiting its function.

[082] ERK inhibitor activity may be assayed in vitro, in vivo or in a cell line. In vitro assays include assays that determine inhibition of either the kinase activity or ATPase activity of activated ERK. Alternate in vitro assays quantitate the ability of the inhibitor to bind to ERK and may be measured either by radiolabelling the inhibitor prior to binding, isolating the inhibitor/ERK complex and determining the amount of radiolabel bound, or by running a competition experiment where new inhibitors are incubated with ERK bound to known radioligands. One may use any type or isoform of ERK, depending upon which ERK type or isoform is to be inhibited. An example of measuring ERK inhibitory activity is described in EP 1317453 B1.

[083] A MEK polypeptide (e.g. Gene accession numbers 5604 or 5605; Refseq RNA

Accessions NMJ)Q2755.3 or NMJ330662.3; protein NP_002746.1 or NP_109587.1), protein or peptide is to indicate a polypeptide having serine/threonine protein kinase activity. For example MEK1 (e.g. Genbank Accession NO: NP002746) and MEK2 (e.g. Genbank Accession NO: NP109587) phosphorylates and activates MAPK. Another example is MEK3 ((e.g. Genbank Accession NO: NP002747). 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, MKK2 are the same enzyme, known as MEK2. MEK1 and MEK2, together MEK, can phosphorylate serine, threonine and tyrosine residues in protein or peptide substrates. To date, few cellular substrates of MEK isoforms have been identified. The amino acid sequence of MEK enzymes, other proteins mentioned herein, and variations thereof are available in GenBank, accessible via http://www.ncbi.nlm.nih.gov/genbank/ by entering either the numbers mentioned above or entering the relevant protein name.

[084] By MEK biological activity is meant any function of MEK, such as enzymatic

activity, kinase activity, or signaling the MAPK/ERK pathway.

[085] By MEK inhibitor is meant a compound that reduces the biological activity of MEK; or that reduces the expression of an mRNA encoding a MEK polypeptide; or that reduces the expression of a MEK polypeptide. A MEK inhibitor can inhibit one member, several members or all members of the family of MEK kinases. In one embodiment the MEK inhibitor is a selective inhibitor.

[086] Preferred MEK inhibitors, already known in the art, include but are not limited to the MEK inhibitors PD184352 and PD98059, inhibitors of MEK1 and MEK2 U0126 (see Favata, M., et al., Identification of a novel inhibitor of mitogen-activated protein kinase. 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). In particular, PDI 84352 (Allen, Lee et al Seminars in Oncology, Oct. 2003, pp. 105-106, vol. 30) has been found to have a high degree of specificity and potency when compared to other known MEK inhibitors, and may thus be preferred. A preferred MEK inhibitor GSK1 120212/Trametinib (GlaxxoSmithKline) has been approved for treatment of BRAF mutant melanoma under the name Mekinist.

MEK162 (Novartis) is also preferred. Other MEK inhibitors and classes of MEK inhibitors are described in Zhang et al. (2000) Bioorganic & Medicinal Chemistry Letters; 10:2825- 2828. [087] Further MEK inhibitors are for example described in Tecle et al Medicinal Chemistry Letters Volume 19, Issue 1 , 1 January 2009, Pages 226-229;

WO2009018238, WO2007/044084, WO2005/051300, WO2011/095807,

WO2008124085, WO2009018233, WO20071 13505, US201 1105521 , WO201 1067356, WO2011067348, US2010004247, and US2010130519. Reference is made to said documents with respect to their content regarding MEK inhibitors, and methods for making the same. GSK1120212 is an example of a further MEK inhibitor.

[088] The MEK inhibitor may also preferably be selected from AZD6244, 4-(4-Bromo-2- fluorophenylamino)-N-(2-hydroxyethoxy)- 1 ,5-dimethyl-6-oxo- 1 ,6-dihydropyridazine-3- carboxamide or 2-(2-fluoro-4-iodophenylamino)-N-(2-hydroxyethoxy)- 1 ,5-dimethyl-6- oxo-l,6-dihydropyridine-3-carboxamide. In one embodiment the MEK inhibitor is selected from AZD6244 or a pharmaceutically acceptable salt thereof. In one embodiment the MEK inhibitor is AZD6244 hydrogen sulphate salt. AZD6244 hydrogen sulphate salt and derivates thereof may be synthesized according to the process described in

WO2007/076245.

[089] In another embodiment the MEK inhibitor is selected from 6-(4-Bromo-2-chloro- phenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylic acid (2-hydroxy- ethoxy)-amide or a pharmaceutically acceptable salt thereof. In one embodiment the MEK inhibitor is 6-(4-Bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H- benzoimidazole-5- carboxylic acid (2-hydroxy-ethoxy)-amide hydrogen sulphate salt. 6-

(4-Bromo-2-chloro- phenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylic acid (2-hydroxy- ethoxy)-amide hydrogen sulphate salt may be synthesized according to the process described in International Patent Publication Number WO2007/076245.

[090] Furthermore, according to the invention the MEK inhibitor may be selected from the group consisting of certain experimental compounds, some of which are currently in

Phase 1 or Phase II studies, namely PD-325901 (Phase 1 , Pfizer), XL518 (Phase 1 , Genentech), PD-184352 (Allen and Meyer Semin Oncol. 2003 Oct;30(5 Suppl 16): 105- 16.), PD- 318088 (Tecle et al nic & Medicinal Chemistry Letters Volume 19, Issue 1 , 1 January 2009, Pages 226-229), AZD6244 (Phase II, Dana Farber, AstraZeneca) and Cl- 1040 (Lorusso et al Journal of clinical oncology 2005, vol. 23, no23, pp. 5281-5293).

