WO2013109142A1

WO2013109142A1 - Combined pdk and mapk/erk pathway inhibition in neoplasia

Info

Publication number: WO2013109142A1
Application number: PCT/NL2013/050021
Authority: WO
Inventors: Daniel Simon Peeper; Joanna KAPLON
Original assignee: Stichting Het Nederlands Kanker Instituut
Priority date: 2012-01-16
Filing date: 2013-01-16
Publication date: 2013-07-25

Abstract

The current inventors have found a new method for treatment of malignant neoplasia. In particular the invention relates to the field of treating neoplasia in subject in need thereof and that involve (activated) MAPK/ERK pathway proteins, like BRAF melanomas and the like. Compositions for use in such treatment are provided which make use of pyruvate dehydrogenase kinase (PDK) inhibitors.

Description

Combined PDK and MAPK/ERK pathway inhibition in neoplasia.

Technical Field The current disclosure relates to the field of treating neoplasia in subject in need thereof, in particular neoplasia that involve (activated) MAPK/ERK pathway proteins, like BRAF melanomas and the like.

Background of the invention

Cancer, malignant neoplasm, is a heterogeneous group of conditions characterized by abnormal proliferation of cells (neoplasia). Cancer is a leading cause of death.

Skin neoplasm relates to neoplasia of cell originating from the skin. 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)

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.

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. The treatment typically includes surgical removal of the melanoma, adjuvant treatment, chemo- and immunotherapy, and/or radiation therapy. The chance of a cure is greatest when the melanoma is discovered while it is still small and thin, and can be removed entirely. Approximately 40-60% of (cutaneous) melanomas carry a mutation in a specific protein kinase referred to as BRAF. 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. 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)).

Briefly, in healthy cells, the MAPK pathway is activated as follows. RAS receptor-ligand binding results in cytoplasmic BRAF protein being localized to the intracellular membrane surface by binding directly to RAS (Jaiswal et al., Mol Cell Biol. 14(10):6944- 53 (1994)), which, in turn, results in BRAF phosphorylation. Once phosphorylated, BRAF serine/threonine kinase activity is activated and the activated enzyme phosphorylates MEK, which is also referred to as MAPKK. MEK phosphorylation, in turn, activates its kinase activity, and it in turn phosphorylates ERK, which is also referred to as MAPK. Upon phosphorylation, ERK is translocated into the nucleus, where it phosphorylates transcription factors and thereby stimulates transcription of various genes involved in cell growth, differentiation and apoptosis (Peyssonnaux et al, Biol Cell. 93 (1-2) -.53-62 (2001)).

The pathway includes many other proteins, next to the above-mentioned MAPK (originally called ERK), BRAF and MEK (MAPKK). The latter one are examples of proteins that communicate by adding phosphate groups to a neighboring protein, which acts as an "on" or "off switch in the MAPK/ERK pathway.

In various cancers, as document above for BRAF, the MAPR/ERK pathway is dysregulated (see also Flaherty 201 1 : Nature Reviews: Drug Discovery 10:811), for example one or more of the proteins in the pathway may be mutated, leading the protein to be stuck in the "on" or "off position. One example is that in BRAF tumors, e.g. melanomas, mutated BRAF proteins typically have elevated kinase activity and as such may cause uncontrolled activation of the MAPK pathway, leading to uncontrolled proliferation of malignant tumor cells. Also the other components of the MAPK/ERK pathway are considered as important targets in attempts to control cancers (e.g. see Poulikakos et al. 2011 Science Signaling 4(166): 1) or Roberts et al.2007 Oncogene 26:3291). Drugs that reverse such "on" or "off" switch, and/or reduce the expression of a nucleic acid encoding a protein that is part of the pathway, and/or reduce expression of the protein that is part of the pathway and/or reduce the activity of a protein that is part of the pathway are being investigated as potential new drugs in cancer treatments.

One such drug is Vemurafenib (PLX4032), which is a potent inhibitor of mutated BRAF (Bollag et al Nature 2010;467:596-9). PLX4032 has remarkable clinical activity in patients with melanomas that contain the V600E mutation in BRAF (Flaherty et al N Engl J Med 2010; 363:809-19). However, although profound, responses to a BRAF inhibitor such as PLX4032 are temporary, and typically resistance to PLX4032 develops over time (reviewed in Solit et al. 201 1 ; N Engl J Med 364:8, and see also Flaherty 2011 : Nature Reviews: Drug Discovery 10:811)). To date, no adequate solution for overcoming PLX4032 resistance in melanoma patients has been proposed. Like for BRAF, development of resistance against inhibitors of other protein incolved in the MAPK/ERK pathway, in particular has been reported (e.g. see Poulikakos et al. 201 1 Science Signaling 4(166): 1) or Roberts et al.2007 Oncogene 26:3291

Description of the invention

Definitions

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 invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994); March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., J. Wiley & Sons (New York, N.Y. 1992); and Sambrook and Russel, Molecular Cloning: A Laboratory Manual 3rd ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, N.Y. 2001) provide one skilled in the art with a general guide to many of the terms used in the present application. 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. Indeed, the present invention is in no way limited to the methods and materials described. For purposes of the present invention, the following terms are defined below.

As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise. The term "and/or" indicates indicate that one or more of the stated cases may occur. In other words, a stated case may either occur alone or in combination with at least one of the stated cases, up to with all of the stated cases. The term and/or discloses each stated case alone, as well as the specific combination of a stated case with at least one of the other stated cases, up to with all of the stated cases.

By "alteration" is meant a change (increase or decrease) in the expression levels of a polynucleotide or polypeptide, or activity of the protein as detected by standard art known methods such as those described above. As used herein, an alteration includes a 5% or 10% change, a 25% change, a 40% change, a 50%, a 100% change, or greater change, or any change within the range of the values provided.

The term "amelioration" refers to a reduction of at least one sign and/or symptom of a specific disease or condition. Treatment refers to reduction of at least one sign and/or symptom of a disease or condition to reduce or eliminate at least one sign and/or symptom of the disease or condition, or to prevent or delay progression of the disease or condition. Amelioration and treatment need not be considered separate interventions, but instead can be considered a continuum of therapeutic interventions. "Beneficial results" may include, but are in no way limited to, lessening or alleviating the severity of the disease condition, preventing the disease condition from worsening, curing the disease condition and prolonging a patient's life or life expectancy. The disease conditions may relate to or may be modulated by the central nervous system. "Cancer" and "cancerous", as used herein, refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Cancer is also referred to as malignant neoplasm.

As used herein, "changed as compared to a control reference sample" is understood as having a level or activity of an analyte, or in a whole organism change of physical

characteristics or signs or symptoms of a disease, to be detected at a level that is statistically different than a sample from a normal, untreated, or control sample. Methods to select and test control samples are within the ability of those in the art.

Control samples typically include a cell or an animal of the same type that has not been contacted with an active agent or been subjected to a particular treatment, and has optionally been contacted with a carrier or subjected to a sham treatment. Control samples also include a cell or an animal not subjected to an agent or treatment to induce a specific disease or condition.

"Chemotherapy" as used herein, refers to the use of chemicals, such as pharmaceuticals or drugs, in the treatment of a disease condition, such as cancer.

"Chemotherapeutic agents" denote particular chemicals, such as pharmaceuticals or drugs, which are used to effect chemotherapy.

The phrase "in combination with" is intended to refer to all forms of administration that provide a first agent together with a second agent, such as a second inhibitory molecule or a chemotherapeutic agent, where the two are administered concurrently or sequentially in any order. For two or more agents to be administered in combination with each other, the agents need not be administered simultaneously or in the same formulation. Agents administered in combination with each other simultaneously present or have biological activity in the subject to which the agents are delivered. Determination of the presence of a agent in a subject can be readily determined by empirical monitoring or by calculations using known

pharmacokinetic properties of the agents.

In general "A therapeutic combination" is understood as a combination of one or more active drug substances, i.e. compounds having a therapeutic utility. Typically, each such compound in the therapeutic combinations will be present in a pharmaceutical composition comprising that compound and a pharmaceutically acceptable carrier. The compounds in a therapeutic combination of the present invention may be administered simultaneously or separately, as part of a regimen

By "control" is meant a standard or reference condition.

By "decreases" or "inhibit" or "lower" is meant a reduction by at least about 5% relative to a reference level. A decrease may be by 5%, 10%, 15%, 20%, 25% or 50%, or even by as much as 75%, 85%, 95% or more, or any change within the range of the values provided. The terms "disease" or "condition" are commonly recognized in the art and designate the presence of at least one sign and/or symptom in a subject or patient that are generally recognized as abnormal. Diseases or conditions may be diagnosed and categorized based on pathological changes (e.g., hyperproliferation). Signs may include any objective evidence of a disease such as changes that are evident by physical examination of a patient or the results of diagnostic tests that may include, among others, laboratory tests. Symptoms are subjective evidence of disease or a patient condition, e.g., the patient's perception of an abnormal condition that differs from normal function, sensation, or appearance, which may include, without limitations, physical disabilities, morbidity, pain, and other changes from the normal condition experienced by a subject.

By "an effective amount" is meant the amount of an agent 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 neoplasia 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. For example, the term effective amount refers to a dosage or amount that is sufficient to reduce, halt, or slow tumor progression to result in alleviation, lessening or amelioration of symptoms in a patient or to achieve a desired biological outcome, e.g., slow or stop tumor growth or reduction or disappearance of a tumor. Thus, in connection with the administration of an agent which 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. An agent can be effective against a specific disease or condition without being effective against the disease or condition for all subjects. Methods of selection of patient populations for treatment with the agents of the invention is an aspect of the invention. When reference is made to the "expression level" of a gene, the expression level is to be understood as the amount of RNA transcript that is transcribed by a gene and/or the amount of protein that may be translated from an RNA transcript, e.g. mRNA. For example, for genes which encode a miRNA, the expression level may be determined through quantifying the amount of RNA transcript which is expressed, e.g. using standard methods such as quantitative PCR of a mature miRNA, microarray, or Northern blot. Alternatively, the expression level may also be determined through measuring the effect of a miRNA on a target mRNA. For example, a 3'UTR sequence comprising a target miRNA target sequence may be incorporated in a reporter gene, e.g. luciferase, this way, the expression level of a miRNA gene may be indirectly measured by measuring the amount of Luciferase expression which may be controlled by a particular miRNA. Furthermore, in case the 3'UTR sequence is from a particular gene, the amount of Luciferase expression also correlates with the expression level of the particular gene. As such, when determining the expression level of a miRNA as described above (e.g. directly through qPCR or indirectly with a reporter gene construct), in addition, the expression level of the miRNA may be correlated to the expression level of a target gene of the particular miRNA. The amount of protein that may be translated from an RNA transcript may also be measured to determine the expression level of a gene. Methods are known in the art, such as ELISA and Western blot. Alternatively, the expression level of a protein may also be indirectly measured. For example, in case the expression level of an RNA transcription factor needs to be determined, a reporter gene construct may be used which comprises the RNA transcription factor binding site. The expression level may than be subsequently determined by measuring the reporter gene construct expression, e.g. in case GFP is used, green fluorescence intensity may be determined as a measure of the expression level of the RNA transcription factor.

"Formulations" or "compositions" 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 formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient/therapeutic which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated and the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred per cent, this amount (in weight) will range from about 1 per cent to about ninety-nine percent of active ingredient, preferably from about 5 per cent to about 70 per cent, most preferably from about 10 per cent to about 30 per cent.

The term "gene" means a DNA sequence comprising a region (transcribed region), which is transcribed into an RNA molecule (e.g. an mRNA) in a cell, operably linked to suitable regulatory regions (e.g. a promoter). A gene may thus comprise several operably linked sequences, such as a promoter, a 5' leader sequence comprising e.g. sequences involved in translation initiation, a (protein) coding region (cDNA or genomic DNA) and a 3' non- translated sequence comprising e.g. transcription termination sites.

By "inhibits a neoplasia" is meant slows, decreases, or stabilizes the growth, proliferation, or metastasis of a neoplasia, or increases apoptosis.

"Mammal," as used herein, refers to any member of the class Mammalia, including, without limitation, humans and nonhuman 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.

By "neoplasia" is meant any disease that is caused by or results in inappropriately high levels of cell division, inappropriately low levels of apoptosis, or both. For example, cancer is a neoplasia. Neoplasia includes solid tumors and non-solid tumors or blood tumors. Examples of cancers include, without limitation, leukemias, lymphoma .sarcomas and carcinomas (e.g. colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, lung cancer, melanoma, lymphoma, non-Hodgkin lymphoma, colorectal cancer, malignant melanoma, thyroid cancer, papillary thyroid carcinoma, lung cancer, non-small cell lung carcinoma, and adenocarcinoma of lung).

A "nucleic acid" may include any polymer or oligomer of pyrimidine and purine bases, preferably cytosine, thymine, and uracil, and adenine and guanine, respectively (See Albert L. Lehninger, Principles of Biochemistry, at 793-800 (Worth Pub. 1982) which is herein incorporated by reference in its entirety for all purposes). The present invention contemplates any deoxyribonucleotide, ribonucleotide or peptide nucleic acid component, and any chemical variants thereof, such as methylated, hydroxymethylated or glycosylated forms of these bases, and the like. The polymers or oligomers may be heterogenous or homogenous in composition, and may be isolated from naturally occurring sources or may be artificially or synthetically produced. In addition, the nucleic acids may be DNA or RNA, or a mixture thereof, and may exist permanently or transitionally in single-stranded or double-stranded form, including homoduplex, heteroduplex, and hybrid states.

