Cancer is one of the leading causes of death in developed countries. Cancer is a heterologous class of diseases characterized by uncontrolled cell division and the ability of these cells to invade other tissues, either by direct growth into adjacent tissue or by migration of cells to distant sites. The proliferative properties of the cells initially give rise to a tumor or neoplasm. A tumor is considered a cancer when its cells acquire the ability to invade other tissues e.g. by forming secondary tumors at other sites in the body.

The unregulated growth is caused by mutations in DNA of, for example, genes that control cell division or cell growth. The presence of one or more of these mutations, which can be inherited or acquired, can lead to uncontrolled cell division and cancer.

At least 100 different genes have been identified which, when mutant, may result in cancer, and many of these genes play a role in the regulation of cell divisions, a highly complicated process involving multiple and parallel pathways. Multiple mutational mechanisms may cause cancer and the mechanisms may differ between one tissue and another (Cancer Medicine, 5th edition, Bast et al , B. C. Decker Inc., Hamilton, Ontario).

Cancer can cause many different symptoms, depending on the site and character of the malignancy and whether there is metastasis. A definitive diagnosis usually includes biological and biochemical determinations on tissue obtained by biopsy. Once diagnosed, cancer is usually treated with surgery, chemotherapy and/or radiation. Despite recent advances in understanding the cellular mechanisms involved in cancer and, consequently, improved diagnosis and treatment of cancer, the available drug therapies for metastatic disease are unfortunately often only palliative in nature and seldom offer a long-term cure.

Cancer cells often have an addiction to the signals generated by the cancer-causing genes (oncogenes). Cancer drugs that selectively inhibit the products of activated oncogenes can therefore have a dramatic effect on cancer cell viability. Treatment with such targeted drugs provides significant clinical results for patients with, for example, Non-Small Cell Lung Cancer (NSCLC) characterized by activating mutations in EGFR or by translocations of the ALK kinase. Unfortunately, this approach has not been successful in all type of cancers. Anti-cancer therapies using targeted drugs are frequently ineffective due to resistance of the tumor cells to therapy. Resistance may be acquired during therapy. Acquired resistance to the therapy often manifests either a diminished amount of tumor regression for the same dose of a drug or the need for an increased dose for an equal amount of tumor regression. Alternatively, resistance may be intrinsic, i.e. not acquired of induced by the anti-cancer therapy. When, the resistance is intrinsic the tumor cells already originally lack sensitivity to one or more anti-cancer drugs.

There are many well-documented instances where cancers in subjects have acquired resistance to a targeted drug inhibitor which initially had successfully been used in these subjects to treat the cancer. A cancer in which acquired resistance is often observed is melanoma.

Melanoma has an incidence and mortality in Europe of about 100.000 and 22.000 persons in Europe alone. Treatment of melanoma 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 by surgery. More than 50% of (cutaneous) melanomas carry a mutation in the protein kinase referred to as BRAF (RAF is an acronym for Rapidly Accelerated Fibrosarcoma). In approximately 90% the mutation in the gene results in the substitution of glutamic acid for valine at codon 600 (BRAF V600E). Other known mutations include BRAF V600K and BRAF V600R. These mutations in BRAF causes proliferation and survival of melanoma cells through activation of the MAPK pathway (Davies et al Nature 2002; 417:949-54; Curtin et al N Engl J Med 2005;353:2135-47). The MAPK 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)).

The discovery of the common BRAFV600E mutation in melanoma has resulted in the development of targeted therapies, which are initially associated with significant clinical benefits; the small molecule inhibitor vemurafenib, specifically targeting the mutant BRAF kinase has become standard of care for subjects diagnosed with mutant BRAF metastatic melanoma. However, although this compound initially reduces tumor burden dramatically, eventually melanomas become resistant and subjects progress in the disease (Wagle et al. J Clin Oncol. 29(22) :3085-96 (2011); Fedorenko et al. BJC 1 12, 217-226 (2015)). Indeed, it is generally accepted that (acquired) resistance to drugs acting as inhibitors of the MAPK pathway, for example in melanoma, almost invariably limits the duration of clinical benefit of treatments with single compounds and/or combinations (Seton-Rogers et al. Nature Reviews Cancer 14, 7 (2014); Van Allen et al. Cancer Discov. http://dx.doi.org/10.1158/2159- 8290.CD-13-0617 (2013)). The current standard-of care for the treatment of melanoma is combination treatment with MEK and BRAF inhibitors (reviewed by Richman in Expert Opin. Pharmacother. (2015) 16(9): 1285-97. doi: 10.1517/14656566.2015.1044971).

In light of this, methods for treatment of cancers that are of may acquire resistance to drug treatment, in particular to MAPK pathway inhibitors would be highly desirable, but are not yet readily available. There is an urgent need to develop better strategies to treat such cancers. Such a treatment could have a dramatic impact on the health of individuals suffering from cancer. Accordingly, the technical problem underlying the present invention can be seen in the provision of a method complying with any of the aforementioned needs. The technical problem is solved by the embodiments characterized in the claims and herein below.

Description

Drawings

Figure 1. Limited sensitivity of resistant BRAF-mutant melanoma cells to drug withdrawal. (A) Colony formation assay (8 days) of PLX4032-resistant BRAF-mutant melanoma cell lines on continued drug exposure (2 μΜ PLX4032) versus drug withdrawal. (B) Colony formation assay (8 days) of double-resistant (DR) lines derived from single-resistant lines (R). Cells were cultured under continued drug exposure (2μΜ PLX4032 and 200 nM PD0325901) or drug withdrawal. Figure 2. PMA stimulates MAPK signaling in BRAF-mutant melanoma. (A) Western blot of markers for MAPK pathway activation in two BRAF-mutant melanoma cell lines. Cells were treated for the indicated times with 10 nM PMA. Ras-GTP was blotted from precipitates of an RBD-agarose pulldown. (B) Colony formation assay (8 days) of BRAF-mutant melanoma cells exposed to 10 nM PMA, 2 μΜ PLX4032, or the combination.

Figure 3. Resistant BRAF-mutant melanoma cells are sensitive to PMA after drug withdrawal. (A) Colony formation assays of parental (P) and resistant (R) BRAF-mutant melanoma treated for 8 days with no drug, 2 μΜ PLX4032 or 10 nM PMA. BRAF-mutant melanoma cells were cultured in the presence of PLX4032 before the start of the 8 day colony formation (B) Western blot analysis of phospho-ERK in response to 1 hour treatment with 10 nM PMA or 2 μΜ PLX4032 in parental cells and resistant cells after PLX4032 withdrawal. GAPDH is used as a loading control.

Figure 4. Effect of Prostratin on BRAF inhibitor resistant melanoma cells after drug withdrawal. Colony formation assay of A375 (parental) and A375R (PLX4032- resistant) treated with 2 μΜ Prostratin or 10 nM PMA in the presence or absence of 2 μΜ PLX4032. 20,000 cells were seeded per well in a 6-wells plate and grown with the indicated drugs for 7 days. Resistant A375R were grown in the presence of 2 μΜ PLX4032 before the start of the experiment.