[091] In one embodiment the MEK inhibitor is selected from any one of AZD6244

(Example 10 of WO03/077914) or a pharmaceutically acceptable salt thereof, 2-(2- fluoro-4-iodophenylamino)-N-(2-hydroxyethoxy)- 1 ,5-dimethyl-6-oxo- 1 JS- dihydropyridine-3-carboxamide or a pharmaceutically acceptable salt thereof, 4-(4- Bromo-2- fluorophenylamino)-N-(2-hydroxyethoxy)- 1 ,5-dimethyl-6-oxo- 1 ,6- dihydropyridazine-3- carboxamide or a pharmaceutically acceptable salt thereof, PD- 0325901 (Pfizer), PD- 184352 (Pfizer), XL-518 (Exelixis), AR-1 19 (Ardea Biosciences, Valeant Pharmaceuticals), AS- 701 173 (Merck Serono), AS-701255 (Merck Serono), 360770-54-3 (Wyeth).

[092] Examples of drugs that inhibit MEK include PD-0325901 (Pfizer), AZD-8330

(AstraZeneca), RG-7167 (Roche/Chugai), RG-7304 (Roche), CIP-137401

(Cheminpharma), WX-554 (Wilex; UCB), SF-2626 (Semafore Pharmaceuticals Inc), RO-

5068760 (F Hoffmann-La Roche AG), RO-4920506 (Roche), G-573 (Genentech) and G- 894 (Genentech), N-acyl sulfonamide prodrug GSK-2091976A (GlaxoSmithKline), Bl- 847325 (Boehringer Ingelheim), WYE-130600 (Wyeth/Pfizer), ERK1-624, ERK1-2067, ERK1-23211 , AD-GL0001 (ActinoDrug Pharmaceuticals GmbH), selumetinib

(AZD6244), trametinib, TAK-733, Honokiol, MEK-162, derivates, and salts thereof.

[093] Examples of MEK-inhibitors are shown in Table 4

Table 4: MEK-inhibitors

compound name CAS number formula

10Z-Hymenialdisine, SK&F

108752 82005-12-7 C1 1 H10BrN5O2

Arctigenin 7770-78-7 C21 H2406

AST03026, Pimasertib,

MSC1936369B 1236699-92-5 C15H 15FIN303

AZD6244, ARRY-14288,

Selumetinib 606143-52-6 C17H15BrCIFN403

AZD8330, ARRY-424704,

ARRY-704 869357-68-6 C16H 17FIN304

CI-1040, PD 184352 212631-79-3 C17H14CIF2IN202

GDC-0973, XL518, XL 518,

XL-518 934660-93-2 C22H22F3IN202

GSK1120212, JTP-74057,

trametinib, Mekinist 871700-17-3 C26H23FIN504

MEK162, Arry-162 606143-89-9 C17H15BrF2N403

PD 0325901 391210-10-9 C16H14F3IN204

PD 184352 212631-79-3 C17H14CIF2IN202

PD 198306 212631-61-3 C18H16F3IN202

PD 318088 391210-00-7 C16H13BrF3IN204

PD 98059 167869-21-8 C16H13N03

RDEA119, Refametinib,

BAY 869766, BAY86-9766 923032-37-5 C19H20F3IN2O5S

R04987655 R05126766

SL327 305350-87-2 C16H12F3N3S

TAK-733, TAK733 1035555-63-5 C17H15F2IN404

U0126 1173097-76-1 C18H16N6S2

WX-554

[094] In another embodiment the MEK inhibitor may inhibit (gene) expression of MEK, for example by interfering with mRNA stability or translation. In one embodiment the MEK inhibitor is selected from small interfering RNA (siRNA), which is sometimes known as short interfering RNA or silencing RNA, or short hairpin RNA (shRNA), which is sometimes known 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.

[095] The MEK inhibitor according to the present invention may be a binding agent such as an antibody which specifically binds MEK, thereby inhibiting its function.

[096] A number of assays for identifying kinase inhibitors, including MEK inhibitors, are known, for example from Downey et al. (1996) J Biol Chem.; 271 (35): 21005- 21011 or EP2496575.

[097] The person skilled in the art will understand that any combination as disclosed herein may be present in the combination as disclosed herein.

[098] The combination therapy disclosed herein is useful in the treatment of patients having a cancer selected from the group consisting of NRAS-, KRAS- and BRAF- mutated cancers. However, the combination is in particular useful in the treatment of patients having melanoma, i.e. NRAS-mutated melanoma, KRAS-mutated-melanoma or

BRAF-mutated melanoma. In a preferred embodiment the combination treatment is for patients with NRAS-mutated melanoma. The cancer, e.g. melanoma, e.g. NRAS mutant melanoma, maybe a naive cancer (previously untreated with anti-cancer drugs, e.g. with an inhibitor on the MAPK/ERK pathway; another inhibitor, chemotherapy or Radiation therapy), or may be a cancer that is resistant or acquired resistance as a consequence of prior treatment (e.g. with an inhibitor on the MAPK/ERK pathway; another inhibitor, chemotherapy or Radiation therapy), as can be witnessed from the examples disclosed herein. The findings support a combination therapy of inhibitors of the MAPK/ERK pathway (ERK-, RAF and/or MEK-inhibitors) and p90RSK inhibitors for NRAS, KRAS and/or BRAF mutated cancers, in particular NRAS mutant melanoma, both treatment- naive and with acquired resistance to MAPK/ERK pathway, e.g. ERK pathway, targeting therapies. [099] In some embodiments, the combination disclosed herein and the use of the disclosed combination in the treatment of the type of cancers disclosed herein may further be combined with other drugs or treatments, for example with (the use of) chemotherapy and/or radio therapy.