"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. "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.

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 cell.

"RNA interference" refers to a process of sequence-specific, post-transcriptional gene silencing (PTGS). RNA interference (RNAi) is a system within living cells that takes part in controlling which genes are active and how active they are. RNAs are the direct products of genes, and these small RNAs can bind to other specific RNAs (mRNA) and decrease their activity, for example by preventing a messenger RNA from producing a protein. RNA molecules capable of RNA interference include, without limitation, siRNA, shRNA, and miRNA.

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

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 be referred to as patients.

A subject "suffering from or suspected of suffering from" a specific disease, condition, or syndrome has at least one risk factor and/or presents with at least one sign or symptom of the disease, condition, or syndrome such that a competent individual would diagnose or suspect that the subject was suffering from the disease, condition, or syndrome.

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.

Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 20 is understood to include any number, combination of numbers, or sub-range from the group including 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20.

"At least" a particular value is understood to mean that 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, etc.

"Less than" or "up to" and the like is understood as the range from zero up to and including the value provided. For example, "less than 5" or "up to 5" is understood as 0, 1 , 2, 3, 4, 5. Unless specifically stated or obvious from context, as used herein, the terms "a", "an", and "the" are understood to be singular or plural.

About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, 0.5%, 0.1 %, 0.05%, or 0.01 % of the stated value.

Throughout this application, various references are cited in parentheses to describe more fully the state of the art to which this invention pertains. Full bibliographic information for each citation is found at the end of the specification, immediately preceding the claims. The disclosure of these references are hereby incorporated by reference into the present disclosure in their entirety.

Detailed description

In a first aspect the disclosure relates to a method of treating or preventing a neoplasia in a subject, the method comprising administrating to a subject in need of such treatment an effective amount of a PDK inhibitor, preferably a PDK-1 inhibitor, and an effective amount of an inhibitor of a protein of the MAPK/ERK pathway.

It was surprisingly found that the combination of both a PDK inhibitor and a inhibitor of a protein of the MAPK/ERK pathway may be beneficial in the treatment of neoplasia.

In particular, it was found that addition of a PDK inhibitor to the treatment of neoplasia with an inhibitor of (a protein of) the MAPK/ERK-pathway leads to further sensitization of the neoplastic cells, for example as reflected by decreased cell viability. Of particular importance is that it was found that neoplastic cells that show resistance to the treatment by a

MAPK/ERK pathway inhibitor (see Examples, in particular a BRAF inhibitor) exhibited even greater sensitization in comparison to cells that are sensitive to the MAPK/ER pathway inhibitor. Within the context of the current disclosure "sensitization" indicates an increased sensitivity of the cells towards a treatment. Sensitization can for example be reflected in cell viability. In case a particular treatment, in comparison to another treatment, leads to decreased cell viability (of the neoplastic cells), this is a clear indication of sensitization.

In other words, it was found that by reducing the activity of PDK by an inhibitor, for example by reducing gene expression, protein expression, of by reducing the specific enzymatic activity of PDK, and by reducing the activity of (a protein of) the MAPK/ERK pathway, for example by reducing gene expression, protein expression, or by reducing the specific enzymatic activity of PDK, neoplastic cells, for example tumors like melanoma, can be treated better in comparison to reducing the activity of a protein of the MAPK/ERK pathway (e.g. BRAF) alone, or in comparison to reducing the activity of PDK alone alone.

The enzymatic activity of an enzyme can, for example be inhibited by allowing small molecules or antibodies or any other type of molecule to interact with the enzyme is such manner that the enzyme can not, or only to a limited amount, perform its undesired function, for example by binding close to or in a site in the protein normally used for ligand binding, or by using a compound that modifies the tertiary structure of the protein. Alternatively, and as document above, the inhibition may comprise modifying the target enzyme/protein such that it stuck (to a certain degree) in its "on" or "off" position (e.g. being or not being phosphorylated).

To date, four PDK (also referred to as PDHK) isoforms have been identified in human Mitochondria, being PDK-1 , PDK-2, PDK-3, and PDK-4 (Popov et al 1997 Adv. Second Messenger Phosphoprotein Res. 31 , 105-11 1 ; Kolobova et al 2001 Biochem. J. 358, 69-77; Korotchkina and Patel 2001 J. Biol. Chem.

276, 37223-37229).

Pyruvate dehydrogenase kinase (PDK) isoforms PDK1 , 2, 3, or 4 are molecular switches that downregulate the pyruvate dehydrogenase complex (PDC) by reversible phosphorylation in mitochondria.

The pyruvate dehydrogenase complex (PDC) is a member of the highly conserved

mitochondrial a-ketoacid dehydrogenase complexes comprising the PDC, the branched-chain a-ketoacid dehydrogenase complex (BCKDC), and the a-ketoglutarate dehydrogenase complex (Patel and Roche 1990 FASEB J. 4, 3224-3233; Reed 2001 J. Biol. Chem. 276,

28329-28336). PDC catalyzes the oxidative decarboxylation of pyruvate to give rise to acetyl- CoA, linking glycolysis to the Krebs cycle. The phosphorylation of specific serine residues by PDK results in the inactivation of PDC, whereas the dephosphorylation by pyruvate dehydrogenase phosphatise (PDP) restores PDC activity (Harris et al 2001 Adv. Enzyme Regul. 41 , 269-288; Holness and Sugden 2003 Biochem. Soc. Trans. 31 , 1 143-1151).

In particular, PDK, e.g. PDK-1 , phosphorylates and inactivates pyruvate dehydrogenase (PDH). Pyruvate dehydrogenase performs the first two reactions within the pyruvate dehydrogenase complex (PDC): a decarboxylation of pyruvate and a reductive acetylation of lipoic acid. Lipoic acid is covalently bound to dihydrolipoamide acetyltransferase E2, which is the second catalytic component enzyme of PDC. The reaction catalyzed by pyruvate dehydrogenase is considered to be the rate-limiting step for the pyruvate dehydrogenase complex.

Given these structural and functional similarities (all isoforms share significant structural similarities (i.e, 66-74% amino acid identity); all isoforms phosphorylate PDH), and although PDK-1 is preferred, any PDK isoform can be substituted for PDK1 in the methods, uses and products of the invention. In addition, compounds that inhibit a PDK isoform are generally useful in the current invention.

A PDK polypeptide or peptide is to indicate a polypeptide having pyruvate dehydrogenase kinase activity and having at least 85% amino acid identity to the amino acid sequence of human PDK1 , PDK2 (e.g. Genbank Accession NO: AAH40478) , PDK3 (e.g. Genbank Accession NO: AAH15948), or PDK4 (e.g. Genbank Accession NO: NP002603). These amino acid sequence of PDK enzymes, other proteins mentioned herein, and variations thereof are available in GenBAnk, accessible via http:/7www.ncbi.nlm.nih.gov/genbank^/ by entering either the numbers mentioned above or entering the relevant protein name.

By PDK biological activity is meant any function of a pyruvate dehydrogenase kinase, such as enzymatic activity, kinase activity, inhibition of the tricarboxylic acid cycle, the enhancement of cell survival under hypoxic conditions, or inhibition of PDH activity. By PDK1 polypeptide is meant a polypeptide having substantial identity to the amino acid sequence provided at GenBank Accession No. NP-002601 , or an active fragment thereof.

By PDK1 nucleic acid molecule is meant a nucleic acid sequence encoding a PDK1 polypeptide. One exemplary nucleic acid sequence is provided at GenBank Accession No. NM-002610.

By PDK1 biological activity is meant any function of PDK1 , such as enzymatic activity, kinase activity, inhibition of the tricarboxylic acid cycle, the enhancement of cell survival under hypoxic conditions, or the inhibition of PDH activity.

By PDK inhibitor is meant a compound that reduces the biological activity of PDK1 , 2, 3, or 4; or that reduces the expression of an mRNA encoding a PDK polypeptide; or that reduces the expression of a PDK polypeptide, , for example by 5%, 10%, 15%, 20%, 25% or 50%, or even by as much as 75%, 85%, 95% or more in comparison to a control situation.

By PDK1 polypeptide is meant a polypeptide having substantial identity to the amino acid sequence provided at GenBank Accession No. NP-002601 , or an active fragment thereof. By PDK1 nucleic acid molecule is meant a nucleic acid sequence encoding a PDK1 polypeptide. One exemplary nucleic acid sequence is provided at GenBank Accession No. NM-002610.

By "PDK1 inhibitor" is meant a compound that reduces the biological activity of PDK1 , that reduces the expression of an mRNA encoding a PDK1 polypeptide; or that reduces the expression of a PDK1 polypeptide, for example by 5%, 10%, 15%, 20%, 25% or 50%, or even by as much as 75%, 85%, 95% or more in comparison to a control situation.

Pyruvate dehydrogenase kinase inhibitors are known in the art and are described, for example, by Mann et al., Biochimica et Biophysica Acta 1480:283-292, 2000; Kato et al Structure. 2007 August; 15(8): 992-1004; Mayer Biochem Soc Trans. 2003 Dec;31 (Pt 6):1 165-7; WO2006042062, US2012004284 (for example in Table 1 thereof),

WO2005019451 and US20090209618. Reference is made to said documents with respect to PDK inhibitor, PDK-1 inhibitor, PDK-2 inhibitor, PDK-3 inhibitor, and/or PDK-4 inhibitor and methods of making the same. Known pyruvate dehydrogenase kinase inhibitors suitable in the current invention include dichloroacetate, halogenated acetophones (e.g., dichloroacetophenone) (Mann et al., supra), adenosine 5'[[beta],[gamma]-imido] triphosphate (Mann et al., supra), substituted triterpenes (Mann et al., supra), lactones (Mann et al., supra), monochloroacetate (Whitehouse et al., Biochem J 141 : 761-774, 1974), dichloroacetate (Whitehouse et al., supra), trichloroacetate (Whitehouse et al., supra), difluoroacetate (Whitehouse et al., supra), chlorofluoroacetic acid, 2-chloropropionate (Whitehouse et al., supra), 2,2'-dichloropropionate (Whitehouse et al., supra), chloropropionate, 3-chloropropionate (Whitehouse et al., supra), and 3,3,3-trifluoro-2- hydroxy-2-methylpropionamide (Mann et al., supra), SDZ048-619 (Novartis), SDZ060-01 1 (Novartis), and SDZ225-066 (Novartis, Aicher et al., supra), radicicol oxime or radicicol, AZD7545 ((2R)-N-{4-[4-(dimethylcarbamoyl)phenylsulfonyl]-2-chlorophenyl}-3,3,3-trifluoro-2- hydroxy-2-methylpropanamide; Katio et al, supra) an anilide derivative of (R)-3,3,3-trifluoro-2- hydroxy-2-methylpropionic acid, a secondary amine of (R)-3,3,3-trifluoro-2-hydroxy-2- methylpropionic acid, or an inner lipoyl domain of dihydrolipoyl acetyltransferase. Other suitable PDK-I inhibitors are those described in in particular W01999/062506 (preferred), W01999/44618, WO2009/067767 and US201 1/0207694. See for further details on AZD7545, and its synthesis for example W01999/062506, Mayers et al 2005 Biochem. Soc. Trans. 33, 367-370; Mayers et al 2005 Biochem. Soc. Trans. 33, 367-370. Further examples of PDK inhibitors include dichloroacetate, 2,2-dichloroacetophenone, and (+)-l-N-[2,5-(,S',JR)- dimethyl-4-N-(4-cyanobenzoyl)piperazine]-(R ) -3,3,3-triiluoro-2- hydroxy-2- methylpropanamide), see for example WO2006042062.

One preferred PDK1 inhibitor is AZD7545, (+)-1-N-[2,5-(S,R)-dimethyl-4-N-(4- cyanobenzoyl)piperazine]-(R)-3,3,3-trifluoro-2-hydroxy-2-methylpropanamide (Aicher et al., supra) AZD7545 is disclosed in W01999/062506 as one of many inhibitors having corresponding molecular structure. The compounds are also suitable as PDK inhibitors for use in the current invention. Other preferred PDK inhibitors are dichloroacetate and 2,2-dichloroacetophenone (see for example Kato et al supra).

The skilled person knows that variations of the above-mentioned compounds may have equivalent function and may thus be used as PDK-1 inhibitor in the context of the current invention.

As documented above, a preferred PDK-1 inhibitor is AZD7545, and the term AZD7545 is to be construed as including modified versions of AZD7545 having PDK inhibitor activity. AZ12 and Nov3r are PDK inhibitors known to be related to AZD7545 (Knoechel et al 2006

Biochemistry 45, 402-415).

In particular examples, the PDK (or PDK-1) inhibitor is a small interfering nucleotide sequence capable of inhibiting PDK (or PDK-1) activity, such as siRNA using one or more small double stranded RNA molecules. For example, PDK 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 PDK activity of such small interfering nucleotide sequence, such as described in US 2009/0209618, for example by determining whether the candidate small interfering nucleotide sequence decreases PDK activity. In one embodiment, the inhibitory nucleic acid molecule is a PDK1 siRNA, for example having the following sequence: 5'-CUACAUGAGUCGCAUUUCAdTdT-3.' (see US2009/0209618 for this and other examples).

The PDK (e.g. PDK-1) inhibitor according to the present invention may be a binding agent such as an antibody which specifically binds PDK (e.g. PDK-1), thereby inhibiting its function.