Figure 5. PMA sensitivity of resistant BRAF-mutant melanoma after prolonged withdrawal. (A) Colony formation assay (8 days) of A375R and mel888R cells comparing sensitivity to no drug, PMA (10 nM) and PLX4032 (2 μΜ) between resistant cells cultured on 2 μΜ PLX4032 or after 6 or 12 day drug withdrawal. (B) Colony formation assay of A375 cells overexpressing EGFR with no drug, 2 μΜ PLX4032 or 10 nM PMA in the presence or absence of 20 ng/mL EGF. Figure 6. Double-resistant BRAF-mutant melanoma cells are hypersensitive to PMA. (A) Colony formation assay of A375DR and mel888DR treated with different combination of 2 μΜ PLX4032 and 100 or 200 nM PD0325901 in the presence or absence of 10 nM PMA.

Figure 7. Colony formation assay of mel888 (parental), mel888R (PLX4032-resistant) untreated or treated with 2 μΜ Prostratin. 200 nM PD0325901 or the combination. Resistant mel888R cells and mel888DR were grown in the presence of 2 μΜ PLX4032 or 2 μΜ PLX4032 and 200 nM PD0325901 , respectively, before the start of the experiment. 20,000 cells were seeded in a 6-wells plate and grown with the indicated drugs for 7 days. Colonies were visualized with crystal violet staining.

Figure 8. Colony formation assay of BRAF inhibitor resistant mel888R cells after drug withdrawal and treatment with either 10nM PMA or 2 μΜ Prostratin fro the indicated times. After the indicated times, medium containing PMA and Prostratin were removed and replaced with medium alone. After 10 days the colonies were visualized with crystal violet staining.

Figure 9. Colony formation assay (8 days) of A375R cells treated with 0.5 μΜ GF109203X (PKCi) or 100 nM PD0325901 (MEKi) in the presence or absence of 10 nM PMA. Definitions

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

"A," "an," and "the": these singular form terms include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a cell" includes a combination of two or more cells, and the like. "About" and "approximately": these terms, when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1 %, and still more preferably ±0.1 % from the specified value, as such variations are appropriate to perform the disclosed methods. "Acquired resistance": this term indicates that a cancer has acquired reduced sensitivity or has become resistant to the effects of a drug after being exposed to it, or a drug targeting the same mechanism or pathway, for a certain period of time. Acquired resistance to the therapy with a drug often manifests either a diminished amount of tumor regression for the same dose of a drug or the need for an increased dose for an equal amount of tumor regression. The term also indicates that a cancer may also become resistant to a first drug after being exposed to a second drug targeting the same mechanism of pathway in the cancer cell. For example, resistance may be acquired to a first ERK-inhibitor due to exposure to a second ERK-inhibitor (and to which the cancer will also have developed resistance). Alternatively, resistance may be intrinsic, i.e. not acquired of induced by the anti-cancer therapy. When, the resistance is intrinsic the tumor cells already originally lack sensitivity to one or more anticancer drugs. Since the resistance can be intrinsic or acquired the observed reduction in sensitivity is either compared to fully sensitive "normal" cancer cells, which are responsive to the therapeutically effective dosage of the applied anticancer drug and/or compared to the original sensitivity upon therapy onset.

"Comprising": this term is construed as being inclusive and open ended, and not exclusive. Specifically, the term and variations thereof mean the specified features, steps or components are included. These terms are not to be interpreted to exclude the presence of other features, steps or components.

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

"Drug": this relates to a chemical entity or biological product, or combination of chemical entities or biological products, administered to a person (for example, intravenous, intramuscular, subcutaneous, intradermal, intraperitoneal, intrathecal, intrapleural, intrauterine, rectal, oral, vaginal, topical, intratumor) to treat or prevent or control a disease or condition. The chemical entity or biological product is preferably, but not necessarily a low molecular weight compound, but may also be a larger compound, for example, an oligomer of nucleic acids, amino acids, or carbohydrates including without limitation proteins, oligonucleotides, ribozymes, DNAzymes, glycoproteins, siRNAs, lipoproteins, aptamers, and modifications and combinations thereof. Drug may have many modes of actions. The drugs referred to herein are generally inhibitors (of the activity) of enzyme(s) or activators (of the activity) of enzymes.

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

"Subject": this is to indicate the organism to be treated. The subject may be any subject in accordance with the present invention, including, e.g., mammals, such as dogs, cats, horses, rats, mice, monkeys, and humans. Preferably the subject is a human patient. Detailed Description

It is contemplated that any method, use or composition described herein can be implemented with respect to any other method, use or composition described herein. Embodiments discussed in the context of methods, use and/or compositions of the invention may be employed with respect to any other method, use or composition described herein. Thus, an embodiment pertaining to one method, use or composition may be applied to other methods, uses and compositions of the invention as well. The inventors of the present invention have surprisingly found that substances that activate protein kinase C (PKC) are effective in the treatment of a mitogen-activated protein kinase (MAPK) pathway inhibitor resistant cancer. In other words, the inventors surprisingly found that PKC activators are effective in the treatment of cancer in subjects who are not or no longer responding to MAPK-pathway inhibitor therapies.

Thus, according to a first aspect, the present invention provides for the use of a protein kinase C activator in the treatment of a mitogen-activated protein kinase pathway inhibitor resistant cancer is a subject.

Protein kinase C (PKC) is a family of non-receptor serine-threonine protein kinases. PKC is involved in controlling the function of other proteins through the phosphorylation of hydroxyl groups of serine and threonine amino acid residues on these proteins. PKC enzymes in turn are activated by signals such as increases in the concentration of diacylglycerol (DAG) or calcium ions (Ca2+). PKC enzymes play important roles in several signal transduction cascades.

Since the discovery of PKC (Kikkawa et al. (1982) J Biol. Chem. 257: 13341), and its identification as a major receptor of phorbol esters (Ashendel et al. (1983) Cancer Res., 43: 4333), a multitude of physiological signaling mechanisms have been ascribed to this enzyme, including roles in receptor desensitization, in modulating membrane structure events, in regulating transcription, in mediating immune responses, and in regulating cell growth.

The PKC family consists of fifteen isozymes in humans and is divided into three subfamilies, based on their second messenger requirements: conventional (or classical), novel, and atypical. ΡΚΰμ is sometime referred to as a fourth subfamily; it resembles the "novel" PKC isoforms but differs by having a putative transmembrane domain.

As mentioned, PKC are thought to have a role in signal transduction in response to physiological stimuli (Nishizuka (1989) Cancer 10: 1892), as well as in neoplastic transformation and differentiation (Glazer (1994) Protein Kinase C. J.F. Kuo, ed., Oxford U. Press (1994): 171-198). Recently, Dowling et al. (Cancers 2015, 7: 1271-1291) reviewed the role of PKC as a target in anti-cancer treatment. They conclude that research into the role of PKC in cancer was primarily based on the assumption that increased PKC activation and expression may promote carcinogen induced tumorigenesis, but that increasing evidence suggests that PKC can act as both tumor suppressors and oncogenes. The authors conclude that a refocus and revisit of the pathways upstream of PKCs presents a more favorable approach to targeting this group of kinases. The prior art is however silent on the role of PKC in mitogen-activated protein kinase pathway inhibitor resistant cancers.