[100] Melanoma is, next to basal cell cancer and squamous cell cancer, one of the three most serious types of skin cancer. Skin cancer is the most commonly diagnosed type of cancer. Although less common than the other two types, melanoma causes the majority (75%) of deaths related to skin cancer (Jerant et al 2000 Am Fam Physician 62 (2): 357-68, 375-6, 381-2). Worldwide, about 160,000 new cases of melanoma are diagnosed yearly. It occurs more frequent in women than in men and is particularly common among Caucasians living in sunny climates, with high rates of incidence in Australia, New Zealand, North America, Latin America, and northern Europe (Parkin et al 2005 CA Cancer J Clin 55 (2): 74-108). According to a WHO report, melanoma cause about 48,000 deaths worldwide per year (Lucas et al 2006 Environmental Burden of Disease Series. 13. World Health Organization. ISBN 92 4 159440 3).

[101] The first sign of melanoma often is a change in size, shape, color or feel of a mole, which may accordingly have turned into a malignant tumor of melanocytes. The present invention relates to combinations suitable for treating or lessening the severity of melanoma, including subtypes of melanoma such as but not limited to superficial spreading melanoma, nodular melanoma, lentigo maligna melanoma, acral lentiginous melanoma, and choroidal intraocular melanoma, ocular intraocular melanoma, and uveal intraocular melanoma.

[102] Melanocytes are cells that normally produce the pigment melanin, which is

responsible for brownish tint of skin. They predominantly occur in skin, but are also found in other parts of the body, such as in the bowel and the eye (uveal melanoma).

Melanoma can occur in any part of the body where melanocytes are present.

[103] Cutaneous melanomas have mutations in the NRAS GTPase in 15% of cases (NRAS mutated cancer, NRAS mutated melanoma; Kelleher et. al (2012 Cancer J.18(2): 132-136)). Compared to melanomas with BRAF mutations, or melanomas "wild- type" for BRAF and NRAS, melanomas with NRAS mutations are more likely to be thicker tumors and to have a higher mitotic rate. Preclinical studies indicate that melanoma cells with NRAS mutations are dependent on NRAS for survival and proliferation, making NRAS an attractive therapeutic target in melanoma. However, to date, therapeutic strategies for NRAS mutant melanomas have not been realized in the art.

[104] In one embodiment disclosed herein, in the combination, the p90RSK-inhibitor is BI-D1870, the RAF-inhibitor is selected from the group consisting of PLX4720

(Vemurafenib), and GSK2118436 (Dabrafenib), the ERK-inhibitor is selected from the group consisting of SCH772984 and VTX-11 e, and/or the MEK-inhibitor is selected from the group consisting of GSK1120212 (Trametinib) and MEK162.

[105] Also provided is a p90RSK-inhibitor for use in treatment of a cancer selected from the group consisting of NRAS-, KRAS- and BRAF-mutated cancer, preferably NRAS-mutated cancer, wherein the p90RSK-inhibitor is administrated simultaneously, separately or sequentially with an inhibitor of another protein of the MAPK/ERK pathway. In a preferred embodiment there is provided for the p90RSK-inhibitor for use in treatment of a cancer selected from the group consisting of NRAS-, KRAS- and BRAF- mutated cancer, preferably NRAS-mutated cancer, wherein said inhibitor of another protein of the MAPK/ERK pathway is selected from the group consisting of a RAF- inhibitor, an ERK-inhibitor and a MEK-inhibitor.

[106] Likewise is provided for a RAF-inhibitor for use in treatment of a cancer selected from the group consisting of NRAS-, KRAS- and BRAF-mutated cancer, preferably NRAS-mutated cancer, wherein the RAF-inhibitor is administrated simultaneously, separately or sequentially with a p90RSK-inhibitor.

[107] Accordingly, there is provided for an ERK-inhibitor for use in treatment of a

cancer selected from the group consisting of NRAS-, KRAS- and BRAF-mutated cancer, preferably NRAS-mutated cancer, wherein the ERK-inhibitor is administrated

simultaneously, separately or sequentially with a p90RSK-inhibitor.

[108] In addition, there is provided for a MEK-inhibitor for use in treatment of a cancer selected from the group consisting of NRAS-, KRAS- and BRAF-mutated cancer, preferably NRAS-mutated cancer, wherein the MEK-inhibitor is administrated

simultaneously, separately or sequentially with a p90RSK-inhibitor.

[109] In a preferred embodiment disclosed herein, in the combination, the p90RSK- inhibitor is BI-D1870, the RAF-inhibitor is selected from the group consisting of PLX4720

(Vemurafenib), and GSK2118436 (Dabrafenib), the ERK-inhibitor is selected from the group consisting of SCH772984 and VTX-11 e, and/or the MEK-inhibitor is selected from the group consisting of GSK1120212 (Trametinib) and MEK162.