Pyruvate dehydrogenase kinase catalytic activity may be assayed by measuring NADH formation by the pyruvate dehydrogenase multienzyme complex (PDC) (Mann et al., supra), by measuring PDH phosphorylation (see US2009209618), by measuring PDH activity (Aicher et al. J. Med. Chem. 43:236-249, 2000) or as described by Kato et al, Structure 15, 992-1004, 2007.

In a preferred embodiment, the protein of the MAPK/ERK pathway is selected from the group consisting of BRAF, MEK and ERK, and combination of two or three thereof. Preferably the protein is BRAF (B-raf).

A BRAF polypeptide or peptide is to indicate a polypeptide having serine/threonine protein kinase activity, e.g. BRAF kinase phosphorylates and activates MEK (MEK1 and MEK2, and having at least 85% amino acid identity to the amino acid sequence of human BRAF (e.g. Genbank Accession NO: NP004324). These amino acid sequence of BRAF 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.

By BRAF biological activity is meant any function of BRAF, such as enzymatic activity, kinase activity, or signaling the MAPK/ERK pathway.

By BRAF inhibitor is meant a compound that reduces the biological activity of BRAF; or that reduces the expression of an mRNA encoding a BRAF polypeptide; or that reduces the expression of a BRAF polypeptide, for example by 5%, 10%, 15%, 20%, 25% or 50%, or even by as much as 75%, 85%, 95% or more in comparison to a control situation.

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). Any BRAF inhibitor, including any pharmaceutical agent having BRAF inhibitory activity may be utilized in the present invention. Such 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, WO2006053201 , 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.

Preferred BRAF 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), PLX-4720, 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), gefitinib (e.g. Iressa, AstraZeneca and Teva), imatinib (e.g. as marketed by Novartis as Gleevex or Glivec), lapatinib (e.g. from

GlaxoSmithKline under trade names Tykerb or Tyverb), sorafenib (e.g. from Bayer and Onyx pharmaceuticals as Nexavar) and sunitinib (e.g. Sutent by Pfizer), or a derivative thereof.

Preferably, the derivative of the BRAF inhibitor is a salt. Thus, according to the invention the BRAF inhibitor may be selected from the group consisting of dasatinib, erlotinib

hydrochloride, dabrafenib, gefitinib, imatinib mesilate, lapatinib, sorafenib tosylate and sunitinib malate. Preferably the BRAF 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).

In one embodiment the BRAF 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 BRAF inhibitor is selected from any one of the BRAF inhibitors disclosed in WO2008120004. Other Braf inhibitors include GSK2118436, 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).

In particular examples, the BRAF inhibitor is a small interfering nucleotide sequence capable of inhibiting BRAF activity, such as siRNA using one or more small double stranded RNA molecules. For example, BRAF 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 BRAF 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.

The BRAF 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.

A BRAF inhibitor has BRAF inhibitor activity, or in other words reduces activated (or mutated) BRAF activity, which activity may be verified by the following assay. For example, using the procedure set out below of a BRAF in vitro ELISA assay.

Activity of human recombinant, purified wild type His-BRAF protein kinase can be determined in vitro using an enzyme-linked immunosorbent assay (ELISA) assay format, which measures phosphorylation of the BRAF substrate, human recombinant, purified 5 His-derived

(detagged) MEK. The reaction utilizes 2.5 nM BRAF, 0.15 μΜ MEK and 10 μΜ adenosine triphosphate (ATP) in 40 mM N-(2-hydroxyethyl)piperazine-N'-(2- ethanesulfonic acid hemisodium salt (HEPES)3 5 mM 1 ,4-dithio-DL-threitol (DTT), 10 mM MgCI2, 1 mM ethylenediaminetetraacetic acid (EDTA) and 0.2 M NaCI (Ix HEPES buffer), with or without compound at various concentrations, in a total reaction volume of 25 μΙ in 384 well plates. BRAF and compound were preincubated in Ix HEPES buffer for 1 hour at 25 0C. Reactions are initiated with addition of MEK and ATP in Ix HEPES buffer and incubated at 25 °C for 50 minutes and reactions are stopped by addition of 10 μ1 175 mM EDTA (final concentration 50 mM) in Ix HEPES buffer. 5 μΙ of the assay mix was then diluted 1 :20 into 50 mM EDTA in Ix HEPES buffer, transferred to 384 well black high protein binding plates and incubated overnight at 4 °C. Plates were washed in tris buffered saline containing 0.1 % Tween20 (TBST), blocked with 50 μΙ Superblock (Pierce) for 1 hour at 25 °C, washed in TBST, incubated with 50 μΙ rabbit polyclonal anti-phospho-MEK antibody (Cell Signaling) diluted 1 :1000 in TBS for 2 hours at 25 °C, washed with TBST, incubated with 50 μΙ goat anti-rabbit horseradish peroxidase -linked antibody (Cell Signaling) diluted 1 :2000 in TBS for 1 hour at 25 °C and washed with TBST. 50 μΙ of fluorogenic peroxidase substrate (Quantablu - Pierce) is added and following incubation for 45-60 minutes, 50 μΙ QuantabluSTOP (Pierce) was added. Blue fluorescent product was detected at excitation 325 and emission 420 using a TECAN Ultra plate reader. Data can be graphed and IC50S calculated using Excel Fit (Microsoft). When tested in the above in vitro assay, a BRAF inhibitor according to the present invention will reduce BRAF activity by for example by 5%, 10%, 15%, 20%, 25% or 50%, or even by as much as 75%, 85%, 95% or more in comparison to the situation without BRAF inhibitor.

A MEK polypeptide (e.g. EC 2.7.12.2) or peptide is to indicate a polypeptide having serine/threonine protein kinase activity, e.g. MEK1 (e.g. Genbank Accession NO: NP002746) and MEK2 (e.g. Genbank Accession NO: NP109587) phosphorylates and activates MAPK, and having at least 85% amino acid identity to the amino acid sequence of human MEK1 , 2 or MEK3 ((e.g. Genbank Accession NO: NP002747). These amino acid sequence of MEK enzymes, other proteins mentioned herein, and variations thereof are available in GenBAnk, accessible via http:/Avww.ncbi. nlm.nih.gov/genbank/ by entering either the numbers mentioned above or entering the relevant protein name. By MEK biological activity is meant any function of MEK, such as enzymatic activity, kinase activity, or signaling the MAPK/ERK pathway.

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, for example by 5%, 10%, 15%, 20%, 25% or 50%, or even by as much as 75%, 85%, 95% or more in comparison to a control situation. A MEK inhibitor can inhibit one member, several members or all members of the family of MEK kinases.

Preferred MEK inhibitors, already known in the art, and suitable for use in the current invention, include but are not limited to the MEK inhibitors PD184352 and PD98059, inhibitors of MEKI 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. Other MEK inhibitors and classes of MEK inhibitors are described in Zhang et al. (2000) Bioorganic & Medicinal Chemistry Letters; 10:2825-2828.

Further suitable 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, WO2007113505, US2011 105521 , WO2011067356, 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. 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 synthesised according to the process described in WO2007/076245.

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 synthesised according to the process described in International Patent Publication Number WO2007/076245.

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 CI-1040 (Lorusso et al Journal of clinical oncology 2005, vol. 23, no23, pp. 5281-5293). 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). In one embodiment the MEK inhibitor is selected from AZD6244 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 or 4-(4-Bromo- 2-fluorophenylamino)-N-(2-hydroxyethoxy)- 1 ,5-dimethyl-6-oxo- 1 ,6-dihydropyridazine-3- carboxamide or a pharmaceutically acceptable salt thereof as described below. In one embodiment the MEK inhibitor is selected from AZD6244 or a pharmaceutically acceptable salt thereof or 2-(2-fluoro-4-iodophenylamino)-N-(2-hydroxyethoxy)- 1 ,5-dimethyl-6-oxo- 1 JS- dihydropyridine-3-carboxamide or a pharmaceutically acceptable salt thereof, as described below. In one embodiment the MEK inhibitor is AZD6244 hydrogen sulphate salt. AZD6244 hydrogen sulphate salt may be synthesised according to the process described in WO07/076245.

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.

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.

A number of assays for identifying kinase inhibitors, including MEK inhibitors, are known. For example, Davies et al (2000) describe kinase assays in which a kinase is incubated in the presence of a peptide substrate and radiolabeled ATP. Phosphorylation of the substrate by the kinase results in incorporation of the label into the substrate. Aliquots of each reaction are immobilised on phosphocellulose paper and washed in phosphoric acid to remove free ATP. The activity of the substrate following incubation is then measured and provides an indication of kinase activity. The relative kinase activity in the presence and absence of candidate kinase inhibitors can be readily determined using such an assay. Downey et al. (1996) J Biol Chem.; 271 (35): 21005- 21011 also describes assays for kinase activity which can be used to identify kinase inhibitors. 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 to ERK1 (e.g. Genbank Accession NO: NP001035145) or ERK2 (e.g. Genbank Accession NO: NP002736) . These amino acid sequence of ERK enzymes, other proteins mentioned herein, and variations thereof are available in GenBAnk, accessible via

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

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.

By ERK inhibitor is meant a compound that reduces the biological activity of ERK; or that reduces the expression of an mRNA encoding a ERK polypeptide; or that reduces the expression of an ERK polypeptide, for example by 5%, 10%, 15%, 20%, 25% or 50%, or even by as much as 75%, 85%, 95% or more in comparison to a control situation. An ERK inhibitor can inhibit one member, several members or all members of the family of ERK kinases .

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).

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).

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):495-506), U0126 (Favata, M., et al., Identification of a novel inhibitor of mitogen-activated protein kinase. J. Biol. Chem. 273, 18623, 1998), PD098059 (Alessi et al J Biol Chem. 1995 Nov 17;270(46):27489-94), or PD184352 (Allen and Meyer Semin Oncol. 2003 Oct;30(5 Suppl 16): 105-16). In some embodiments the ERK inhibitor is U0126.

Preferably the ERK inhibitor is U0126 or a pharmaceutically acceptable salt or solvate thereof, or an analogue thereof, wherein the analogue has ERK inhibitory activity.

Alternatively the inhibitor may be PD98059 or an analogue thereof, wherein the analogue has ERK inhibitory activity. Alternatively the ERK inhibitor is PD0325901 (Henderson et al Mol Cancer Ther July 2010 9; 1968). Further suitable ERK inhibitors may be found in patentdocuments 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 MEK inhibitors, and methods for making the same.

Other suitable ERK inhibitors include 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059), 1 ,4-diamino-2,3-dicyano-1 ,4-bis(2-aminophenylthio)butadiene (U0126), and Z-& E-a-(amino-((4-aminophenyl)thio)methylene)-2-(trifluoromethyl)benzeneacetonitrile (MEK1/2) (US201 11 10916).

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. 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. 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.

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.

In another embodiment, there is provided a method according to any one of the previous claims wherein the subject is a subject that acquired resistance against treatment with a BRAF, MEK or ERK inhibitor.

Various groups report on the occurrence of resistance to inhibitors of BRAF, MEK or ERK inhibitors in the treatment of neoplasia, and combination therapy has been suggested (see Solit et al 201 1 N Engl J Med 364:8). As already documented above, it was now found that the combination of a PDK inhibitor according to the invention, and a inhibitor of the

MAPK/ERK pathway, in particular of ERK, BRAF and/or MEK, leads to better treatment response due to further sensitization of the neoplastic cells.

It is therefore a particularly preferred embodiment of the current invention to combine such inhibitors in the treatment of neoplasia, in particular cancer, in particular in melanoma, including BRAF mutant V600K or BRAF mutant V600R melanoma , and even more in particular in BRAF mutant (V600E) melanoma. Preferably the BRAF inhibitor in

PLX4720/vemuranefib. Preferably, the PDK inhibitor in AZD7545. In a further embodiment of the invention there is provided a method according to the invention wherein the PDK inhibitor is selected from the group consisting of dichloroacetate, halogenated acetophones (e.g., dichloroacetophenone), adenosine 5'[[beta],[gamma]-imido] triphosphate, substituted triterpenes, lactones, monochloroacetate, dichloroacetate, trichloroacetate, difluoroacetate, chlorofluoroacetic acid, 2-chloropropionate, 2,2'- dichloropropionate, chloropropionate, 3-chloropropionate, 3,3,3-trifluoro-2-hydroxy-2- methylpropionamide , SDZ048-619 (Novartis), SDZ060-01 1 (Novartis), and SDZ225-066 (Novartis), , radicicol oxime or radicicol, AZD7545 ((2R)-N-{4-[4- (dimethylcarbamoyl)phenylsulfonyl]-2-chlorophenyl}-3,3,3-trifluoro-2-hydroxy-2- methylpropanamide), an anilide derivative of (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid, a secondary amine of (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid, or an inner lipoyl domain of dihydrolipoyl acetyltransferase, a PDK-I inhibitors disclosed in

W01999/062506, a PDK-I inhibitors disclosed in W01999/44618, a PDK-I inhibitors disclosed in WO2009/067767, a PDK-I inhibitors disclosed in US2011/0207694, dichloroacetate, 2,2- dichloroacetophenone, and (+)-l-N-[2,5-(,S',JR)-dimethyl-4-N-(4-cyanobenzoyl)piperazine]-(R ) -3,3,3-triiluoro-2- hydroxy-2-methylpropanamide), a PDK-I inhibitors disclosed in

WO2006042062, preferably a PDK-I inhibitors disclosed in W01999/062506, even more preferably AZD7545 and derivates thereof as disclosed in W01999/062506, ((2R)-N-{4-[4- (dimethylcarbamoyl)phenylsulfonyl]-2-chlorophenyl}-3,3,3-trifluoro-2-hydroxy-2- methylpropanamide), or (+)-1-N-[2,5-(S,R)-dimethyl-4-N-(4-cyanobenzoyl)piperazine]-(R)- 3,3,3-trifluoro-2-hydroxy-2-methylpropanamide.