This invention relates to contacting a PKC activator with cancer cells of a subject in a manner sufficient to increase the amount and/or activity of a PKC in the cancer cell. Preferably the contacting with the PKC activator is sufficient to stimulate the activity of PKC in the cancer cells of the subject.

Activators of PKC are known to the skilled person. One example is PMA, as described in the examples. The PKC activator that may be used in the invention may, for example, be a macrocyclic lactone or a benzolactam. The PKC activator may also be a pyrrolidinone. The macrocyclic lactone may, for example, be bryostatin, e.g. bryostatin-1 , -2, -3, -4, -5, -6, -7, -8, -9, -10, -1 1 , -12, -13, -14, -15, -16, -17, or - 18. The macrocyclic lactone may also be neristatin, for example neristatin- 1. Another example is ingenol mebutate. A preferred example is Prostratin (12-Deoxyphorbol-13-acetate;

(1 aR, 1 bS,4aR,7aS,7bR,8R,9aS)-4a,7b-dihydroxy-3-(hydroxymethyl)-1 ,1 ,6,8-tetramethyl-5- oxo-1 , 1 a, 1 b,4,4a,5,7a,7b,8,9-decahydro-9aH-cyclopropa[3,4]benzo[1 ,2-e]azulen-9a-yl acetate). Prostratin is a protein kinase C activator found in the bark of the mamala tree of Samoa, Homalanthus nutans (Euphorbiaceae). Synthesis of Prostrating and related compounds have been described (Science (2008) 320(5876): 649-652). For an overview on Prostratin, reference is made to Miana (2015) Mini Rev Med Chem. 5(13):1 122-30.

As will also be appreciated by one of ordinary skill in the art, the PKC activators are amenable to combinatorial synthetic techniques and thus libraries of the compounds can be generated to optimize pharmacological parameters, including, but not limited to efficacy and safety of the compositions. For a further discussion of known PKC modulators see for example W01997043268, US5652232; US6043270; US6080784 and others. The cancer in the subject is a cancer that is resistant to mitogen-activated protein kinase pathway inhibitors (MAPK inhibitors). The cancer in the patient is a cancer that has reduced sensitivity or is insensitive to the effect of drugs that are inhibitors of the mitogen-activated protein kinase pathway. The resistance maybe acquired resistance of may be intrinsic resistance. Preferably the resistance of the cancer is a resistance that has been acquired as the consequence of prior treatment of the subject with mitogen-activated protein kinase pathway inhibitors.

The progression of cancer may be monitored by methods well known to the skilled person. For example, the progression may be monitored by way of visual inspection of the cancer, such as, by means of X-ray, CT scan or MRI. Alternatively, the progression may be monitored by way of tumor biomarker detection. In one embodiment, the subject is monitored at various time points throughout the treatment of the cancer. For example, the progression of a cancer may be monitored by analyzing the progression of cancer at a second time point and comparing this analysis to an analysis at a first time point. An increased growth of the cancer indicates progression of the cancer and resistance to the treatment.

Acquired resistance to mitogen-activated protein kinase pathway inhibitors is a common phenomenon observed in subjects treated with such inhibitors. Resistance of a cancer to treatment with mitogen-activated protein kinase pathway inhibitors is readily recognized by the skilled person, for example by observing a diminished amount of tumor regression for the same dose of a drug or the need for an increased dose for an equal amount of tumor regression. Resistance may be acquired in response to treatment with single mitogen- activated protein kinase pathway inhibitors and/or in response to treatments involving more than one mitogen-activated protein kinase pathway inhibitor or treatments with combinations including at least one mitogen-activated protein kinase pathway inhibitor (e.g. combined RAF/EG FR or RAF/MEK inhibition; see for example Ahronian (2015) Cancer Discov. 5(4): 358-367). In daily practice, resistance to, for example vemurafenib (an inhibitor targeting mutated B-raf; see below), develops on average within 7 months of initial use, as is witnessed by a decrease in initial favorable response to the drug (i.e. less tumor regression, or return of tumor growth after initial stabilization, or as determined by measuring response or expression/activity of marker genes or proteins (e.g. of the MAPK pathway) of cell obtained from the subject by biopsies; see for example Trunzer et al. J Clin Oncol. 2013 May 10;31 (14):1767-74. doi: 10.1200/JCO.2012.44.7888). Thus, despite the promising signs of efficacy demonstrated by clinical trials of mitogen- activated protein kinase pathway inhibitors, either a single drug, or as part of combination, many of those patients deriving initial benefit from therapy ultimately develop resistance to treatment and show disease progression.

Attempts to overcome or prevent resistance of a cancer to treatment with mitogen-activated protein kinase pathway inhibitors include treatment with combinations of mitogen-activated protein kinase pathway inhibitors and PI3K/mTOR inhibitors. A number of combinations of MEK and PI3K/mTOR pathway inhibitors combinations have entered early phase clinical trials, however their benefit in the setting of BRAF/MEK inhibitor resistance remains untested (see. e.g. Gowrishankar in "Melanoma - From Early Detection to Treatment" (2013), edited by Guy Huynh Thien Due, ISBN 978-953-51-0961-7, DOI: 10.5772/53629).

The current inventors have found that cancers that show resistance to mitogen-activated protein kinase pathway inhibitors are discontinued for the treatment with the mitogen- activated protein kinase pathway inhibitor and are at the same time treated with an activator of PKC respond very well to such treatment, as shown in the examples. It was surprisingly found that such cells have become very sensitive to activation of PKC by the PKC activator. The PKC activator is effective in the treatment of cancers that do not, or to a limited extent, respond to inhibitors of enzymes that form part of the mitogen-activated protein kinase pathway. The mitogen-activated protein kinase pathway (MAPK pathway) is one of the most studied pathways in cancer biology, well known to the skilled person and sometimes also referred to as the MAPK/ERK pathway, the RAS-RAF-MEK-ERK pathway, or the RAS-RAF- MEK-ERK-RSK pathway.

The MAPK pathway, as used herein, is a chain or pathway of proteins in the cell that communicates a signal from a receptor on the surface of the cell to the DNA in the nucleus of the cell. In the MAPK pathway, activated RAS activates the protein kinase activity of RAF kinase, RAF kinase phosphorylates and activates MEK (MEK1 and MEK2), and MEK phosphorylates and activates a mitogen-activated protein kinase (MAPK; ERK). MAPK phosphorylates ribosomal protein S6 kinase (RSK).

Within the context of the current invention, a MAPK pathway inhibitor is a compound that inhibits signaling through the MAPK pathway, preferably by inhibiting the activity of one of the proteins forming the chain or pathway. The MAPK pathway inhibitor may do so by, for example, reducing the biological activity of a protein of the pathway, or my reducing expression of an mRNA encoding a protein of the pathway, or my reducing the expression of a protein of the pathway. For example, with a RAF inhibitor, for example a BRAF inhibitor, is meant a compound that may reduce the biological activity of RAF, for example BRAF; or that may reduce the expression of an mRNA encoding a RAF polypeptide, for example BRAF; or that may reduce the expression of a RAF polypeptide, for example BRAF. As will be understood by the skilled person, the above is likewise applicable with respect to inhibitors of the other proteins of the MAPK pathway.