[1 10] In a preferred embodiment of the use, the cancer is NRAS mutated melanoma.

[1 11] The inhibitors for the combination therapy as disclosed herein may be

administered to the patient either simultaneously, separately or sequentially with the other drug(s) of the combination. For example, in practice the product leaflet of the p90RSK-inhibitor may suggest the simultaneous, separate or sequential use of the p90RSK-inhibitor with an inhibitor of the another protein of the MAPK/ERK-pathway, preferably an ERK inhibitor and/or a MEK-inhibitor and/or a RAF-inhibitor.

[1 12] In another example, in practice the product leaflet of the inhibitor of another protein of the MAPK/ERK pathway (preferably an ERK inhibitor and/or a MEK-inhibitor and/or a RAF-inhibitor) may suggest the simultaneous, separate or sequential use of the inhibitor of a protein of the MAPK/ERK pathway with a p90RSK-inhibitor.

[1 13] As explained above, the new use of the combination of inhibitors is not limited to combinations administered separately, but also includes the compositions obtained by physical association of the drugs and in either case a synergistic effect is obtained.

[1 14] As used herein "simultaneous" administration 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 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 indicates that the administration of a first drug if followed, immediately or in time, by the administration of the second drug.

[1 15] The combination of drugs disclosed herein will preferably be administered to the patient in a form that is suitable for administration to the patient and in a dose that is efficacious, i.e. in an effective amount.

[1 16] The current disclosure thus relates, in these aspects, to a combination therapy, wherein during the therapy the patient is treated with a drug that is an inhibitor of p90RSK in combination with (another) inhibitor that inhibits another protein of the MAPK ERK pathway (i.e. not p90RSK), preferably an ERK-inhibitor, a MEK-inhibitor, and/or a RAF-inhibitor.

[1 17] In another aspect of the invention there is provided for a product comprising a p90RSK-inhibitor and an inhibitor of another protein of the MAPK/ERK pathway, as a combined preparation for simultaneous, separate or sequential use in treatment of a cancer selected from the group consisting of NRAS-, KRAS- and BRAF-mutated cancer, preferably NRAS-mutated cancer, even more preferably NRAS-mutated melanoma. In a preferred embodiment, said inhibitor of the another protein of the MAPK/ERK pathway is selected from the group consisting of a RAF-inhibitor, an ERK-inhibitor, and/or a MEK- inhibitor. In another preferred embodiment, in the product, the p90RSK-inhibitor is Bl- D1870, the RAF-inhibitor is selected from the group consisting of PLX4720

(Vemurafenib), and GSK21 18436 (Dabrafenib), the ERK-inhibitor is selected from the group consisting of SCH772984 and VTX-11 e, and/or the MEK-inhibitor is selected from the group consisting of GSK1120212 (Trametinib) and MEK162.

[1 18] In another aspect of the invention there is provided for use of a p90RSK-inhibitor in the manufacture of a medicament for the treatment of a cancer selected from the group consisting of NRAS-, KRAS- and BRAF-mutated cancer, preferably NRAS- mutated cancer, wherein the treatment comprises the simultaneous, separate or sequential administration of the p90RSK-inhibitor and an inhibitor of another protein of the MAPK/ERK pathway.

[1 19] Also provided is for the use of an inhibitor of a protein of the MAPK/ERK

pathway, wherein the protein is another protein than p90RSK, in the manufacture of a medicament for the treatment of a cancer selected from the group consisting of NRAS-,

KRAS- and BRAF-mutated cancer, preferably NRAS-mutated cancer, wherein the treatment comprises the simultaneous, separate or sequential administration of a p90RSK-inhibitor and the inhibitor of the another protein of the MAPK/ERK pathway.

[120] Preferably, said inhibitor of another protein of the MAPK/ERK pathway is

selected from the group consisting of a RAF-inhibitor, an ERK-inhibitor, and/or a MEK- inhibitor. Preferably, the cancer is melanoma, preferably NRAS-mutated melanoma. In another preferred embodiment, in the use, the p90RSK-inhibitor is BI-D1870, the RAF- inhibitor is selected from the group consisting of PLX4720 (Vemurafenib), and

GSK21 18436 (Dabrafenib), the ERK-inhibitor is selected from the group consisting of SCH772984 and VTX-11 e, and/or the MEK-inhibitor is selected from the group consisting of GSK1120212 (Trametinib) and MEK162.

[121] In a last aspect, there is provided for a method for the treatment of a cancer selected from the group consisting of NRAS-, KRAS- and BRAF-mutated cancer, preferably NRAS-mutated cancer, wherein the method comprises the simultaneous, separate or sequential administering to a patient of a p90RSK-inhibitor and an inhibitor of another protein of the MAPK/ERK pathway. Preferably, said inhibitor of another protein of the MAPK/ERK pathway is selected from the group consisting of a RAF- inhibitor, an ERK-inhibitor, and/or a MEK-inhibitor. Preferably, the cancer is melanoma, preferably NRAS-mutated melanoma. In another preferred embodiment, in the method, the p90RSK-inhibitor is BI-D1870, the RAF-inhibitor is selected from the group consisting of PLX4720 (Vemurafenib), and GSK2118436 (Dabrafenib), the ERK-inhibitor is selected from the group consisting of SCH772984 and VTX-11 e, and/or the MEK- inhibitor is selected from the group consisting of GSK1120212 (Trametinib) and

MEK162.