In another embodiment there is provided a method according to the invention wherein the inhibitor of a protein of the MAPK/ERK pathway is selected from

-a BRAF inhibitor selected from the group consisting of A BRAF inhibitor as disclosed in WO 2010/114928, A BRAF inhibitor as disclosed in WO 2005/123696, A BRAF inhibitor as disclosed in WO2007/002325, A BRAF inhibitor as disclosed in WO 2006/003378, A BRAF inhibitor as disclosed in WO 2006/024834, A BRAF inhibitor as disclosed in WO

2006/024836, A BRAF inhibitor as disclosed in WO 2006/040568, A BRAF inhibitor as disclosed in WO 2006/067446 or A BRAF inhibitor as disclosed in WO 2006/079791 , A BRAF inhibitor as disclosed in WO02/24680, or A BRAF inhibitor as disclosed in WO03/022840, A BRAF inhibitor as disclosed in WO 2005/047542, A BRAF inhibitor as disclosed in AU 2003/286447, A BRAF inhibitor as disclosed in US 2004/0096855, A BRAF inhibitor as disclosed in AU2002/356323, A BRAF inhibitor as disclosed in WO2005089443, A BRAF inhibitor as disclosed in WO2006053201 , A BRAF inhibitor as disclosed in US20050267060, A BRAF inhibitor as disclosed in WO2008120004, A BRAF inhibitor as disclosed in

US20090181371 , A BRAF inhibitor as disclosed in WO2008120004, Vemurafenib, PLX4720, PLX4032 (RG7204), GDC-0879, PLX-4720, Sorafenib Tosylate, Nexavar, dasatinib, dabrafenib , erlotinib, gefitinib, imatinib, lapatinib, sorafenib or sunitinib, or active derivatives thereof, A BRAF inhibitor as disclosed in WO2006/024834, A BRAF inhibitor as disclosed in WO2006/067446, A BRAF inhibitor as disclosed in PCT/GB2006/004756, CHIR-265, XL281 (Exelixis) or PLX4032.

-a MEK inhibitor selected from the group consisting of PD184352, PD98059, U0126 , SL327, PDI 84352, a MEK inhibitor disclosed in WO2009018238, a MEK inhibitor disclosed in WO2007/044084, a MEK inhibitor disclosed in WO2005/051300, a MEK inhibitor disclosed in WO2011/095807, a MEK inhibitor disclosed in WO2008124085, a MEK inhibitor disclosed in WO2009018233, a MEK inhibitor disclosed in WO2007113505, a MEK inhibitor as disclosed in WO03/077914, a MEK inhibitor disclosed in US201 1105521 , a MEK inhibitor disclosed in WO2011067356, a MEK inhibitor disclosed in WO201 1067348, a MEK inhibitor disclosed in US2010004247, a MEK inhibitor disclosed in US2010130519, AEZS-131 .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-carboxamid, 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, 6-(4-Bromo-2-chloro-phenylamino)-7-fluoro-3- methyl-3H-benzoimidazole-5- carboxylic acid (2-hydroxy-ethoxy)-amide hydrogen sulphate salt, PD-325901 , XL518, PD-184352, PD- 318088, AZD6244, CI-1040, 4-(4-Bromo-2- fluorophenylamino)-N-(2-hydroxyethoxy)- 1 ,5-dimethyl-6-oxo- 1 ,6-dihydropyridazine-3- carboxamide or a pharmaceutically acceptable salt thereof, AR-1 19, AS- 701173, AS-701255, 360770-54-3 and/or

-a ERK inhibitor selected from the group consisting of an ERK-inhibitor as disclosed in WO2002058687, SL-327, U0126, PD098059, PD184352, PD0325901 , an ERK-inhibitor as disclosed in WO2002058687, an ERK-inhibitor as disclosed in AU2002248381 , an ERK- inhibitor as disclosed in US20050159385, an ERK-inhibitor as disclosed in US2004102506, an ERK-inhibitor as disclosed in US2005090536, an ERK-inhibitor as disclosed in

US2004048861 , an ERK-inhibitor as disclosed in US20100004234, an ERK-inhibitor as disclosed in HR20110892, an ERK-inhibitor as disclosed in WO201 1163330, an ERK-inhibitor as disclosed in TW200934775, an ERK-inhibitor as disclosed in EP2332922, an ERK- inhibitor as disclosed in WO201 1041 152 , an ERK-inhibitor as disclosed in US201 1038876, an ERK-inhibitor as disclosed in WO2009146034, an ERK-inhibitor as disclosed in

HK11 17159, an ERK-inhibitor as disclosed in WO2009026487, an ERK-inhibitor as disclosed in WO2008115890, PH-797804 (Bioorg Med Chem Lett 201 1 Jul 1 ;21 (13):4066-71) an ERK- inhibitor as disclosed in US2009186379, an ERK-inhibitor as disclosed in WO2008055236, an ERK-inhibitor as disclosed in US2007232610, an ERK-inhibitor as disclosed in

WO2007025090, an ERK-inhibitor as disclosed in US2007049591 , 2-(2-amino-3- methoxyphenyl)-4H-1-benzopyran-4-one (PD98059), 1 ,4-diamino-2,3-dicyano-1 ,4-bis(2- aminophenylthio)butadiene (U0126), and Z-& E-a-(amino-((4-aminophenyl)thio)methylene)-2- (trifluoromethyl)benzeneacetonitrile (MEK1/2) (US2011 110916),

-and combinations thereof. Also provided is a method according to the invention wherein the neoplasia is a malignant neoplasia, preferably a malignant neoplasia that involves a disturbance in the MAPK/ERK pathway, preferably a malignant neoplasia selected from the group consisting of lymphoma, non-Hodgkin lymphoma, colorectal cancer, malignant melanoma, thyroid cancer, papillary thyroid carcinoma, lung cancer, non-small cell lung carcinoma, and adenocarcinoma of lung.

Preferably, the malignant neoplasia is a melanoma, including BRAF V600K or BRAF V600R melanoma, preferably a BRAF (V600E) melanoma, and other BRAF(V600E) tumors, or tumors with alternative activation of the BRAF/MEK/ERK pathway.

As already documented above, also provided is a method according to the invention wherein the PDK inhibitor is an inhibitory nucleic acid molecule that reduces PDK gene and/or protein expression, preferably PDK-1 expression. For example, the inhibitory nucleic acid molecule is a small interfering RNA (siRNA, antisense RNA, short hairpin RNA or another nucleic acid inhibitor of PDK expression (e.g. (e.g., PDK1 , 2, 3, 4).

By "inhibitory nucleic acid molecule" is meant a double-stranded RNA, antisense RNA, or siRNA, or portion thereof that reduces the amount of mRNA or protein encoded by a gene of interest. Preferably, the reduction is by at least 5%, more desirable by at least 10%, 25%, or even 50%, relative to an untreated control. Methods for measuring both mRNA and protein levels are well-known in the art.. The siRNA may contain a modified backbone, for example, phosphorothioate, phosphorodithioate, or other modified backbones known in the art, or may contain non-natural internucleoside linkages. As already documented above, also provided is method according to the invention wherein the inhibitor of a protein of the MAPK/ERK pathway is an inhibitory nucleic acid molecule that reduces expression of BRAF, MEK or ERK. For example, the inhibitory nucleic acid molecule is a small interfering RNA (siRNA, antisense RNA, short hairpin RNA or another nucleic acid inhibitor of expression of a BRAF, MEK or ERK protein/gene, as exemplified above.

For example, there is provided a method according to the invention wherein the PDK inhibitor decreases expression of a PDK polypeptide, decreases the biological activity of a PDK polypeptide, and/or decreases the expression of a PDK nucleic acid molecule.. In another example, there is provided a method according to the invention wherein the inhibitor of a protein of the MAPK/ERK pathway decreases expression of a BRAF, MEK or ERK polypeptide, decreases the biological activity of a BRAF, MEK or ERK polypeptide, and/or decreases the expression of a BRAF, MEK or ERK nucleic acid molecule. Alternatively, and in accordance with the disclosure above, there is provided an effective amount of a PDK inhibitor, preferably a PDK-1 inhibitor, and an effective amount of an inhibitor of a protein of the MAPK ERK pathway for use in treatment or prevention of a neoplasia in a subject.

Alternatively there is provided for use of an effective amount of a PDK inhibitor, preferably a PDK-1 inhibitor, and an effective amount of an inhibitor of a protein of the MAPK/ERK pathway for the manufacture of a medicament for treatment or prevention of a neoplasia in a subject.

In another embodiment there is provided for the effective amount of a PDK inhibitor and the effective amount of a protein of the MAPK/ERK pathway or use thereof according to the invention wherein the protein of the MAPK/ERK pathway is selected from the group consisting of BRAF, MEK and ERK, and combination of two or three thereof.

In another embodiment there is provided for the effective amount of a PDK inhibitor and the effective amount of a protein of the MAPK/ERK pathway or use thereof according to the invention wherein the subject is a subject that acquired resistance against treatment with a BRAF, MEK or ERK inhibitor.

In another embodiment there is provided for the effective amount of a PDK inhibitor and the effective amount of a protein of the MAPK/ERK pathway or use thereof according to the invention wherein the PDK inhibitor is selected from the group consisting of dichloroacetate, halogenated acetophones (e.g., dichloroacetophenone), adenosine 5'[[beta],[gamma]-imido] triphosphate, substituted triterpenes, lactones, monochloroacetate, dichloroacetate, trichloroacetate, difluoroacetate, chlorofluoroacetic acid, 2-chloropropionate, 2,2'- dichloropropionate, chloropropionate, 3-chloropropionate, 3,3,3-trifluoro-2-hydroxy-2- methylpropionamide , SDZ048-619 (Novartis), SDZ060-01 1 (Novartis), and SDZ225-066 (Novartis), , radicicol oxime or radicicol, AZD7545 ((2R)-N-{4-[4- (dimethylcarbamoyl)phenylsulfonyl]-2-chlorophenyl}-3,3,3-trifluoro-2-hydroxy-2- methylpropanamide), an anilide derivative of (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid, a secondary amine of (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid, or an inner lipoyl domain of dihydrolipoyl acetyltransferase, a PDK-I inhibitors disclosed in

W01999/062506, a PDK-I inhibitors disclosed in W01999/44618, a PDK-I inhibitors disclosed in WO2009/067767, a PDK-I inhibitors disclosed in US201 1/0207694, dichloroacetate, 2,2- dichloroacetophenone, and (+)-l-N-[2,5-(,S',JR)-dimethyl-4-N-(4-cyanobenzoyl)piperazine]-(R ) -3,3,3-triiluoro-2- hydroxy-2-methylpropanamide), a PDK-I inhibitors disclosed in

In another embodiment there is provided for the effective amount of a PDK inhibitor and the effective amount of a protein of the MAPK ERK pathway or use thereof according to the invention wherein the inhibitor of a protein of the MAPK/ERK pathway is selected from -a BRAF inhibitor selected from the group consisting of A BRAF inhibitor as disclosed in WO 2010/114928, A BRAF inhibitor as disclosed in WO 2005/123696, A BRAF inhibitor as disclosed in WO2007/002325, A BRAF inhibitor as disclosed in WO 2006/003378, A BRAF inhibitor as disclosed in WO 2006/024834, A BRAF inhibitor as disclosed in WO

2006/024836, A BRAF inhibitor as disclosed in WO 2006/040568, A BRAF inhibitor as disclosed in WO 2006/067446 or A BRAF inhibitor as disclosed in WO 2006/079791 , A BRAF inhibitor as disclosed in WO02/24680, or A BRAF inhibitor as disclosed in WO03/022840, A BRAF inhibitor as disclosed in WO 2005/047542, A BRAF inhibitor as disclosed in AU

2003/286447, A BRAF inhibitor as disclosed in US 2004/0096855, A BRAF inhibitor as disclosed in AU2002/356323, A BRAF inhibitor as disclosed in WO2005089443, A BRAF inhibitor as disclosed in WO2006053201 , A BRAF inhibitor as disclosed in US20050267060, A BRAF inhibitor as disclosed in WO2008120004, A BRAF inhibitor as disclosed in

US20090181371 , A BRAF inhibitor as disclosed in WO2008120004, Vemurafenib, PLX4720, PLX4032 (RG7204), GDC-0879, PLX-4720, Sorafenib Tosylate, Nexavar, dasatinib, dabrafenib , erlotinib, gefitinib, imatinib, lapatinib, sorafenib or sunitinib, or active derivatives thereof, A BRAF inhibitor as disclosed in WO2006/024834, A BRAF inhibitor as disclosed in WO2006/067446, A BRAF inhibitor as disclosed in PCT/GB2006/004756, CHIR-265, XL281 (Exelixis) or PLX4032,

-a MEK inhibitor selected from the group consisting of PD184352, PD98059, U0126 , SL327, PDI 84352, a MEK inhibitor disclosed in WO2009018238, a MEK inhibitor disclosed in

WO2007/044084, a MEK inhibitor disclosed in WO2005/051300, a MEK inhibitor disclosed in WO2011/095807, a MEK inhibitor disclosed in WO2008124085, a MEK inhibitor disclosed in WO2009018233, a MEK inhibitor disclosed in WO2007113505, a MEK inhibitor as disclosed in WO03/077914, a MEK inhibitor disclosed in US201 1105521 , a MEK inhibitor disclosed in WO2011067356, a MEK inhibitor disclosed in WO2011067348, a MEK inhibitor disclosed in US2010004247, a MEK inhibitor disclosed in US2010130519, 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-carboxamid, 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, 6-(4-Bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H- benzoimidazole-5- carboxylic acid (2-hydroxy-ethoxy)-amide hydrogen sulphate salt, PD- 325901 , XL518, PD-184352, PD- 318088, AZD6244, CI-1040, 4-(4-Bromo-2- fluorophenylamino)-N-(2-hydroxyethoxy)- 1 ,5-dimethyl-6-oxo- 1 ,6-dihydropyridazine-3- carboxamide or a pharmaceutically acceptable salt thereof, AR-1 19, AS- 701173, AS-701255, 360770-54-3, and/or

-an ERK inhibitor selected from the group consisting of an ERK-inhibitor as disclosed in WO2002058687, SL-327, U0126, PD098059, PD184352, PD0325901 , an ERK-inhibitor as disclosed in WO2002058687, an ERK-inhibitor as disclosed in AU2002248381 , an ERK- inhibitor as disclosed in US20050159385, an ERK-inhibitor as disclosed in US2004102506, an ERK-inhibitor as disclosed in US2005090536, an ERK-inhibitor as disclosed in

-and combinations thereof.