Preferably the MAPK pathway inhibitor is an inhibitor of a protein of the MAPK pathway, preferably an inhibitor of RAS protein, an inhibitor of RAF protein, an inhibitor of MEK protein, an inhibitor of ERK protein and/or an inhibitor of RSK protein.

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

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

A RAF protein is a polypeptide belonging to the RAF kinase family. RAF kinases are a family of three serine/threonine-specific protein kinases that are related to retroviral oncogenes. The three RAF kinase family members are ARAF (A-RAF; for example Genbank Accession NO: NP001243125), BRAF (B-RAF; (for example, Genbank Accession NO: NP004324)) and CRAF (C-RAF; (e.g. Gene accession number 5894; Refseq RNA Accessions NM_002880.3 ; protein NP_002871.1), and are well-known to the skilled person.

RAF kinase inhibitors are known to the skilled person. Non-limitative examples include the compounds GW5074, BAY 43-9006, CHIR-265 (Novartis), Vemurafenib, PLX4720 (Tsai et al. 2008 PNAS 105(8):3041), PLX4032 (RG7204), GDC-0879 (Klaus P. Hoeflich et al. Cancer Res.2009 April 1 ;69:3042-3051), sorafenib tosylate (e.g. from Bayer and Onyx Pharmaceuticals as Nexavar), dasatinib (also known as BMS-354825, e.g. as produced by Bristol-Myers Squibb and sold under the trade name Sprycel), AZ628 from Genentech, LGX818 from Novartis, dabrafenib (e.g. Tafinlar™ capsule, made by GlaxoSmithKline, LLC), GSK2118436, BGB659 (Yao et al. 2015 Cancer Cell 28(3): 370-383) and LY3009120 (Peng et al. 2015 Cancer Cell 28(3): 384-398). Preferred examples of RAF inhibitors include Vemurafenib (Roche/Plexxikon), Dabrafenib (GSK), LGX818 (Novartis), TAK-632 (Takeda), MLN2480 (Takeda/Millennium), PLX4032- 4720 (Plexxikon). A MEK polypeptide (e.g. Gene accession numbers 5604 or 5605; Refseq RNA Accessions NM_002755.3 or NM_030662.3; protein NP_002746.1 or NP_109587.1) is a polypeptide having serine/threonine protein kinase activity. For example MEK1 (e.g. Genbank Accession NO: NP002746) and MEK2 (e.g. Genbank Accession NO: NP109587) phosphorylates and activates MAPK (ERK). Another example is MEK3 ((e.g. Genbank Accession NO: NP002747). MEK comprises both MEK1 and MEK2: MAP/ERK kinase 1 , MEK1 , PRKMK1 , MAPKK1 , MAP2K1 , MKK1 are the same enzyme, known as MEK1 , MAP/ERK kinase 2, MEK2, PRKMK2, MAPKK2, MAP2K2, MKK2 are the same enzyme, known as MEK2. MEK1 and MEK2, together MEK, can phosphorylate serine, threonine and tyrosine residues in protein or peptide substrates.

Examples of MEK inhibitors, include but are not limited to the MEK inhibitors PD184352 and PD98059, inhibitors of MEKI and MEK2 U0126 (see Favata, M., et al., J. Biol. Chem. 273, 18623, 1998) and SL327 (Carr et al Psychopharmacology (Berl). 2009 Jan;201 (4):495-506), and those MEK inhibitors discussed in Davies et al (2000) (Davies et al Biochem J. 351 , 95- 105). Another example is PDI 84352 (Allen, Lee et al Seminars in Oncology, Oct. 2003, pp. 105-106, vol. 30) or trametinib, which has been approved for treatment of BRAF mutant melanoma under the name Mekinist. MEK162 (Novartis) is another example. Other known MEK inhibitors may be selected from PD-325901 (Pfizer), XL518 (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; WO2007/076245.) and CI-1040 (Lorusso et al Journal of clinical oncology 2005, vol. 23, no23, pp. 5281-5293), selumetinib (AZD6244), TAK-733, or Honokiol. Preferred examples of MEK inhibitors include Trametinib (GSK), Cobimetinib (GDC-0973) (Genentech/Exelixis), MEK162 (Novartis/Array BioPharma), AZD6244 (AstraZeneca/Array BioPharma), R05126766 (Roche/Chugai), GDC-0623 (Genentech/Chugai), PD0325901 (Pfizer), and Selumetinib. An ERK protein is a polypeptide having serine/threonine protein kinase activity, e.g. ERK phosphorylates and activates MAP (microtubule-associated proteins), and having at least 85% amino acid identity to the amino acid sequence of a human ERK, e.g. to ERK1 (e.g. Gene accession number 5595; Refseq RNA Accessions NM_001040056.2; protein NP_001035145.1) or ERK2 (e.g. Gene accession number 5594; Refseq RNA Accessions NM_002745.4 ; protein NP_002736.3). ERK inhibitors are known to the skilled person, and includes such inhibitors as disclosed in WO2002058687, for example SL-327 (Carr et al Psychopharmacology (Berl). 2009 Jan;201 (4):495-5060). Further ERK inhibitors may be found in WO2002058687, AU2002248381 , US20050159385, US2004102506, US2005090536, US2004048861 , US20100004234, HR20110892, WO201 1163330, TW200934775, EP2332922, WO2011041152, US2011038876, WO2009146034, HK11 17159, WO2009026487, WO2008115890, US2009186379, WO2008055236, US2007232610, WO2007025090, and US2007049591. Further non-limiting examples or ERK-inhibitors include BVD-523, FR 180204 (CAS No. 865362-74-9), Hypothemycin (CAS no. 76958-67-3), MK-8353, SCH9003531 , Pluripotin (CAS no. 839707-37-8), SCH772984 (CAS no. 942183-80-4), and VX-11 e (Cas no. 896720-20-0).

Preferred examples of ERK inhibitors include SCH772984 (Merck/Schering- Plough), VTX11e (Vertex) and GDC-0994 (Roche/Genentech). An RSK protein (e.g. EC 2.7.11.1 ; e.g. Gene accession numbers 6195, 6197, 6196, or 27330; Refseq RNA Accessions NM_001006665, NM_002953, NM_004586, NM_001006932, NM_021 135, or NM_014496 ; protein NP_001006666.1 , NP_004577.1 , NP_001006933.1 , or NP_05531 1.1) is a polypeptide of the ribosomal s6 kinase (rsk) a family of protein kinases. There are two subfamilies of rsk, p90RSK, also known as MAPK-activated protein kinase-1 (MAPKAP-K1), and p70RSK, also known as S6-H1 Kinase or simply S6 Kinase. The RSK protein is a MAP kinase activated protein kinase (MAPKAP kinase) and described in, e.g., Leukemia, 17: 1263-1293 (2003). RSK is phosphorylated and activated by Erk1 and ERK-2 in response to many growth factors, polypeptide hormones and neurotransmitters. RSK inhibitors are known to the skilled person and include, for example, Kaempherol-3-0-(4'- O-acetyl-a-L-rhamnopyranoside), or such inhibitors as disclosed in EP1845778.