[122] The treatment of the patient includes treatment in the first line or second line, or third line. In particular the disclosure herein can advantageously be used in patients that, e.g. in monotherapy, show reduced response to the use of an MEK-inhibitor, a ERK- inhibitor, a RAF-inhibitor, either from the start, or after a certain period of treatment with the MEK-inhibitor, ERK-inhibitor, and/or RAF-inhibitor, for example patients that are resistant to such inhibitor.

[123] The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The described embodiments are to be considered in all respects as illustrative only and not restrictive. The scope of the invention is, therefore, indicated by the appended claims rather than by the foregoing description. All changes which come within the meaning and range of equivalency of the claims are to be embraced within their scope.

[124] A list of preferred inhibitors,

BRaf inhibitor: PLX4720 (Vemurafenib), GSK21 18436 (Dabrafenib)

MEK inhibitor: GSK1120212 (Trametinib), MEK162

ERK inhibitor: SCH772984, VTX-11 e

p90RSK inhibitor: BI-D1870

Examples

Example 1

Summary

[125] Malignant melanoma is an example of a highly aggressive cancer with rising incidence worldwide and, once metastasized, is notoriously difficult to treat. The most commonly mutated genes in cutaneous melanoma are BRAF and NRAS, whereas oncogenic KRAS is also observed in a minority of the cases. Targeted therapies are now used for BRAF, but are lacking for NRAS. MEK inhibitors show promise as future drugs for the treatment of NRAS mutant melanoma, but will probably be insufficient as single agents due to a limited response rate and toxicity issues. In fact, NRAS and BRAF mutant melanoma patients develop resistance to MEK inhibitor MEK162 already after a median of 3.7 and 3.3 months, respectively (Ascierto et al. (2013), Lancet Oncol. 14, 249-256). It is known that intrinsic and acquired resistance to targeted agents can limit their therapeutic effect.

[126] We screened a NRAS mutant melanoma cell line to identify kinases that become essential for cell survival and growth when cells are treated with a MEK inhibitor. We validated p90RSK2 as a hit in a panel of treatment-naive NRAS mutant melanoma cell lines with shRNAs and extended the validation to a larger panel of NRAS or BRAF mutant melanoma cell lines with a p90RSK inhibitor (BI-D1870), which inhibits all 4 isoforms equally. p90RSK inhibition sensitizes melanoma cells to inhibitors targeting the MAPK/ERK pathway at different points in the signaling cascade (BRaf, MEK, or ERK).

[127] In addition, we also validated p90RSK as drug target in cell line models of acquired resistance. To this end we created resistant sublines of NRAS mutant melanoma by long-term culture in the presence of inhibitors of the MAPK/ERK pathway. ERK phosphorylation is required for survival and growth. In sensitive melanoma cell lines MEK or ERK inhibitors inhibit ERK phosphorylation. Therefore, sensitive lines cannot survive or grow in presence of MEK or ERK inhibitors. The ERK inhibitor resistant melanoma sublines we created have reactivated ERK signaling, i.e. the levels of phosphorylated p90RSK in the presence of ERK inhibitor in these resistant sublines are as high as in untreated parental cell lines. Therefore, these resistant sublines are able to survive and grow in the presence of MEK or ERK inhibitors. Since these ERK inhibitor resistant sublines show MAPK/ERK pathway reactivation to a level seen in their treatment-naive counterparts, it stands to reason that they remain dependent on the MAPK/ERK pathway. We found that the ERK inhibitor resistant melanoma sublines of NRAS mutant melanoma can be killed by a combination treatment of ERK or MEK inhibitor with p90RSK inhibitor, just like their treatment-naive counterparts. Therefore, targeting the MAPK/ERK pathway at multiple points in the signalling cascade is not only effective in, for example, treatment- naive NRAS and BRAF mutant melanoma cell lines, but also in MAPK/ERK pathway inhibitor resistant sublines of e.g. NRAS mutant melanoma. Simultaneous inhibition of several components of a pathway instead of a single component can be beneficial, because it inhibits the pathway more effectively. Clinical experience with BRaf^V600E and MEK inhibitors has shown that almost complete pathway inhibition (i.e. reduction in ERK activity by about 90%) is required to achieve a therapeutic effect. Inhibition of a single pathway component can even have paradoxical activation effects by relieving feedback inhibition of the same pathway via down- regulation of the activity of this component. Furthermore, the therapeutic effect on the tumor and dose-limiting toxicities are intimately linked for MAPK/ERK pathway inhibitors. Such side effects have prompted patients to stop treatment even though an effect on tumor growth was apparent (Ascierto et al. (2013), Lancet Oncol. 14, 249-256). Importantly, the combination treatments we propose could provide a therapeutic window: they would allow to lower the dose of the individual drugs in a fashion that will reduce side effects and retain therapeutic activity.

Materials and methods

Inhibitors

[128] We used the MEK inhibitor GSK1120212 (Selleck), ERK inhibitor SCH772984 (Merck), pan-p90RSK inhibitor BI-D1870 (Enzo Life Sciences), and BRaf inhibitor PLX4720 (Selleck).

Cell culture and inhibitor treatments

[129] Melanoma cell line sources: Leiden University Medical Center:Mel 95.03, ATCC:

A375, Other sources: BLM, SK-MEL-147. BRAF mutation: V600E (mel 93.03, A375).

NRAS mutations: Q61 R (BLM, SK-MEL-147).