In another embodiment there is provided for the effective amount of a PDK inhibitor and the effective amount of a protein of the MAPK/ERK pathway or use thereof according to the invention wherein the neoplasia is a malignant neoplasia, preferably selected from the group consisting of a malignant neoplasia that involves a disturbance in the MAPK/ERK pathway, a malignant neoplasia selected from the group consisting of lymphoma, non-Hodgkin lymphoma, colorectal cancer, malignant melanoma, thyroid cancer, papillary thyroid carcinoma, lung cancer, non-small cell lung carcinoma, and adenocarcinoma of lung, preferably melanoma, more preferably BRAF (V600E) melanoma and other BRAF(V600E) tumors, or tumors with alternative activation of the BRAF/MEK/ERK pathway

In another embodiment there is provided for the effective amount of a PDK inhibitor and the effective amount of a protein of the MAPK/ERK pathway or use thereof according to the invention wherein the PDK inhibitor is an inhibitory nucleic acid molecule that reduces PDK expression, preferably PDK-1 expression.

In another embodiment there is provided for the effective amount of a PDK inhibitor and the effective amount of a protein of the MAPK/ERK pathway or use thereof according to the invention and wherein the inhibitor of a protein of the MAPK/ERK pathway is an inhibitory nucleic acid molecule that reduces expression of BRAF, MEK or ERK.

In another embodiment there is provided for the effective amount of a PDK inhibitor and the effective amount of a protein of the MAPK/ERK pathway or use thereof according to the invention wherein the PDK inhibitor decreases expression of a PDK polypeptide, decreases the biological activity of a PDK polypeptide, and/or decreases the expression of a PDK nucleic acid molecule. In another embodiment there is provided for the effective amount of a PDK inhibitor and the effective amount of a protein of the MAPK/ERK pathway or use thereof according to the invention wherein the inhibitor of a protein of the MAPK/ERK pathway expression of a BRAF, MEK or ERK polypeptide, decreases the biological activity of a BRAF, MEK or ERK polypeptide, and/or decreases the expression of a BRAF, MEK or ERK nucleic acid molecule.

In a last aspect there is provided for a pharmaceutical composition comprising an effective amount of a PDK inhibitor, preferably a PDK-1 inhibitor, and an effective amount of an inhibitor of a protein of the MAPK/ERK pathway, preferably BRAF, as defined or disclosed herein.

The current invention may be summarized, non-limiting,, as follows

1. A PDK inhibitor, preferably a PDK-1 inhibitor, and an inhibitor of a protein of the

MAPK/ERK pathway for use in treatment or prevention of a neoplasia in a subject.

2. Use of a PDK inhibitor, preferably a PDK-1 inhibitor, and an inhibitor of a protein of the MAPK/ERK pathway for the manufacture of a medicament for treatment or prevention of a neoplasia in a subject. 3. A PDK inhibitor and a protein of the MAPK/ERK pathway or use thereof according to any of the clauses 1 - 2 wherein the protein of the MAPK/ERK pathway is selected from the group consisting of BRAF, MEK and ERK, and combination of two or three thereof.

4. A PDK inhibitor and a protein of the MAPK/ERK pathway or use thereof according to any of the clauses 1 - 3 wherein the subject is a subject that acquired resistance against treatment with a BRAF, MEK or ERK inhibitor. 5. A PDK inhibitor and a protein of the MAPK/ERK pathway or use thereof according to any of the clauses 1 - 4 wherein the neoplasia is a malignant neoplasia, preferably selected from the group consisting of a malignant neoplasia that involves a

disturbance in the MAPK/ERK pathway, preferably a malignant neoplasia selected from the group consisting of lymphoma, non-Hodgkin lymphoma, colorectal cancer, malignant melanoma, thyroid cancer, papillary thyroid carcinoma, lung cancer, non- small cell lung carcinoma, and adenocarcinoma of lung, preferably melanoma, BRAF V600K melanoma or BRAF V600R melanoma, more preferably BRAF (V600E) melanoma and other BRAF(V600E) tumors, or tumors with alternative activation of the BRAF/MEK/ERK pathway.

6. A PDK inhibitor and a protein of the MAPK/ERK pathway or use thereof according to any of the clauses 1 - 5 wherein the PDK inhibitor is an inhibitory nucleic acid molecule that reduces PDK expression, preferably PDK-1 expression. 7. A PDK inhibitor and a protein of the MAPK/ERK pathway or use thereof according to any of the clauses 1 - 6 wherein the inhibitor of a protein of the MAPK/ERK pathway is an inhibitory nucleic acid molecule that reduces expression of BRAF, MEK or ERK.

8. A PDK inhibitor and a protein of the MAPK/ERK pathway or use thereof according to any of the clauses 1 - 7 wherein the PDK inhibitor decreases expression of a PDK polypeptide, decreases the biological activity of a PDK polypeptide, and/or decreases the expression of a PDK nucleic acid molecule.

9. A PDK inhibitor and a protein of the MAPK/ERK pathway or use thereof according to any of the clauses 1 - 8 wherein the inhibitor of a protein of the MAPK/ERK pathway expression of a BRAF, MEK or ERK polypeptide, decreases the biological activity of a BRAF, MEK or ERK polypeptide, and/or decreases the expression of a BRAF, MEK or ERK nucleic acid molecule. A PDK inhibitor and a protein of the MAPK/ERK pathway or use thereof according to any of the clauses 1 - 9 wherein the PDK inhibitor is selected from the group consisting of dichloroacetate, halogenated acetophones (e.g., dichloroacetophenone), adenosine 5'[[beta],[gamma]-imido] triphosphate, substituted triterpenes, lactones, monochloroacetate, dichloroacetate, trichloroacetate, difluoroacetate,

chlorofluoroacetic acid, 2-chloropropionate, 2,2'-dichloropropionate, chloropropionate, 3-chloropropionate, 3,3,3-trifluoro-2-hydroxy-2-methylpropionamide , SDZ048-619 (Novartis), SDZ060-01 1 (Novartis), and SDZ225-066 (Novartis), , radicicol oxime or radicicol, AZD7545 ((2R)-N-{4-[4-(dimethylcarbamoyl)phenylsulfonyl]-2-chlorophenyl}- 3,3,3-trifluoro-2-hydroxy-2-methylpropanamide), an anilide derivative of (R)-3,3,3- trifluoro-2-hydroxy-2-methylpropionic acid, a secondary amine of (R)-3,3,3-trifluoro-2- hydroxy-2-methylpropionic acid, or an inner lipoyl domain of dihydrolipoyl

acetyltransferase, a PDK-I inhibitors disclosed in W01999/062506, a PDK-I inhibitors disclosed in W01999/44618, a PDK-I inhibitors disclosed in WO2009/067767, a PDK- I inhibitors disclosed in US201 1/0207694, dichloroacetate, 2,2-dichloroacetophenone, and (+)-l-N-[2,5-(,S',JR)-dimethyl-4-N-(4-cyanobenzoyl)piperazine]-(R ) -3,3,3-triiluoro- 2- hydroxy-2-methylpropanamide), a PDK-I inhibitors disclosed in WO2006042062, preferably a PDK-I inhibitors disclosed in W01999/062506, even more preferably AZD7545 and derivates thereof as disclosed in W01999/062506, ((2R)-N-{4-[4- (dimethylcarbamoyl)phenylsulfonyl]-2-chlorophenyl}-3,3,3-trifluoro-2-hydroxy-2- methylpropanamide), or (+)-1-N-[2,5-(S,R)-dimethyl-4-N-(4-cyanobenzoyl)piperazine]- (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropanamide.

A PDK inhibitor and a protein of the MAPK/ERK pathway or use thereof according to any of the clauses 1 - 10 wherein the inhibitor of a protein of the MAPK/ERK pathway is selected from

-a BRAF inhibitor selected from the group consisting of A BRAF inhibitor as disclosed in WO 2010/114928, A BRAF inhibitor as disclosed in WO 2005/123696, A BRAF inhibitor as disclosed in WO2007/002325, A BRAF inhibitor as disclosed in WO

2006/003378, A BRAF inhibitor as disclosed in WO 2006/024834, A BRAF inhibitor as disclosed in WO 2006/024836, A BRAF inhibitor as disclosed in WO 2006/040568, A BRAF inhibitor as disclosed in WO 2006/067446 or A BRAF inhibitor as disclosed in WO 2006/079791 , A BRAF inhibitor as disclosed in WO02/24680, or A BRAF inhibitor as disclosed in WO03/022840, A BRAF inhibitor as disclosed in WO 2005/047542, A

BRAF inhibitor as disclosed in AU 2003/286447, A BRAF inhibitor as disclosed in US 2004/0096855, A BRAF inhibitor as disclosed in AU2002/356323, A BRAF inhibitor as disclosed in WO2005089443, A BRAF inhibitor as disclosed in WO2006053201 , A BRAF inhibitor as disclosed in US20050267060, A BRAF inhibitor as disclosed in WO2008120004, A BRAF inhibitor as disclosed in US20090181371 , A BRAF inhibitor as disclosed in WO2008120004, Vemurafenib, PLX4720, PLX4032 (RG7204), GDC- 0879, PLX-4720, Sorafenib Tosylate, Nexavar, dasatinib, dabrafenib , erlotinib, gefitinib, imatinib, lapatinib, sorafenib or sunitinib, or active derivatives thereof, A

BRAF inhibitor as disclosed in WO2006/024834, A BRAF inhibitor as disclosed in WO2006/067446, A BRAF inhibitor as disclosed in PCT/GB2006/004756, CHIR-265, XL281 (Exelixis) or PLX4032, -a MEK inhibitor selected from the group consisting of PD184352, PD98059, U0126 ,

SL327, PDI 84352, a MEK inhibitor disclosed in WO2009018238, a MEK inhibitor disclosed in WO2007/044084, a MEK inhibitor disclosed in WO2005/051300, a MEK inhibitor disclosed in WO201 1/095807, a MEK inhibitor disclosed in WO2008124085, a MEK inhibitor disclosed in WO2009018233, a MEK inhibitor disclosed in

WO2007113505, a MEK inhibitor as disclosed in WO03/077914, a MEK inhibitor disclosed in US2011 105521 , a MEK inhibitor disclosed in WO2011067356, a MEK inhibitor disclosed in WO201 1067348, a MEK inhibitor disclosed in US2010004247, a MEK inhibitor disclosed in US2010130519, 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-carboxamid, 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, 6-(4-Bromo-2-chloro-phenylamino)-7-fluoro- 3-methyl-3H-benzoimidazole-5- carboxylic acid (2-hydroxy-ethoxy)-amide hydrogen sulphate salt, PD-325901 , XL518, PD-184352, PD- 318088, AZD6244, CI-1040, 4-(4-

Bromo-2- fluorophenylamino)-N-(2-hydroxyethoxy)- 1 ,5-dimethyl-6-oxo- 1 ,6- dihydropyridazine-3- carboxamide or a pharmaceutically acceptable salt thereof, AR- 1 19, AS- 701173, AS-701255, 360770-54-3, and/or

-an ERK inhibitor selected from the group consisting of an ERK-inhibitor as disclosed in WO2002058687, SL-327, U0126, PD098059, PD184352, PD0325901 , an ERK- inhibitor as disclosed in WO2002058687, an ERK-inhibitor as disclosed in

AU2002248381 , an ERK-inhibitor as disclosed in US20050159385, an ERK-inhibitor as disclosed in US2004102506, an ERK-inhibitor as disclosed in US2005090536, an ERK-inhibitor as disclosed in US2004048861 , an ERK-inhibitor as disclosed in US20100004234, an ERK-inhibitor as disclosed in HR201 10892, an ERK-inhibitor as disclosed in WO201 1163330, an ERK-inhibitor as disclosed in TW200934775, an ERK-inhibitor as disclosed in EP2332922, an ERK-inhibitor as disclosed in

WO2011041152 , an ERK-inhibitor as disclosed in US201 1038876, an ERK-inhibitor as disclosed in WO2009146034, an ERK-inhibitor as disclosed in HK11 17159, an ERK-inhibitor as disclosed in WO2009026487, an ERK-inhibitor as disclosed in WO2008115890, PH-797804 (Bioorg Med Chem Lett 201 1 Jul 1 ;21 (13):4066-71) an ERK-inhibitor as disclosed in US2009186379, an ERK-inhibitor as disclosed in WO2008055236, an ERK-inhibitor as disclosed in US2007232610, an ERK-inhibitor as disclosed in WO2007025090, an ERK-inhibitor as disclosed in US2007049591 , 2- (2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059), 1 ,4-diamino-2,3- dicyano-1 ,4-bis(2-aminophenylthio)butadiene (U0126), and Z-& E-a-(amino-((4- aminophenyl)thio)methylene)-2-(trifluoromethyl)benzeneacetonitrile (MEK1/2) (US201 11 10916),

and combinations thereof.