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

The inventors have found that when a cancer displays resistance to inhibitors of the MAPK pathway, for example inhibitors of the different proteins of the MAPK pathway, such as those described above, such cancer is in particular sensitive to treatment with a PKC activator. In addition, it was found that PKC activator treatment of the cancer that displays resistance to the MAPK pathway inhibitor should take place in the absence of any MAPK pathway inhibitor, for example after treatment with the MAPK pathway inhibitor is discontinued.

As will be understood by the skilled person, the amount of the PKC activator, preferably prostratin, that may be administered to the subject is an effective amount. As will be understood by the skilled person, more than one PKC activator may be used. The PKC activator may also be combined with other drugs useful in the treatment of the cancer and/or in alleviating symptoms related to the cancer or the treatment thereof. The skilled person understand how to determine the effective amount, for example, by performing dose-finding studies while monitoring tumor pro- or regression.

The present invention relates to using a PKC activator to treat a cancer which has acquired resistance to a MAPK pathway inhibitor, irrespective of the molecular mechanism responsible for it. The present invention also provides methods of treating a cancer in a subject, comprising administering an effective amount of a PKC activator to said subject having a cancer, wherein said cancer is refractory to a MAPK pathway inhibitor. The term "refractory" means that the cancer (including a tumor and/or any metastasis thereof), upon treatment with at least one MAPK pathway inhibitor, shows no or only weak anti-cancer response (e.g., anti-proliferative response; such as, no or only weak inhibition of tumor growth) after the treatment. Thus, after a subject has been treated with the MAPK pathway inhibitor with success, subsequent treatments show no or little affect, and the cancer can be described as being refractory to the MAPK pathway inhibitor. It is preferred that the cancer that is resistant to a mitogen-activated protein kinase pathway inhibitor, is a BRAF-mutation harboring cancer. In such embodiment, the cancer of the subject is a BRAF-mutated cancer, i.e. a cancer harboring mutation in BRAF. The term "BRAF-mutated cancer" or "BRAF-mutation harboring cancer" is known to the skilled person. As detailed above, BRAF (e.g. Gene accession number 673; Refseq RNA Accessions NM_004333.4 ; protein NP_004324.2), is a member of the RAF family, which includes ARAF and CRAF in humans (Ikawa, Mol Cell Biol. 8(6):2651-4 (1988)). BRAF is a serine/threonine protein kinase and participates in the RAS/RAF/MEK/ERK/RSK mitogen activated protein kinase pathway (MAPK pathway, see Williams & Roberts, Cancer Metastasis Rev. 13(1):105- 16 (1994); Fecher et al 2008 Curr Opin Oncol 20, 183-189 or Cargnello M, Roux PP. Microbiol Mol Biol Rev. 2011 Mar;75(1):50-83). BRAF mutations are found in different types of cancer. For example, approximately 40-60% of (cutaneous) melanomas carry a mutation in the BRAF protein. Approximately 90% of these mutations result in the substitution of glutamic acid for valine at codon 600 (BRAF V600E, although other mutations are also known (e.g. BRAF V600K and BRAF V600R). Such mutation in BRAF typically leads to proliferation and survival of the cancer cells (Davies et al Nature 2002; 417:949-54; Curtin et al N Engl J Med 2005;353:2135-47).

Current treatment of BRAF-mutation harboring cancers includes the use of trametinib and/or vemurafenib. Also the combination of the BRAF inhibitor vemurafenib and the MEK inhibitor cobimetinib demonstrated a statistically significant improvement in overall survival (Larkin (2015) J. Clin Oncol. 33 (suppl; abstr 9006). Another combination used is dabrafenib (a BRAF inhibitor), a BRAF inhibitor, plus trametinib (Mekinist®), a MEK inhibitor.

In accordance with the current invention, BRAF-mutation harboring cancers that are resistant to treatment with MAPK pathway inhibitors, for example, such inhibitors and combinations as described in the previous paragraph are, after discontinuation of the treatment with such MAPK pathway inhibitors, treated with an effective amount of a PKC activator. It was surprisingly found that the BRAF-mutation harboring cancer that is (or acquired) resistance to drugs targeting the MAPK pathway, and combination comprising such drugs, have become or are highly sensitive to PKC activators. By treatment of the cancers with the PKC activator, in accordance with the invention disclosed herein, progression of a cancer as a consequence of the resistance to the MAPK pathway targeting drugs, may be halted and/or regression of the cancer may be obtained.

It is preferred that the protein kinase C activator that is used in the treatment of the mitogen- activated protein kinase pathway resistant cancer is administered to the subject to be treated after any treatment with a mitogen-activated protein kinase pathway inhibitor is discontinued. Without necessarily limiting the invention thereto, it was observed that in particular cancer- treating effects were obtained when the PKC activator was provided after treatment with drugs targeting protein of the MAPK pathway was stopped or discontinued. This embodiment is in particular relevant for cancer that has acquired resistance to a drug targeting the MAPK pathway, e.g. an inhibitor of RAF, MER and/or ERK protein. The skilled person will understand that in this embodiment the treatment with the PKC activator is in the absence of any effective amount of a drug that act as an inhibitor on the MAPK pathway, for example drugs that inhibit enzymes of the MAPK pathway, irrespective of the fact whether the subject was prior treated with said MAPK pathway targeting drug or with any other MAPK pathway targeting drug, or not.

The skilled person understands what is meant with a discontinued treatment and thus understands when, in accordance with the current invention, the PKC activator may be administered to the subject. For example, the PKC activator may be administered to the subject certain time after the last dose of the MAPK pathway targeting drug was administered. The length of the time between the last dose of the MAPK pathway targeting drug and the first dose of the PKC activator will depend on the pharmacodynamics characteristics of the drug compounds used in the treatment regime and can readily be established by the skilled person practicing the invention.

It is also a preference, that the protein kinase C activator for use in the treatment of a mitogen-activated protein kinase pathway inhibitor resistant cancer in a subject is used in a treatment that comprises

a) treatment of the subject with one or more mitogen-activated protein kinase pathway inhibitor(s);

b) discontinuation of the treatment of step a); and

c) treatment of the subject with a protein kinase C activator after the treatment of step a) is discontinued in step b).