[130] All cell lines were cultured in Dulbecco's modified Eagle's Medium (DMEM,

Gibco) supplemented with 9% fetal calf serum (Sigma) and 1 % glutamine. To generate ERK inhibitor resistant sublines, cells were plated on 10cm dishes. Medium containing 1 μΜ SCH772984 was added the next day and changed twice a week. After 2 weeks colonies were picked and expanded in the presence of the inhibitor. In addition, multiple colonies per 10cm dish were trypsinized and expanded to obtain resistant pools.

[131] For short-term viability assays of inhibitors, cells were plated in 96-well format and treated on the next day with a dilution range of one inhibitor with or without a second inhibitor with a concentration as mentioned in the figure. After 3 days, cells were incubated with CellTiter-Blue (Promega) and fluorescence was measured with a TECAN Infinite M200 microplate reader. For proliferation assays, equal cell numbers were plated on 6 well dishes. The next day, cells were treated with one or more inhibitors and refreshed with medium with inhibitors every 3-4 days. Cells were stained with crystal violet after 7-1 1 days of treatment.

[132] For Western blot analysis, cells were plated on 10cm dishes. Cells were treated on the next day with single inhibitors or combinations thereof and harvested 24 hours later.

Immunoblotting and antibodies

[133] Cells pellets were lysed in RIPA buffer (50 mM TRIS pH8.0, 150 mM NaCI, 1 % Nonidet P40, 0.5% sodium deoxycholate, 0.1 % SDS) and the protein concentration was determined using BioRad Protein Assay. Immunoblot analysis was performed using standard techniques on 4-12% Bis-Tris polyacrylamide-SDS gels (NuPAGE). Antibodies used were: phospho-AKT (Ser473, #9271), p42/p44 (#9102), phospho-p42/p44 (Thr202/Tyr204, #9106), MEK (#4694), phospho-MEK (Ser217/221 , 41G9, #9154), PARP (#9542), RSK1/2/3 (p90) (#9355), phospho-S6RP (Ser235/236, #4856), phospho- S6RP (Ser240/244, #2215), and S6RP (#2217) (all from Cell Signaling), phospho-RSK1 (p90) (Thr359/Ser363, #04-419, Millipore), alpha-tubulin (DM 1A, Sigma), AKT1/2 (sc- 8312), (from Santa Cruz), p27 (610241 , BD Transduction Laboratories). Protein detection for Western blotting was performed by fluorescence detection with the Odyssey reader (LI-COR Biosciences).

Results

Treatment-naive melanoma cell lines are sensitive to combinations of MEK or ERK inhibitors with p90RSK inhibitors

[134] Throughout our studies we use a panel of human metastatic melanoma cell lines carrying the activating NRAS codon 61 or BRAF^V600E mutations to model drug/inhibitor sensitivity of treatment- naive melanoma; the panel includes various cell lines available in the prior art. Some cell lines were part of a large collection of low passage lines generated by the Leiden University Medical Center in The Netherlands. None of the lines have been exposed to targeted agents before explantation. NRAS mutation status of exon 3 or BRAF mutation status of exon 15 has been determined by conventional sequencing.

[135] The data suggests that in general single inhibitors would probably be insufficient to treat NRAS mutant melanoma. This was confirmed for a subset of cell lines and inhibitors targeting different pathways in long-term proliferation assays of 7-1 1 days in 6- well format (data not shown).

[136] To identify suitable targets for combination therapies we performed a high- throughput screen with a lentiviral hairpin library targeting and thereby downregulating the expression of all human kinases. We infected a MEK inhibitor-sensitive NRAS^{Q61 R} mutant metastatic melanoma cell line with the library and monitored hairpin abundance with next-generation sequencing. Hairpins targeting genes, which become essential for cell survival and growth in the presence of the MEK inhibitor, are depleted upon MEK inhibitor treatment and genes targeted by several hairpins per gene were scored as a hit. To further validate the hits, we created a sublibrary targeting the top 20 genes and screened 4 NRAS mutant melanoma lines with widely differing sensitivities to MEK inhibition based on the short-term viability assays. One of the top hits emerging from the validation screen was p90RSK, in particular p90RSK2, which is part of a family of four highly homologous ribosomal S6 protein kinases (S6RP) and directly downstream of ERK.

[137] Interestingly, while pan-p90RSK inhibitor BI-D1870 showed only modest activity as single agent in aforementioned viability assays, used at an IC₂o concentration it sensitized the tested NRAS mutant melanoma lines to MEK or ERK inhibitors producing an effect greater than the effect of the individual drugs, in an additive to synergistic fashion in long-term proliferation assays (Figure 1 and Figure 2). We also observed an at effect of the combination of ERK inhibitor and p90RSK inhibitor greater than that of the individual drugs on the proliferation of BRAF mutant melanoma lines (Figure 3). Note that the individual inhibitor concentrations were titrated to have little effect on their own (e.g. in the low nanomolar range for MEK inhibitor GSK1 120212), but produce an effect greater than the effect of the individual drugs, in an additive (co-operative) to synergistic interaction upon combination (Figures 1 , 2, 3). Thus, we validated the hit p90RSK in two independent manners, with genetic and with pharmacological means.