Figures

Figure 1. PDK1 downregulation and PDP2 upregulation correlates with PDH

dephosphorylation in BRAF^E600-induced senescence and it is reversed in senescence abrogation.

A. Cell viability assay of HDFs (Tig3(et)p16i) transduced with vector control or shRNA targeting C/EBP #1 and subsequently with a BRAF^E600-expressing retrovirus. Cells undergoing OIS (upon transduction of BRAF^E600) or bypassing OIS (shC/EBP depletion in combination with BRAF^E600), cycling and C/EBP depleted cells were fixed and stained 1 1 days after exposure to BRAF^E600. B. BrdU incorporation of samples described in A measured 1 1 days after exposure to BRAF^E600. Levels are represented as mean of three independent experiments. Error bars represent s.d. C. Samples from A were analyzed by immunoblotting as indicated. -actin serves as loading control. D. Schematic representation of major biochemical pathways in mammalian cells. PDH, a key enzyme linking glycolysis to oxidative phosphorylation is regulated through a reversible phosphorylation cycle. Phosphorylation of PDH by PDK1 inhibits its action and pushes metabolism into glycolysis while

dephosphorylation of PDH by PDP2 has an opposite effect.

Figure 2. PDK1 overexpression abrogates BRAF^E600-induced senescence.

A. Cell viability assay of HDFs (Tig3(et)p16i) expressing empty vector or PDK1 in the presence and absence of oncogenic BRAF^E600. Cells were fixed and stained 11 days after exposure to BRAF^E600. B. BrdU incorporation of samples described in A measured 8 days after exposure to BRAF^E600. Levels are represented as mean of at least three independent experiments. Error bars represent s.d. C. Representative images of senescence associated (SA)- -galactosidase staining for the cells described in A. Quantification is performed on three independent experiments, with s.d. D. Samples from A were analyzed by

immunoblotting as indicated. Hsp90 serves as loading control. E. PDH activity of the samples described in A measured by DipStick assay. F-H. Regulation of IL8 (F), IL6 (G) and C/EBPa (H) transcripts of the samples described in A as determined by qRT-PCR. Measurements are based on at least three independent experiments and standardized to the BRAF^E600- expressing senescent cells. Figure 2 Suppl. PDK1 overexpression abrogates BRAF^E600-induced senescence independently of p16^INK4A status.

A. Cell viability assay of HDFs (Tig3(et)) expressing empty vector or PDK1 in the presence and absence of oncogenic BRAF^E600. Cells were fixed and stained 1 1 days after exposure to BRAF^E600. B. BrdU incorporation of samples described in A measured 8 days after exposure to BRAF^E600.

Figure 3. PDP2 is required for BRAF -induced senescence.

A. Cell viability assay of HDFs (Tig3(et)p16i) expressing vector control or independent, nonoverlapping shRNAs targeting PDP2 in the presence and absence of oncogenic

BRAF^E600. Cells were fixed and stained 1 1 days after exposure to BRAF^E600. B. BrdU incorporation of samples described in A measured 8 days after exposure to BRAF^E600. Levels are represented as mean of at least three independent experiments. Error bars represent s.d. C. Representative images of senescence associated (SA)- -galactosidase staining for the cells described in A. Quantification is performed on three independent experiments, with s.d. D, G-l. Regulation of PDP2 (D), IL8 (G), IL6 (H) and C/EBPU (I) transcripts of the samples described in A as determined by qRT-PCR. Measurements are based on three independent experiments and standardized to the BRAF^E600 -expressing senescent cells. E. Samples from A were analyzed by immunoblotting as indicated. -actin serves as loading control. E. PDH activity of the samples described in A measured by DipStick assay.

Figure 4. Silencing of PDK1 induces senescence in primary cells.

A. BrdU incorporation of HDFs (Tig3(et)) expressing vector control or two independent, non- overlapping shRNAs targeting PDK1 measured 7 days after transduction with shRNA- expressing lentivirus. Levels are represented as mean of at least three independent experiments. Error bars represent s.d. B. Representative images of senescence associated (SA)- -galactosidase staining for the cells described in A. Quantification is performed on three independent experiments, with s.d. C. Samples from A were analyzed by

immunoblotting as indicated. -actin serves as loading control. D. PDH activity of the samples described in A measured by DipStick assay. E. BrdU incorporation of melanocytes (FM 186ae p16i) expressing vector control or independent, nonoverlapping shRNAs targeting PDK1 measured 7 days after transduction with shRNA-expressing lentivirus. Levels are represented as mean of three independent experiments. Error bars represent s.d. F.

Representative images of senescence associated (SA)- -galactosidase staining (lower panel) and phase contrast (upper panel) for the cells described in E. Quantification is performed on three independent experiments, with s.d. G. Samples from E were analyzed by immunoblotting as indicated. -actin serves as loading control. Figure 6. Depletion of PDK1 sensitizes BRAF mutant melanoma cell lines to the BRAF inhibitor PLX4720.

A. PLX4720 dose response curves of BRAF mutant melanoma cell lines sensitive or resistant to the PLX treatment transduced with vector control or shRNA targeting PDK1. Cells were treated with concentration range of PLX4720 for 3 days and cell viability was measured by cell titer blue assay. B. PLX4720 dose response curves from A normalized to untreated cells. C. Percentage of residual population of PLX4720-resistant melanoma cell lines transduced with vector control or shRNA targeting PDK1. Cells were treated with 40μΜ concentration of PLX4720 for 3 days and percentage of residual population was measured by cell titer blue assay.

Examples

Example 1

Summary

We observed that regulation of pyruvate dehydrogenase (PDH) plays a crucial role in mediating BRAF^E600-lnduced Senescence (BIS). BRAF^E600 acts on PDH via downregulation of pyruvate dehydrogenase kinase isoform 1 (PDK1 or PDHK1) and upregulation of pyruvate dehydrogenase phosphatase (PDP2). We found that interference with these changes abrogates BIS. Specifically, we demonstrate that BIS is efficiently bypassed by either overexpression of PDK1 or shRNA depletion of PDP2. Both lead to the accumulation of phosphorylated PDH, which causes a shift in metabolism from oxidative phosphorylation to aerobic glycolysis, a process also known as the Warburg effect. This regulation is not observed in Ras^v12-induced senescence. PDK1 depletion is sufficient to induce senescence in primary cells. We found that shRNA depletion of PDK1 or inhibition with PDK inhibitors (= reducing expression/activity of PDK-1) sensitizes BRAF mutant melanoma cell lines to BRAF inhibitors, e.g. the new clinical BRAF inhibitor PLX4720/Vemurafenib. This is particularly relevant because melanoma patients treated with this drug invariably show a relapse at 7 months on average after initial strong responses. In several cultured human melanoma cell lines, this corresponds to the presence of a PLX4720-resistant population, which we demonstrate is killed by inhibition of PDK, e.g. depletion of PDK1. Thus, there is an urgent need to combine inhibition of the BRAF/MEK/ERK pathway with inhibition of a second target. Our data show that inhibition of PDK1 e.g. with a small molecule, meet that need.

EXPERIMENTAL PROCEDURES

Cell culture, cell lines, cell proliferation assay

The human diploid fibroblast (HDF) cell line Tig3 expressing the ectopic receptor and hTERT (Tig3 (et)) and all melanoma cell lines were maintained in DMEM with 4.5 mg ml^-1 glucose and 0.11 mg ml^-1 sodium pyruvate, supplemented with 9% fetal bovine serum (PAA), 2 mM glutamine, 100 units ml^-1 penicillin and 0.1 mg ml^-1 streptomycin (GIBCO). Melanocytes were propagated in 254CF medium in the presence of 0.2 mM CaCI₂ and melanocyte growth supplement (Cascade Biologicals).

PDK1 was depleted with short hairpin (sh) RNAs encoded by lentiviral infections. Infections were performed using HEK293T cells as producers of viral supernatants. For infections, filtered (pore size 0.45 mm) viral supernatant, supplemented with 4-8 g ml^"1 polybrene was used. In general, a single infection round of 6 h was sufficient to infect at least 90% of the population.

Cells were infected with shRNAs targeting PDK1 and selected pharmacologically (puromycin). After 5 days of selection, cells were seeded for cell viability assay in 24 well plates and for BrdU incorporation assays or trypan blue exclusion assay into six-well plates and maintained in the selection medium. For cell viability fixation and staining with crystal violet was performed 7 days after infection. Images of cell viability assays reflect representative results of several independent experiments. BrdU labelling was carried out for 3 h followed by fixation. Incorporated BrdU was detected by immunostaining as described in (Serrano et al., 1997) and FACS analysis. Results are represented as mean with SD of three or more independent experiments. Trypan blue exclusion assay was performed 6 days after infection as described previously (Forcet et al, 2001). Results are represented as the mean of at least three independent experiment +/- SD.

PLX4720 dose response analysis

Melanoma cell lines expressing either empty vector of shRNA targeting PDK1 were plated into a 96-well plate and left to grow overnight before being treated with increasing concentrations of PLX4720 in triplicate. After 96 h, the levels of growth inhibition were examined using Cell Titer-Blue viability assay (Promega). Data show the mean of at least three independent experiment +/- SD. shRNAs

Lentiviral knockdown constructs were purchased from Sigma-Aldrich:

shPDK1#3 - TRCN000006261

shPDK1#5 - TRCN000006263

As negative control pLKO-puro without insert was used.

Quantitative real-time RT-PCR

Total RNA was DNase-treated with RQ1 RNase-Free DNase (Promega). Reverse transcription was performed with Superscript II First Strand Kit (Invitrogen). qRT-PCR was performed with the SYBR Green PCR Master Mix (Applied Biosystems) on an ABI PRISM 7700 Sequence detection System.

Primer sets used were as follows:

PDK1 : 5'- CCAAGACCTCGTGTTGAGACC-3' and 5'- AATACAGCTTCAGGTCTCCTTGG-3'; RPL13 was used as control. For analysis, the ΔΤ method was applied. Data are represented as mean + s.d. of three or more independent experiments.

Antibodies

Antibodies used for immunoblotting were β-actin (AC-74; A5316; Sigma), PDHEI a ((9H9AF5); 459400; Invitrogen), phospho-PDHEIa [Ser293] (NB110-93479; Novus Biologicals), PDK1 (KAP-PK1 12; Stressgene)//pct

Measurement of PDH activity

PDH activity was measured using the DipStick assay kit from MitoSciences (MSP90). Cells were lysed in the 10x sample buffer provided by the manufacturer, followed by centrifugation and measurement of the protein concentration with the BioRad Protein Assay. 140 mg of protein lysate was loaded and PDH activity was measured according to the manufacturer's protocol.

Results

PDH dephosphorylation as a function of BRAF^E600 - induced senescence

We aimed to study the metabolic regulation during tumor progression i.e., from senescence to immortalization. We compared BRAF^E600 - senescent human diploid fibroblasts (HDFs) to cycling cells and cells in which senescence is abrogated upon C/EBP beta silencing (Figure 1A&B, Kuilman et al, 2008) for levels/phosphorylation of key rate-limiting enzymes in glycolysis, oxidative phosphorylation, lipid synthesis and the pentose shunt (Figure 1 D). We observed that BRAF^E600-senescent cells display differential phosphorylation of pyruvate dehydrogenase (PDH). PDH is the key enzyme linking glycolysis to oxidative phosphorylation and it is regulated through a reversible phosphorylation cycle (Figure 1 D). Phosphorylation of PDH by pyruvate dehydrogenase kinases (PDK1-4) inhibits its action and pushes metabolism into glycolysis, while dephosphorylation of PDH by pyruvate dehydrogenase phosphatases (PDP1&2) has the opposite effect. PDH phosphorylation was reduced in cells undergoing BRAF^E600- induced senescence when compared to cycling cells and phosphorylation was restored when senescence was abrogated by C/EBP beta depletion (Figure 1C). This change in phosphorylation of PDH correlated with levels of PDK1 and PDP2 proteins. PDK1 was downregulated and PDP2 was upregulated in BRAF^E600-induced senescence but not in cycling cells or cells in which senescence was abrogated by C/EBP beta depletion (Figure 1 C).