The above use of the PKC activator is in particular for use in situation wherein the cancer has acquired resistance to at least one MAPK pathway inhibitor. In the practice of the invention, a subject, in particular a subject having a cancer characterized by the presence of a mutated BRAF, is initially treated in line with the accepted treatment in the art, for example with a combination of a BRAF inhibitor and a MEK inhibitor, or with only a BRAF inhibitor or MEK inhibitor. During treatment normally regression/progression of the cancer is monitored. Once it is observed that the subject does not respond anymore, or once response to the treatment is reduced, treatment with the MAPK pathway targeting drugs is discontinued. After the treatment is discontinued, the patient is provided with a PKC activator as according to the invention. In a preferred embodiment, the treatment with the MAPK pathway inhibitors (step a)), prior to the treatment with the PKC activator, comprises a treatment with a BRAF inhibitor, a MEK inhibitor or a combination of at least a BRAF inhibitor and a MEK inhibitor.

As shown in the Examples, cancers that acquired resistance to in particular the above- indicated drugs are very sensitive to treatment with a PKC activator after treatment with MAPK pathway inhibitor(s) is discontinued.

As described above, the invention is not in particular limited to any PKC activator. Indeed, the inventors have shown that the effect of the PKC activators can be completely rescued by addition of a PKC inhibitor, proving that the observed effect is via PKC activation per se. However it is preferred the PKC activator is phorbol-12-myristate-13-acetated (PMA), Bryostatin or Prostratin. In particular the latter two are preferred.

The skilled person will understand the PKC activator may be provided in the form of a pharmaceutical composition comprising the PKC activator and one or more pharmaceutical excipients. As explained above, the amount of the PKC activator will depend on the particular PKC activator employed in the invention and may be established using routine experimentation available in the art, including for example ascending dose studies and the like.

Although the use of the protein kinase C activator in the treatment of a mitogen-activated protein kinase pathway inhibitor resistant cancer in a subject is not in particular limited to any specific type of cancer, it is preferred the cancer is melanoma, colon cancer, papillary thyroid carcinoma, ovarian carcinoma, astrocytoma, ganglioglioma, craniopharyngioma, Langerhans cell histiocytosis, hairy cell leukemia, or ameloblastoma.

The skilled person readily recognized these different types of cancer, and for which the current invention is in particular suitable. Even more preferred the above-mentioned cancers are BRAF-mutation harboring cancers, as discussed in detail herein.

Most preferably the cancer is melanoma, preferably BRAF-mutated melanoma. According to a preference, there is provided for the protein kinase C activator for use in the treatment of a mitogen-activated protein kinase pathway inhibitor resistant cancer in a subject as disclosed herein wherein the mitogen-activated protein kinase pathway inhibitor is a RAS inhibitor, RAF inhibitor, MEK inhibitor, ERK inhibitor and/or RSK inhibitor and/or wherein the treatment further comprises the use of an Receptor Tyrosine Kinases inhibitor, preferably a EGFR inhibitor, HER2 inhibitor, HER3 inhibitor, Met inhibitor, Axl inhibitor or PDGF inhibitor. According to this preference, the cancer is resistant to, for example has acquired resistance, to a RAS inhibitor, a RAF inhibitor, a MEK inhibitor, an ERK inhibitor and/or a RSK inhibitor. The inhibitor to which the cancer is resistant, for example to which the cancer acquired resistance may be any of the known inhibitors of RAS, RAF, MEK, ERK and/or RSK inhibitors mentioned herein.

In addition, the mitogen-activated protein kinase pathway inhibitor treatment may further comprise the use of a Receptor Tyrosine Kinases inhibitor, preferably an EGFR inhibitor, a HER family inhibitor, Met inhibitor, Axl inhibitor or PDGF inhibitor. In this embodiment, the cancer is prior treated with a combination of at least one mitogen-activated protein kinase pathway inhibitor and a further inhibitor of a Receptor Tyrosine Kinases inhibitor, preferably an EGFR inhibitor (Gefitinib, Erlotinib), HER family inhibitor (Trastuzumab, Lapatinib, Pertuzumab, Afatinib, Dacometinib), Met inhibitor (XL184 (Cabozantinib), Tivantinib) , Axl inhibitor (BGB324, TP-0903) or PDGF inhibitor (Imatinib, Sunitinib, Sorafinib). The skilled person is aware of such inhibitors and is aware on how these inhibitors may be used in the treatment of cancer, in particular in combination with any mitogen-activated protein kinase pathway inhibitor (Prahhalad et al. 2012. Nature 483 (7387): 100-103, Sun et al. 2014 Cell Rep. 10; (1): 86-93)

As discussed herein, the protein kinase C activator is, when the cancer is first treated with a mitogen-activated protein kinase pathway inhibitor, to preferably to be administered to a subject after the treatment with the mitogen-activated protein kinase pathway inhibitor is discontinued. As will be understood by the skilled person, the time between the last treatment with the mitogen-activated protein kinase pathway inhibitor and the first treatment with the PKC activator will depend on, for example, the kind of mitogen-activated protein kinase pathway inhibitor used in the prior treatment and/or the PKC activator to be administered to the subject. However, it is in general preferred that the protein kinase C activator for use in the treatment of a mitogen-activated protein kinase pathway inhibitor resistant cancer in a subject as disclosed herein is administered within a period of 1 - 28 days after treatment of the subject with a mitogen-activated protein kinase pathway inhibitor is discontinued. In other words, there is at least a period of 1 day (24 hours) between the final administration of the mitogen- activated protein kinase pathway inhibitor and the first administration of the PKC activator. Preferably the period is within 1 - 14 days after treatment, even more preferably within 1 - 7 days after treatment with the mitogen-activated protein kinase pathway inhibitor is discontinued. The PKC activator may be administered to the subject 1 , 2, 3, 4, 5, 6... , 1 1 , 12, 13...20, 21 , 22, 23...28 days after the last treatment with the mitogen-activated protein kinase pathway inhibitor.

With respect to the PKC activator, in some embodiments, PKC activator administration is repeated more than once; in some embodiments the PKC activator administration is repeated at regular intervals. In other embodiments the interval is between 6 hours and two weeks, between 12 hours and one week, or between one day and three days. In one embodiment, the administration of the PKC activator is continued for a fixed period of time. In one embodiment, the administration of the PKC activator is repeated for a period greater than one day. In another embodiment, the administration of the PKC activator is repeated for a period of between one day and one month, or is repeated for a period greater than one month. The treatment with the PKC activator may be a combined treatment with other drugs useful in the treatment of the cancer in the subject.

In the treatments disclosed herein progression of cancer in a subject may be monitored at a time point after the subject has initiated the treatment, for example the treatment with the mitogen-activated protein kinase pathway inhibitor. As discussed herein progression of the cancer, while being treated with mitogen-activated protein kinase pathway inhibitor, is indicative of cancer that is resistant to such mitogen-activated protein kinase pathway inhibitor and, according to the invention, the treatment with the mitogen-activated protein kinase pathway inhibitor should be discontinued and be replaced by treatment with the PKC activator.

Administering effective amounts of the PKC activator according to the invention can treat one or more aspects of the cancer disease, including, but not limited to, causing tumor regression; causing cell death; causing apoptosis; causing necrosis; inhibiting cell proliferation; inhibiting tumor growth; inhibiting tumor metastasis; inhibiting tumor migration; inhibiting tumor invasion; reducing disease progression; stabilizing the disease; reducing or inhibiting angiogenesis; prolonging subject survival; enhancing subject's quality of life; reducing adverse symptoms associated with cancer; and reducing the frequency, severity, intensity, and/or duration of any of the aforementioned aspects.