[138] Western blot analysis revealed that MEK, ERK and p90RSK inhibitors reduced levels of S6RP phosphorylation and total levels of Cyclin D1 and increased levels of apoptosis markers like cleaved caspase 3, cleaved PARP, Bim and cell cycle inhibitors like p27 (data not shown). MEK or ERK inhibitor resistant sublines of NRAS mutant melanoma are sensitive to a combination of ERK and p90RSK inhibitors

[139] To model resistance of NRAS mutant melanoma to agents targeting the MAPK pathway, we generated sublines resistant to ERK inhibitor SCH772984. We seeded multiple cell lines on 10cm dishes and changed the culture medium containing 1 μΜ of the inhibitor twice a week. After 2 weeks small colonies of a few cells with different morphology from the surrounding cells with senescence-like flat and broad morphology were picked and propagated. In addition, pools of resistant cells were generated by collecting the cells from colonies by short trypsinization, which left the senescent-like cells on the plate.

[140] Initial characterization of ERK inhibitor resistant sublines of, e.g. BLMindicated a much reduced sensitivity to the ERK inhibitor across all doses, with a significantly increased residual population. These cell lines show levels of phosphorylated p90RSK in the presence of the inhibitors that are as high as in untreated parental cell lines and S6RP, a substrate for p90RSK, is phosphorylated at serine 235, 236, 240, and 240 in the presence of ERK inhibitor (Figure 6). The sublines did not have increased expression of AKT and were as sensitive to the dual PI₃K/mTOR inhibitor PI-103 as the parental line, suggesting no increased dependence on the PI₃K pathway. Taken together these results suggest that the resistant sublines have reactivated MAPK/ERK signaling instead of upregulating PI₃K signaling.

[141] These findings strongly suggested that not only treatment-naive NRAS mutant melanoma lines, but also NRAS mutant melanoma sublines with an acquired resistance to MAPK pathway inhibitors could be amenable to combination treatment with MEK and p90RSK inhibitors, because they have activated MAPK/ERK signaling and might depend on it for growth and survival. Indeed, similarly to the parental line BLM, which shows an increased effect of the combination of MEK or ERK inhibitors with a p90RSK inhibitor on cell viability in long-term assays compared to single agents, also the ERK inhibitor resistant sublines of BLM respond to the combination of ERK and p90RSK inhibitors (Figure 4). Importantly, a response to a combination of MEK and p90RSK inhibitors was also observed for ERK inhibitor resistant sublines of BLM (Figure 5).

[142] We demonstrated efficacy of combinations of MEK or ERK inhibitors with p90RSK inhibitors in both treatment-naive and MAPK/ERK pathway resistant NRAS mutant melanoma lines and combinations of BRaf, MEK, or ERK inhibitors with p90RSK inhibitors in treatment-naive BRAF mutant melanoma lines. Our findings suggest that such inhibitor combinations will be effective in BRAF mutant melanoma with intrinsic or acquired resistance to drugs targeting the MAPK/ERK pathway like the clinically approved Vemurafenib and Dabrafenib. One major cause of resistance to these drugs is the occurrence of activating NRAS mutations, which lead to MAPK/ERK pathway reactivation and are identical to the mutations found in the NRAS mutant melanoma lines we used for the drug and genetic screens.

Claims

Claims

1. A combination of a p90RSK- inhibitor and an inhibitor of another protein of the

MAPK/ERK pathway in the treatment of a cancer selected from the group consisting of NRAS-, KRAS- and BRAF-mutated cancer, preferably a NRAS-mutated cancer.

2. The combination of claim 1 wherein said inhibitor of another protein of the MAPK/ERK pathway is selected from the group consisting of a RAF-inhibitor, an ERK-inhibitor and a MEK-inhibitor.

The combination of any one of claims 1 - 2, wherein the cancer is melanoma, preferably NRAS-mutated melanoma, or wherein the cancer is melanoma, preferably NRAS- mutated melanoma, that is resistant to a RAF-inhibitor, an ERK-inhibitor and/or a MEK- inhibitor.

4. The combination of any one of claims 1 - 3, wherein

a. the p90RSK-inhibitor is BI-D1870;

b. the RAF-inhibitor is selected from the group consisting of PLX4720

(Vemurafenib), and GSK2118436 (Dabrafenib);

c. the ERK-inhibitor is selected from the group consisting of SCH772984 and VTX- 1 1e ; and/or

d. the MEK-inhibitor is selected from the group consisting of GSK1120212 (Trametinib) and MEK162 .

5. A p90RSK-inhibitor for use in treatment of a cancer selected from the group consisting of NRAS-, KRAS- and BRAF-mutated cancer, preferably NRAS-mutated cancer, wherein the p90RSK-inhibitor is administrated simultaneously, separately or sequentially with an inhibitor of another protein of the MAPK/ERK pathway.

6. The p90RSK-inhibitor for use in treatment of a cancer of claim 5, wherein said inhibitor of another protein of the MAPK/ERK pathway is selected from the group consisting of a

RAF-inhibitor, an ERK-inhibitor and a MEK-inhibitor.

7. A RAF-inhibitor for use in treatment of a cancer selected from the group consisting of NRAS-, KRAS- and BRAF-mutated cancer, preferably NRAS-mutated cancer, wherein the RAF-inhibitor is administrated simultaneously, separately or sequentially with a p90RSK-inhibitor.

8. An ERK-inhibitor for use in treatment of a cancer selected from the group consisting of NRAS-, KRAS- and BRAF-mutated cancer, preferably NRAS-mutated cancer, wherein the ERK-inhibitor is administrated simultaneously, separately or sequentially with a p90RSK-inhibitor.