A critical role of PDH in mediating 01 S

Next we investigated whether regulation of PDH activity has a causal role in mediating OIS. First we analyzed if overexpression of PDK1 can restore PDH phosphorylation inhibiting its activation and abrogate senescence. Although p16^INK4A is not strictly required for BRAF^E600- induced senescence in HDFs, it might contribute to in the context of additional factors. Therefore HDFs (Tig3) expressing shRNA targeting p16^INK4A (Tig3(et)/p16i) were used. Polyclonal cell lines expressing either PDK1 or carrying control vector were generated and these were subsequently transduced with BRAF^E600-expressing retrovirus. Ectopic expression of PDK1 rescued the decrease in PDH phosphorylation and reduced the increase in activity observed upon BRAF^E600 expression (Figure 2D and Figure 2E). Notably that caused efficient abrogation of BRAF^E600 -induced arrest as evident from the cell viability and BrdU incorporation assays (Figure 2A&B). Restored proliferation was not due to the loss of BRAF^E600 expression or a difference in downstream MEK ERK pathway activation (Figure 2D). Moreover it was independent of the p16^INK4A status as it was seen irrespective of p16^INK4A depletion (Figure 2 Suppl).

OIS abrogation upon PDK1 overexpression was associated with a marked reduction in (SA)- beta-galactosidase activity (Figure 2C) and p15^INK4B protein level (Figure 2D). In addition IL6 and IL8 transcript levels were significantly reduced in these cells when compared to OIS cells (Figure 2 F&G) indicating a link between PDK1 and the senescence-messaging secretome (SMS). Interestingly, induction of the C/EBPbeta transcript in the presence of BRAF^E600 was also reduced upon PDK1 overexpression (Figure 2H), which together with upregulation of PDK1 upon C/EBPbeta silencing (Figure 1 C) suggests the existence of a feedback loop between these two.

To get further evidence that abrogation of BRAF -induced senescence upon PDK1 overexpression is mediated through regulation of PDH we modulated PDP2 levels in the presence or absence of BRAF^E600. As PDP2 has the opposite effect on PDH than PDK1 , we expected that PDP2 depletion restores PDH phosphorylation inhibiting its activation and abrogates senescence. To silence PDP2 two non-overlapping shRNAs (shPDP2#1 and shPDP2#4) giving strong depletion of the PDP2 mRNA (Figure 3D) and protein (Figure 3E) were used.

Both cell viability and BrdU incorporation assays demonstrated that depletion of PDP2 effectively abrogated BRAF^E600 -induced arrest (Figure 3A&B). This could not be explained by loss of BRAF^E600 expression or difference in activation of its downstream effectors (Figure 3E). Consistent with the effect on proliferation, (SA)-beta-galactosidase activity (Figure 3C), p15^INK4B protein levels (Figure 3E) and transcript levels of SMS components IL6 and IL8 (Figure 3G &H) were reduced when compared to OIS cells. Similarly to abrogation of senescence upon PDK1 overexpression, PDP2 depletion reduced C/EBPbeta transcript induction in presence of BRAF^E600 suggesting the existence of a feedback loop (Figure 3I). Importantly abrogation of BRAF^E600 -induced senescence upon PDP2 silencing correlated with the restoration of PDH phosphorylation and the reduction in its activity (Figure 3E and F), which strongly suggests the critical role of PDH in mediating OIS.

PDK1 depletion is sufficient to induce senescence in primary cells

Next we examined whether PDH dephosphorylation upon PDK1 silencing is sufficient to induce senescence. To deplete PDK1 two non-overlapping shRNAs (shPDK1#3 and shPDK1#5) were used. PDK1 silencing inhibited proliferation of HDFs (Tig3(et)) and melanocytes as measured in BrdU incorporation assay (Figure 4A & E). That arrest shared a number of characteristics with senescence including the increase in (SA)-B-galactosidase activity (Figure 4B & F), decrease in PCNA levels and upregulation of several cell cycle regulators including p16^INK4A, p15^INK4B and p27 and to smaller extent p53 levels (Figure 4C & G). That correlated with the decrease in PDH phosphorylation and the increase in its activity (Figure 4C & D) emphasizing the importance of PDH regulation and metabolic rewiring for senescence.

Depletion of PDK.1 induces cell death in BRAF mutant melanoma cell lines but not in primary cells.

Since senescence represents an important antitumor mechanism, we aimed to study the relevance of PDH-PDK1-PDP2 pathway regulation in cancer setting. To address this question we depleted PDK1 in BRAF mutant melanoma cell lines (BRAF MT) and primary cells and analyzed the effect on cell fitness. In a cell viability assay we observed a significant decrease in cell viability upon PDK1 depletion in BRAF mutant melanoma cell lines, but not in primary cells . The effect on cell viability could not be explained by differences in proliferation because both BRAF mutant melanoma cell lines and primary cells arrested as measured in BrdU incorporation assay. On the contrary it was caused by induction of cell death specifically in BRAF mutant melanoma cell lines but not in primary cells as quantified by trypan blue exclusion assay. Importantly, this difference in viability was not due to the difference in the PDK1 silencing efficiency between melanoma and primary cell lines. Moreover the same effect on cell viability was observed when 2 independent shRNAs targeting PDK1 were used, excluding off target effects.

Increase in PDH activity upon PDK1 depletion was observed in BRAF mutant cell lines.

Depletion of PDK1 sensitizes BRAF mutant melanoma cell lines to the BRAF inhibitor PDC4720

Although preclinical studies with a novel RAF-selective inhibitor PLX4032/Vemurafenib demonstrated high (80%) anti-tumor response rate among patients with BRAF^E600-positive melanomas, drug resistance occurrence is frequently observed. Several studies showed that resistance arises either from co-activation of parallel or downstream survival and proliferation pathways and/or compensatory activation of alternative survival pathways. This demonstrates the need for combinational therapy inhibiting multiple survival pathways to obtain potent antitumor effects. As BRAF mutant melanoma cell lines are sensitive to PDK1 silencing we asked whether PDK1 depletion could sensitize BRAF mutant melanoma cell lines to PLX4720 treatment (a preclinical analogue of PLX4032). We selected 4 BRAF^E600 melanoma cell lines sensitive to PLX4720 and 4 BRAF^E600 melanoma cell lines moderately resistant to the treatment (percentage of residual population of cells at 40μΜ PLX4720 concentration is higher than 20). In both sensitive and resistant cell lines PDK1 was depleted and sensitization was measured over a PLX4720 dose response (Figure 6 A&B). In all cell lines tested PDK1 depletion significantly shifted the IC50 for PLX4720 to the left (meaning increasing the toxicity; Figure 6 A). In PLX4720-sensitive cell lines additive sensitization was observed, indicating that the response observed was the sum of the effect of PDK1 depletion and PLX4720 treatment individually (Figure 6 A&B). On the other hand, strikingly, all PLX4720- resistant cell lines exhibited synergistic sensitization, showing a greater response than the sum of the two treatments alone (Figure 6 A&B). Importantly, PDK1 depletion not only significantly shifted the PLX4720 response curve but also eliminated residual population present at high PLX4720 dose in PLX4720-resistant cells (Figure 6 C). This finding is of particular interest in view of the clinical observation that Vemurafenib treatment invariably leads to the emergence of resistant tumors.

Hence, the availability of a PDK1 inhibitor may have clinical utility in the context of (BRAF or MEK/ERK) inhibition.

Claims

1. An effective amount of a PDK inhibitor, preferably a PDK-1 inhibitor, and an effective amount of an inhibitor of a protein of the MAPK/ERK pathway for use in treatment or prevention of a neoplasia in a subject.

2. Use of an effective amount of a PDK inhibitor, preferably a PDK-1 inhibitor, and an effective amount of an inhibitor of a protein of the MAPK/ERK pathway for the manufacture of a medicament for treatment or prevention of a neoplasia in a subject.

3. The effective amount of a PDK inhibitor and the effective amount of a protein of the MAPK/ERK pathway or use thereof according to any of the claims 1 - 2 wherein the protein of the MAPK/ERK pathway is selected from the group consisting of BRAF, MEK and ERK, and combination of two or three thereof.

4. The effective amount of a PDK inhibitor and the effective amount of a protein of the MAPK/ERK pathway or use thereof according to any of the claims 1 - 3 wherein the subject is a subject that acquired resistance against treatment with a BRAF, MEK or ERK inhibitor.

5. The effective amount of a PDK inhibitor and the effective amount of a protein of the MAPK/ERK pathway or use thereof according to any of the claims 1 - 4 wherein the neoplasia is a malignant neoplasia, preferably selected from the group consisting of a malignant neoplasia that involves a disturbance in the MAPK/ERK pathway, preferably a malignant neoplasia selected from the group consisting of lymphoma, non-Hodgkin lymphoma, colorectal cancer, malignant melanoma, thyroid cancer, papillary thyroid carcinoma, lung cancer, non-small cell lung carcinoma, and adenocarcinoma of lung, preferably melanoma, BRAF V600K melanoma or BRAF V600R melanoma, more preferably BRAF (V600E) melanoma and other BRAF(V600E) tumors, or tumors with alternative activation of the BRAF/MEK/ERK pathway.

6. The effective amount of a PDK inhibitor and the effective amount of a protein of the MAPK/ERK pathway or use thereof according to any of the claims 1 - 5 wherein the PDK inhibitor is an inhibitory nucleic acid molecule that reduces PDK expression, preferably PDK-1 expression.

7. The effective amount of a PDK inhibitor and the effective amount of a protein of the MAPK/ERK pathway or use thereof according to any of the claims 1 - 6 wherein the inhibitor of a protein of the MAPK/ERK pathway is an inhibitory nucleic acid molecule that reduces expression of BRAF, MEK or ERK.

8. The effective amount of a PDK inhibitor and the effective amount of a protein of the MAPK/ERK pathway or use thereof according to any of the claims 1 - 7 wherein the

PDK inhibitor decreases expression of a PDK polypeptide, decreases the biological activity of a PDK polypeptide, and/or decreases the expression of a PDK nucleic acid molecule. 9. The effective amount of a PDK inhibitor and the effective amount of a protein of the MAPK/ERK pathway or use thereof according to any of the claims 1 - 8 wherein the inhibitor of a protein of the MAPK/ERK pathway expression of a BRAF, MEK or ERK polypeptide, decreases the biological activity of a BRAF, MEK or ERK polypeptide, and/or decreases the expression of a BRAF, MEK or ERK nucleic acid molecule.

10. The effective amount of a PDK inhibitor and the effective amount of a protein of the MAPK/ERK pathway or use thereof according to any of the claims 1 - 9 wherein the PDK inhibitor is selected from the group consisting of dichloroacetate, halogenated acetophones (e.g., dichloroacetophenone), adenosine 5'[[beta],[gamma]-imido] triphosphate, substituted triterpenes, lactones, monochloroacetate, dichloroacetate, trichloroacetate, difluoroacetate, chlorofluoroacetic acid, 2-chloropropionate, 2,2'- dichloropropionate, chloropropionate, 3-chloropropionate, 3,3,3-trifluoro-2-hydroxy-2- methylpropionamide , SDZ048-619 (Novartis), SDZ060-01 1 (Novartis), and SDZ225- 066 (Novartis), , radicicol oxime or radicicol, AZD7545 ((2R)-N-{4-[4- (dimethylcarbamoyl)phenylsulfonyl]-2-chlorophenyl}-3,3,3-trifluoro-2-hydroxy-2- methylpropanamide), an anilide derivative of (R)-3,3,3-trifluoro-2-hydroxy-2- methylpropionic acid, a secondary amine of (R)-3,3,3-trifluoro-2-hydroxy-2- methylpropionic acid, or an inner lipoyl domain of dihydrolipoyl acetyltransferase, a PDK-I inhibitors disclosed in W01999/062506, a PDK-I inhibitors disclosed in

W01999/44618, a PDK-I inhibitors disclosed in WO2009/067767, a PDK-I inhibitors disclosed in US2011/0207694, dichloroacetate, 2,2-dichloroacetophenone, and (+)-l- N-[2,5-(,S',JR)-dimethyl-4-N-(4-cyanobenzoyl)piperazine]-(R ) -3,3,3-triiluoro-2- hydroxy-2-methylpropanamide), a PDK-I inhibitors disclosed in WO2006042062, preferably a PDK-I inhibitors disclosed in W01999/062506, even more preferably AZD7545 and derivates thereof as disclosed in W01999/062506, ((2R)-N-{4-[4-

(dimethylcarbamoyl)phenylsulfonyl]-2-chlorophenyl}-3,3,3-trifluoro-2-hydroxy-2- methylpropanamide), or (+)-1-N-[2,5-(S,R)-dimethyl-4-N-(4-cyanobenzoyl)piperazine]- (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropanamide.