As exemplified in the Examples the current inventors have surprisingly found that cancer, in particular mitogen-activated protein kinase pathway inhibitor resistant cancer, in particular BRAF-mutation harboring mitogen-activated protein kinase pathway inhibitor resistant cancers can be effectively treated with PKC activators. In particular, the PKC activator is provided to a subject (contacted with the cancer) under conditions that the cancer is not at the same time treated with a MAPK pathway inhibitor. As also exemplified in the Examples, there is unexpected decrease in the number of cancer cells after the treatment with the PKC activator. However, at the same time it was found that small populations of cells remain even after the treatment with the PKC activator. Surprisingly it was found that these cells, after the treatment with the PKC activator are sensitive to further treatment with a MAPK pathway inhibitor.

Therefore, in a preferred embodiment of the invention there is provide for the protein kinase C activator for use in the treatment of a mitogen-activated protein kinase pathway inhibitor resistant cancer in a subject as disclosed herein wherein further mitogen-activated protein kinase pathway inhibitors are administered after treatment of the subject with the protein kinase C activator is discontinued.

In other words, in this embodiment, a subject having a mitogen-activated protein kinase pathway inhibitor resistant cancer is first treated with a PKC activator for a time sufficiently to treat the cancer, and that may cause the cancer to regress. After a certain period of time, the treatment with the PKC activator is discontinued, after which the remaining cancer is further treated with a MAPK pathway inhibitor, for example as disclosed herein.

Again, the timing and period of treatment of each individual step can be established by the skilled person using routine experimentation.

Thus, in such embodiments, a subject having a cancer, preferably a BRAF-mutation harboring cancer, is first treated with a treatment comprising the use of MAPK pathway inhibitors (e.g. RAS, MEK and/or ERR inhibitors). According to the invention, once it observed the cancer has acquired resistance to the MAPK pathway inhibitor (the subject now has, within the context of the current invention, a mitogen-activated protein kinase pathway inhibitor resistant cancer) treatment with the MAPK pathway inhibitor is discontinued. After the treatment comprising the MAPK pathway inhibitor is discontinued, the subject is provided with treatment comprising a PKC activator. Also during this stage cancer progression/regression may be monitored. After a certain period of time, for example, once a reduction in the therapeutic effect of the treatment comprising the PKC activator is observed, and in this embodiment of the invention, treatment with the PKC activator is discontinued.

Once the treatment with the PKC activator is discontinued, a further treatment may be provided to the patient, comprising the further use of MAPK pathway inhibitors. The skilled person understands such treatment may be repeated several times if so desired in view of the treatment of the subject (MAPK pathway inhibitor - PKC activator - MAPK pathway inhibitor - PKC activator ).

According to another aspect of the invention there is provided a protein kinase C activator for use in the treatment of cancer in a subject, wherein the treatment comprises administering the protein kinase C activator to cancer after the cancer has acquired resistance to treatment with a mitogen-activated protein kinase pathway inhibitor, preferably wherein the cancer is a BRAF-mutation harboring cancer. In this embodiment, the subject is first treated with a MAPK pathway inhibitor until the cancer acquires resistance to the MAPK pathway inhibitor, for example, as determined as disclosed herein. Next, treatment with the MAPK pathway inhibitor is discontinued. After the treatment with the MAPK pathway inhibitor is discontinued, the subject, now having a mitogen-activated protein kinase (MAPK) pathway inhibitor resistant cancer is treated with a PKC activator, as disclosed herein.

In a preferred embodiment, there is provided the protein kinase C activator for use in the treatment of cancer in a subject as disclosed herein, wherein the treatment comprises a) treatment of the subject with one or more mitogen-activated protein kinase pathway inhibitor(s), preferably a BRAF inhibitor, a MEK inhibitor, or a combination of at least a BRAF inhibitor and a MEK inhibitor;

b) discontinuation of the treatment of step a); and

c) treatment of the subject with a protein kinase C activator after the treatment of step a) is discontinued in step b), essentially as already disclosed herein.

Also provided is use of a protein kinase C activator in the manufacture of a medicament a) for the treatment of a mitogen-activated protein kinase pathway inhibitor resistant cancer in a subject, preferably a BRAF-mutation harboring cancer; and/or

b) for the treatment of a cancer in a subject, preferably a BRAF-mutation harboring cancer, wherein the treatment comprises administering the protein kinase C activator to cancer after the cancer has acquired resistance to treatment with a mitogen-activated protein kinase pathway inhibitor.

The skilled person is aware how to manufacture a medicament for the above treatment, for example using common techniques available in the art.

Also provided is a mitogen-activated protein kinase pathway inhibitor for use in the treatment of a cancer in a subject, preferably a BRAF-mutation harboring cancer, wherein the treatment comprises

a) treatment of the subject with the mitogen-activated protein kinase pathway inhibitor;

b) discontinuation of the treatment of step a); and

c) treatment of the subject with a protein kinase C activator after the treatment of step a) is discontinued in step b).

Likewise, there is provided for the use of a mitogen-activated protein kinase pathway inhibitor in the manufacture of a medicament for the treatment of a cancer in a subject, preferably a BRAF-mutation harboring cancer, wherein the treatment comprises administering a protein kinase C activator to cancer after the cancer has acquired resistance to treatment with the mitogen-activated protein kinase pathway inhibitor. According to another aspect there is provided for a method of treatment of a mitogen- activated protein kinase pathway inhibitor resistant cancer in a subject, preferably a BRAF- mutation harboring cancer, the method comprising treatment of the subject with a protein kinase C activator. According to even another aspect there is provided for method of treatment of cancer in a subject, preferably a BRAF-mutation harboring cancer, the method comprising:

a) treatment of the subject with one or more mitogen-activated protein kinase pathway inhibitor(s), preferably a BRAF inhibitor, a MEK inhibitor, or a combination of at least a BRAF inhibitor and a MEK inhibitor;

b) discontinuation of the treatment of step a); and

c) treatment of the subject with a protein kinase C activator after the treatment of step a) is discontinued in step b). Preferably, there is provided for method of treatment of cancer in a subject of any of the previous claims wherein step c) starts within a period of 1 - 28 days after step b).

According to another aspect there is provided Prostratin for use in the treatment of cancer in a subject, preferably for use in the treatment of a mitogen-activated protein kinase pathway inhibitor resistant cancer in a subject.

According to another aspect there is provided for Prostratin for use in the treatment of cancer in a subject as disclosed herein wherein the cancer is a BRAF-mutation harboring cancer. Preferably the cancer is a melanoma.

The skilled person will understand that in all aspect and all embodiment disclosed above, the treatment may further comprise the discontinuation of the treatment with the PKC activator, followed by further treatment with a MAPK pathway inhibitor once the treatment with the PKC activator is discontinued, as detailed herein, including any further repeat steps ((MAPK pathway inhibitor - PKC activator - MAPK pathway inhibitor - PKC activator ). Preferably the step of providing the further MAPK pathway inhibitors to the subject, after the treatment with the PKC activator is discontinued starts within a period of 1 - 28 days after the treatment with the PKC activator is discontinued.