9. A MEK-inhibitor for use in treatment of a cancer selected from the group consisting of NRAS-, KRAS- and BRAF-mutated cancer, preferably NRAS-mutated cancer, wherein the MEK-inhibitor is administrated simultaneously, separately or sequentially with a p90RSK-inhibitor.

10. The p90RSK-inhibitor, RAF-inhibitor, ERK-inhibitor or MEK-inhibitor for use in treatment of a cancer of any one of claims 5 - 9, wherein

a. the p90RSK-inhibitor is BI-D1870;

b. the RAF-inhibitor is selected from the group consisting of PLX4720

(Vemurafenib), and GSK21 18436 (Dabrafenib);

c. the ERK-inhibitor is selected from the group consisting of SCH772984 and VTX- 1 1e ; and/or

d. the MEK-inhibitor is selected from the group consisting of GSK1120212

(Trametinib) and MEK162 .

1 1. The p90RSK-inhibitor, RAF-inhibitor, ERK-inhibitor or MEK-inhibitor for use in treatment of a cancer of any one of claims 5 - 10, wherein the cancer is melanoma, preferably NRAS-mutated melanoma, or wherein the cancer is melanoma, preferably NRAS- mutated melanoma, that is resistant to a RAF-inhibitor, an ERK-inhibitor and/or a MEK- inhibitor.

12. A product comprising a p90RSK-inhibitor and an inhibitor of another protein of the

MAPK/ERK pathway, as a combined preparation for simultaneous, separate or sequential use in treatment of a cancer selected from the group consisting of NRAS- KRAS- and BRAF-mutated cancer, preferably NRAS-mutated cancer, even more preferably NRAS-mutated melanoma.

13. The product of claim 12, wherein said inhibitor of another protein of the MAPK/ERK pathway is selected from the group consisting of a RAF-inhibitor, an ERK-inhibitor and a MEK-inhibitor.

14. The product of any one of claims 12 - 13 wherein

a. the p90RSK-inhibitor is BI-D1870; b. the RAF-inhibitor is selected from the group consisting of PLX4720

(Vemurafenib), and GSK2118436 (Dabrafenib);

c. the ERK-inhibitor is selected from the group consisting of SCH772984 and VTX- 1 1e ; and/or

d. the MEK-inhibitor is selected from the group consisting of GSK1120212

(Trametinib) and MEK162 .

15. Use of a p90RSK-inhibitor in the manufacture of a medicament for the treatment of a cancer selected from the group consisting of NRAS-, KRAS- and BRAF-mutated cancer, preferably NRAS-mutated cancer, wherein the treatment comprises the simultaneous, separate or sequential administration of the p90RSK-inhibitor and an inhibitor of another protein of the MAPK/ERK pathway.

16. Use of an inhibitor of a protein of the MAPK/ERK pathway, wherein the protein is

another protein than p90RSK, in the manufacture of a medicament for the treatment of a cancer selected from the group consisting of NRAS-, KRAS- and BRAF-mutated cancer, preferably NRAS-mutated cancer, wherein the treatment comprises the simultaneous, separate or sequential administration of a p90RSK-inhibitor and the inhibitor of the another protein of the MAPK/ERK pathway.

17. The use any one of claims 15 -16, wherein said inhibitor of the another protein of the MAPK/ERK pathway is selected from the group consisting of a RAF-inhibitor, an ERK- inhibitor and a MEK-inhibitor.

18. The use of any one of claims 15 -17, wherein the cancer is melanoma, preferably NRAS- mutated melanoma, or wherein the cancer is melanoma, preferably NRAS-mutated melanoma, that is resistant to a RAF-inhibitor, an ERK-inhibitor and/or a MEK-inhibitor.

19. The use of any one of claims 15 - 18, wherein

a. the p90RSK-inhibitor is BI-D1870;

b. the RAF-inhibitor is selected from the group consisting of PLX4720 (Vemurafenib), and GSK2118436 (Dabrafenib);

c. the ERK-inhibitor is selected from the group consisting of SCH772984 and VTX-1 1e ; and/or

d. the MEK-inhibitor is selected from the group consisting of GSK1120212

(Trametinib) and MEK162 .

20. A method for the treatment of a cancer selected from the group consisting of NRAS-, KRAS- and BRAF-mutated cancer, preferably NRAS-mutated cancer, wherein the method comprises the simultaneous, separate or sequential administering to a patient of a p90RSK-inhibitor and an inhibitor of another protein of the MAPK/ERK pathway.

21. The method of claim 20 wherein said inhibitor of another protein of the MAPK/ERK

pathway is selected from the group consisting of a RAF-inhibitor, an ERK-inhibitor and a MEK-inhibitor.

22. The method of any one of claims 20 - 22, wherein the cancer is melanoma, preferably NRAS-mutated melanoma, or wherein the cancer is melanoma, preferably NRAS- mutated melanoma, that is resistant to a RAF-inhibitor, an ERK-inhibitor and/or a MEK- inhibitor.

23. The method of any one of claims 20 -23, wherein

a. the p90RSK-inhibitor is BI-D1870;

b. the RAF-inhibitor is selected from the group consisting of PLX4720 (Vemurafenib), and GSK2118436 (Dabrafenib);

c. the ERK-inhibitor is selected from the group consisting of SCH772984 and VTX-1 1e ; and/or

d. the MEK-inhibitor is selected from the group consisting of GSK1120212

(Trametinib) and MEK162.