1. The effective amount of a PDK inhibitor and the effective amount of a protein of the MAPK/ERK pathway or use thereof according to any of the claims 1 - 10 wherein the inhibitor of a protein of the MAPK/ERK pathway is selected from

-a BRAF inhibitor selected from the group consisting of A BRAF inhibitor as disclosed in WO 2010/114928, A BRAF inhibitor as disclosed in WO 2005/123696, A BRAF inhibitor as disclosed in WO2007/002325, A BRAF inhibitor as disclosed in WO 2006/003378, A BRAF inhibitor as disclosed in WO 2006/024834, A BRAF inhibitor as disclosed in WO 2006/024836, A BRAF inhibitor as disclosed in WO 2006/040568, A BRAF inhibitor as disclosed in WO 2006/067446 or A BRAF inhibitor as disclosed in WO 2006/079791 , A BRAF inhibitor as disclosed in WO02/24680, or A BRAF inhibitor as disclosed in WO03/022840, A BRAF inhibitor as disclosed in WO 2005/047542, A BRAF inhibitor as disclosed in AU 2003/286447, A BRAF inhibitor as disclosed in US 2004/0096855, A BRAF inhibitor as disclosed in AU2002/356323, A BRAF inhibitor as disclosed in WO2005089443, A BRAF inhibitor as disclosed in WO2006053201 , A BRAF inhibitor as disclosed in US20050267060, A BRAF inhibitor as disclosed in WO2008120004, A BRAF inhibitor as disclosed in US20090181371 , A BRAF inhibitor as disclosed in WO2008120004, Vemurafenib, PLX4720, PLX4032 (RG7204), GDC- 0879, PLX-4720, Sorafenib Tosylate, Nexavar, dasatinib, dabrafenib , erlotinib, gefitinib, imatinib, lapatinib, sorafenib or sunitinib, or active derivatives thereof, A BRAF inhibitor as disclosed in WO2006/024834, A BRAF inhibitor as disclosed in WO2006/067446, A BRAF inhibitor as disclosed in PCT/GB2006/004756, CHIR-265, XL281 (Exelixis) or PLX4032,

-a MEK inhibitor selected from the group consisting of PD184352, PD98059, U0126 , SL327, PDI 84352, a MEK inhibitor disclosed in WO2009018238, a MEK inhibitor disclosed in WO2007/044084, a MEK inhibitor disclosed in WO2005/051300, a MEK inhibitor disclosed in WO2011/095807, a MEK inhibitor disclosed in WO2008124085, a MEK inhibitor disclosed in WO2009018233, a MEK inhibitor disclosed in

WO2007113505, a MEK inhibitor as disclosed in WO03/077914, a MEK inhibitor disclosed in US201 1105521 , a MEK inhibitor disclosed in WO201 1067356, a MEK inhibitor disclosed in WO201 1067348, a MEK inhibitor disclosed in US2010004247, a MEK inhibitor disclosed in US2010130519, 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-carboxamid, 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, 6-(4-Bromo-2-chloro-phenylamino)-7-fluoro- 3-methyl-3H-benzoimidazole-5- carboxylic acid (2-hydroxy-ethoxy)-amide hydrogen sulphate salt, PD-325901 , XL518, PD-184352, PD- 318088, AZD6244, CI-1040, 4-(4- Bromo-2- fluorophenylamino)-N-(2-hydroxyethoxy)- 1 ,5-dimethyl-6-oxo- 1 ,6- dihydropyridazine-3- carboxamide or a pharmaceutically acceptable salt thereof, AR- 1 19, AS- 701173, AS-701255, 360770-54-3, and/or

WO2011041152 , an ERK-inhibitor as disclosed in US201 1038876, an ERK-inhibitor as disclosed in WO2009146034, an ERK-inhibitor as disclosed in HK1 117159, an ERK-inhibitor as disclosed in WO2009026487, an ERK-inhibitor as disclosed in

WO2008115890, PH-797804 (Bioorg Med Chem Lett 201 1 Jul 1 ;21 (13):4066-71) an ERK-inhibitor as disclosed in US2009186379, an ERK-inhibitor as disclosed in WO2008055236, an ERK-inhibitor as disclosed in US2007232610, an ERK-inhibitor as disclosed in WO2007025090, an ERK-inhibitor as disclosed in US2007049591 , 2- (2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059), 1 ,4-diamino-2,3- dicyano-1 ,4-bis(2-aminophenylthio)butadiene (U0126), and Z-& E-a-(amino-((4- aminophenyl)thio)methylene)-2-(trifluoromethyl)benzeneacetonitrile (MEK1/2) (US201 11 10916),

and combinations thereof.

12. A method of treating or preventing a neoplasia in a subject, the method comprising administrating to a subject in need of such treatment an effective amount of at least one PDK inhibitor, preferably a PDK-1 inhibitor, and an effective amount of at least one inhibitor of a protein of the MAPK/ERK pathway.

13. A method according to claim 12 wherein the protein of the MAPK/ERK pathway is selected from the group consisting of BRAF, MEK and ERK, and combination of two or three thereof.

14. Method according to any one of claims 12 -13 wherein the subject is a subject that acquired resistance against treatment with a BRAF, MEK or ERK inhibitor.

15. A method according to any one claims 12 -14 wherein the neoplasia is a malignant neoplasia, preferably selected from the group consisting of a malignant neoplasia that involves a disturbance in the MAPK/ERK pathway, preferably a malignant neoplasia selected from the group consisting of lymphoma, non-Hodgkin lymphoma, colorectal cancer, malignant melanoma, thyroid cancer, papillary thyroid carcinoma, lung cancer, non-small cell lung carcinoma, and adenocarcinoma of lung, preferably melanoma, , BRAF V600K melanoma or BRAF V600R melanoma, more preferably BRAF (V600E) melanoma and other BRAF(V600E) tumors, or tumors with alternative activation of the BRAF/MEK/ERK pathway.

16. A method according to any one of claims 12 - 15 wherein the PDK inhibitor is an

inhibitory nucleic acid molecule that reduces PDK expression, preferably PDK-1 expression.

17. A method according to any one of claim 12 - 16 wherein the inhibitor of a protein of the MAPK/ERK pathway is an inhibitory nucleic acid molecule that reduces expression of BRAF, MEK or ERK.

18. A method according to any one of the claims 12 - 17 wherein the PDK inhibitor

decreases expression of a PDK polypeptide, decreases the biological activity of a PDK polypeptide, and/or decreases the expression of a PDK nucleic acid molecule.

19. A method according to any one of the claims 12 -18 wherein the inhibitor of a protein of the MAPK/ERK pathway decreases expression of a BRAF, MEK or ERK

polypeptide, decreases the biological activity of a BRAF, MEK or ERK polypeptide, and/or decreases the expression of a BRAF, MEK or ERK nucleic acid molecule.

20. A method according to any one of the claims 12 -19 wherein the PDK inhibitor is

selected from the group consisting of dichloroacetate, halogenated acetophones (e.g., dichloroacetophenone), adenosine 5'[[beta],[gamma]-imido] triphosphate, substituted triterpenes, lactones, monochloroacetate, dichloroacetate, trichloroacetate, difluoroacetate, chlorofluoroacetic acid, 2-chloropropionate, 2,2'-dichloropropionate, chloropropionate, 3-chloropropionate, 3,3,3-trifluoro-2-hydroxy-2-methylpropionamide

, SDZ048-619 (Novartis), SDZ060-01 1 (Novartis), and SDZ225-066 (Novartis), , radicicol oxime or radicicol, AZD7545 ((2R)-N-{4-[4- (dimethylcarbamoyl)phenylsulfonyl]-2-chlorophenyl}-3,3,3-trifluoro-2-hydroxy methylpropanamide), an anilide derivative of (R)-3,3,3-trifluoro-2-hydroxy-2- methylpropionic acid, a secondary amine of (R)-3,3,3-trifluoro-2-hydroxy-2- methylpropionic acid, or an inner lipoyl domain of dihydrolipoyl acetyltransferase, a PDK-I inhibitors disclosed in W01999/062506, a PDK-I inhibitors disclosed in

W01999/44618, a PDK-I inhibitors disclosed in WO2009/067767, a PDK-I inhibitors disclosed in US2011/0207694, dichloroacetate, 2,2-dichloroacetophenone, and (+)-l- N-[2,5-(,S',JR)-dimethyl-4-N-(4-cyanobenzoyl)piperazine]-(R ) -3,3,3-triiluoro-2- hydroxy-2-methylpropanamide), a PDK-I inhibitors disclosed in W02006042062, preferably a PDK-I inhibitors disclosed in W01999/062506, even more preferably AZD7545 and derivates thereof as disclosed in W01999/062506, ((2R)-N-{4-[4- (dimethylcarbamoyl)phenylsulfonyl]-2-chlorophenyl}-3,3,3-trifluoro-2-hydroxy-2- methylpropanamide), or (+)-1-N-[2,5-(S,R)-dimethyl-4-N-(4-cyanobenzoyl)piperazine]- (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropanamide.

21. A method according to any one of the claims 12 -20 wherein the inhibitor of a protein of the MAPK/ERK pathway is selected from

-a BRAF inhibitor selected from the group consisting of A BRAF inhibitor as disclosed in WO 2010/114928, A BRAF inhibitor as disclosed in WO 2005/123696, A BRAF inhibitor as disclosed in WO2007/002325, A BRAF inhibitor as disclosed in WO 2006/003378, A BRAF inhibitor as disclosed in WO 2006/024834, A BRAF inhibitor as disclosed in WO 2006/024836, A BRAF inhibitor as disclosed in WO 2006/040568, A BRAF inhibitor as disclosed in WO 2006/067446 or A BRAF inhibitor as disclosed in WO 2006/079791 , A BRAF inhibitor as disclosed in WO02/24680, or A BRAF inhibitor as disclosed in WO03/022840, A BRAF inhibitor as disclosed in WO 2005/047542, A BRAF inhibitor as disclosed in AU 2003/286447, A BRAF inhibitor as disclosed in US 2004/0096855, A BRAF inhibitor as disclosed in AU2002/356323, A BRAF inhibitor as disclosed in WO2005089443, A BRAF inhibitor as disclosed in WO2006053201 , A BRAF inhibitor as disclosed in US20050267060, A BRAF inhibitor as disclosed in WO2008120004, A BRAF inhibitor as disclosed in US20090181371 , A BRAF inhibitor as disclosed in WO2008120004, Vemurafenib, PLX4720, PLX4032 (RG7204), GDC- 0879, PLX-4720, Sorafenib Tosylate, Nexavar, dasatinib, dabrafenib , erlotinib, gefitinib, imatinib, lapatinib, sorafenib or sunitinib, or active derivatives thereof, A BRAF inhibitor as disclosed in WO2006/024834, A BRAF inhibitor as disclosed in WO2006/067446, A BRAF inhibitor as disclosed in PCT/GB2006/004756, CHIR-265, XL281 (Exelixis) or PLX4032 -a MEK inhibitor selected from the group consisting of PD184352, PD98059, U0126 , SL327, PDI 84352, a MEK inhibitor disclosed in WO2009018238, a MEK inhibitor disclosed in WO2007/044084, a MEK inhibitor disclosed in WO2005/051300, a MEK inhibitor disclosed in WO201 1/095807, a MEK inhibitor disclosed in WO2008124085, a MEK inhibitor disclosed in WO2009018233, a MEK inhibitor disclosed in

WO2007113505, a MEK inhibitor as disclosed in WO03/077914, a MEK inhibitor disclosed in US2011 105521 , a MEK inhibitor disclosed in WO2011067356, a MEK inhibitor disclosed in WO201 1067348, a MEK inhibitor disclosed in US2010004247, a MEK inhibitor disclosed in US2010130519, 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-carboxamid, 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, 6-(4-Bromo-2-chloro-phenylamino)-7-fluoro- 3-methyl-3H-benzoimidazole-5- carboxylic acid (2-hydroxy-ethoxy)-amide hydrogen sulphate salt, PD-325901 , XL518, PD-184352, PD- 318088, AZD6244, CI-1040, 4-(4- Bromo-2- fluorophenylamino)-N-(2-hydroxyethoxy)- 1 ,5-dimethyl-6-oxo- 1 ,6- dihydropyridazine-3- carboxamide or a pharmaceutically acceptable salt thereof, AR- 1 19, AS- 701173, AS-701255, 360770-54-3, and/or

AU2002248381 , an ERK-inhibitor as disclosed in US20050159385, an ERK-inhibitor as disclosed in US2004102506, an ERK-inhibitor as disclosed in US2005090536, an ERK-inhibitor as disclosed in US2004048861 , an ERK-inhibitor as disclosed in US20100004234, an ERK-inhibitor as disclosed in HR201 10892, an ERK-inhibitor as disclosed in WO201 1163330, an ERK-inhibitor as disclosed in TW200934775, an

ERK-inhibitor as disclosed in EP2332922, an ERK-inhibitor as disclosed in

WO2011041152 , an ERK-inhibitor as disclosed in US201 1038876, an ERK-inhibitor as disclosed in WO2009146034, an ERK-inhibitor as disclosed in HK11 17159, an ERK-inhibitor as disclosed in WO2009026487, an ERK-inhibitor as disclosed in WO2008115890, PH-797804 (Bioorg Med Chem Lett 201 1 Jul 1 ;21 (13):4066-71) an

ERK-inhibitor as disclosed in US2009186379, an ERK-inhibitor as disclosed in WO2008055236, an ERK-inhibitor as disclosed in US2007232610, an ERK-inhibitor as disclosed in WO2007025090, an ERK-inhibitor as disclosed in US2007049591 , 2- (2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059), 1 ,4-diamino-2,3- dicyano-1 ,4-bis(2-aminophenylthio)butadiene (U0126), and Z-& E-a-(amino-((4- aminophenyl)thio)methylene)-2-(trifluoromethyl)benzeneacetonitrile (MEK1/2) (US201 11 10916), and combinations thereof.

22. A pharmaceutical composition comprising an effective amount of a PDK inhibitor, preferably a PDK-1 inhibitor, and an effective amount of an inhibitor of a protein of the MAPK/ERK pathway, as defined in any one of the previous claims.

23. A therapeutic pharmaceutical combination comprising a PDK inhibitor and an inhibitor of a protein of the MAPK/ERK pathway, as defined in any one of the previous claims.