As will be clear to the skilled person, the treatment with the PKC activator as disclosed herein does not comprise the simultaneous treatment with a MAPK pathway inhibitor.

Having now generally described the invention, the same will be more readily understood through reference to the following examples which is provided by way of illustration and is not intended to be limiting of the present invention.

Examples

Example 1

Various cancers, including, for example, BRAFV600E-mutant melanomas are strongly driven by MAPK signaling. Inhibition of proteins involved in the MAPK pathway, for example inhibition of BRAF and/or MEK, generally leads to clinical regression of the disease. But even with combinations of inhibitors, e.g. a combination of BRAF and MEK inhibitor, resistance almost invariably occurs. Here we show that upon drug withdrawal (i.e. withdrawal of the inhibitors(s) of the MAPK pathway), exogenous activation of PKC with, for example, PMA, leads to a detrimental growth defects in MAPK-pathway inhibitors resistance cancers, in particular BRAF-inhibitor resistant melanoma cells and colorectal cancer cells.

A common mechanism to BRAF inhibitors in the clinic is the acquisition of an activating mutation in the RAS oncogene. We show that the introduction of a Ras oncogene in BRAF- mutant melanoma cells results in sensitivity to PKC activation with for example PMA. BRAF-mutant melanoma cells that have become resistant to the combination of BRAF and MEK inhibitors also show increased vulnerability to exogenous activation of PKC, for example by PMA, characterized by an induction of senescence and cell cycle defects.

In conclusion, we demonstrate that MAPK-pathway inhibitor resistant cancers, in particular BRAF-mutant melanoma as well as colorectal cancer, gains a specific vulnerability to PKC activation causing MAPK (hyper-)activation.

Our work implies that drugs stimulating PKC and therewith hyper-activate the MAPK pathway are specifically effective against MAPK pathway inhibitor resistant BRAF-mutant cancers such as melanoma and offer a potential therapeutic solution for MAPK inhibitor resistance in such (melanoma) patients.

Materials and Methods Cell culture and reagents

A375, mel888, D10, WM266-4 and AO cell lines were obtained from in-house stocks and authenticated by STR profiling (BaseClear, Leiden). Resistant cell lines were previously described (Muller et al. 2014. Nat Commun.15;5:5712) and authenticated by STR profiling. Patient-derived naive and resistant melanoma lines were provided by the Peeper lab (Dutch Cancer Institute, The Netherlands).

All cell lines were grown in DMEM (Gibco) supplemented with 10% FCS (Gibco) and penicillin/streptomycin (Gibco). Resistant lines were grown in 2 μΜ PLX4032 (vemurafenib; Selleck) or 2 μΜ PLX4032 and 200 nM PD0325901 (Selleck). PMA (12-0- tetradecanoylphorbol-13-acetate) and prostratin were purchased at Sigma-Aldrich. EGF (recombinant human epithelial growth factor) was purchased at BD Biosciences. PKC inhibitor GF109203X was purchased from Selleck. All compounds were reconstituted in DMSO.

Constructs, virus production and infection

pCDH_ER-RasV12_puro was created by cloning ER-RasV12 from Addgene #21 19 into pCDH_puro (System Biosciences #CD510B-1). PLX4032_EGFR_blast was picked from the Broad ORF library. pLKO_shNF1_puro was picked from the Broad TRC collection (TRCN_39717). Constructs were cotransfected with lentiviral packaging vectors (3: 1 : 1 : 1) into 293T producer cells using 4 μg of PEI (Sigma-Aldrich) per μg of plasmid DNA. 12 μg plasmid DNA was used for one 10 cm dish. Medium was refreshed after overnight incubation. After 24 hours virus production, supernatant was collected and filtered. Infections were achieved by applying virus supernatant in 1 :3 - 1 :50 dilution (optimal dilution was determined per construct and cell line) in the presence of 8 μg/mL hexadimethrine bromide (Sigma-Aldrich). Cells were put on puromycin (2 μg/mL) or blasticidin (10 μg/mL) selection 24 hours after infection for 3-5 days.

CellTiter-Blue viability assay and colony formation assays

For 96-hour cell viability assay, 500-1000 cells were plated per well in a 384-wells plate. Drugs were added 4 hours after seeding and viability was measured 96 hours after adding drugs using CellTiter-Blue (Promega). For colony formation assays 20,000 cells were seeded per well of a 6-wells plate and drugs were added the next day. In case of resistant cells grown on PLX4032 or PD0325901 , medium was replaced before drug addition. Plates were fixed with paraformaldehyde after 7 days unless indicated otherwise and stained with crystal violet. Live cell monitoring with apoptosis reporter

5,000 cells were seeded per well of a 96-wells plate. Drugs and CellPlayer Caspase 3/7 live apoptosis reporter assay (Essen Bioscience) were added after 4 hours at the indicated concentrations or 1 : 1000 respectively. Cells were monitored in the IncuCyte Zoom (Essen Bioscience). Images at 48 hours after drug addition are shown.

Western blot and ELISA

All protein samples were collected in RIPA buffer, except samples for Ras-GTP, which were collected and precipitated with RAF1-RBD-agarose beads (Millipore) according to the manufacturer's instructions.

Western blot was performed using standard techniques. Antibodies used: phospho-ERK, phospho-MEK, total ERK, total MEK, phospho-c-Raf, cleaved PARP (Cell Signaling: 4094, 9154, 4695, 9126, 9427, 5625), GAPDH-HRP (Abeam, ab9482), pan-Ras (BD Transduction Laboratories 610001), phospho-EGFR (Life Technologies 44788G), total EGFR, HSP90 (Santa Cruz: sc-03, sc-7947). ELISA on phospho-ERK was executed according to manufacturer's instructions (Cell Signaling, 7246), except the readout, which was achieved using ECL substrate and measurement of luminescence on a Perkin-Elmer Envision. Luminescence signals were normalized to protein input as determined by BCA assay and standardized to the appropriate control sample.

Results

Sensitivity to drug withdrawal is dictated by the degree of resistance

To assess the efficacy of a drug holiday treatment, we determined the effect of drug withdrawal in a panel of BRAF-inhibitor resistant BRAF-mutant melanoma cell lines (acquired resistance). We found these resistant cells to be largely refractory to drug withdrawal (Fig. 1A).

From the BRAF inhibitor resistant lines, A375R and mel88R, we generated BRAF and MEK inhibitor double-resistant (DR) lines through exposure to 2 μΜ PLX4032 and 0.2 μΜ PD0325901 for ~2 months. In contrast to the single-resistant lines, A375DR and mel888DR were sensitive to drug withdrawal (Fig. 1 B). This effect was accompanied by an increase in the levels of phospho-c- Raf and phospho-ERK compared to untreated and drug treated cells. In contrast, drug withdrawal in single resistant cell lines did not lead to a remarkable increase in phospho-ERK compared to the parental cells or drug continuation. The growth inhibitory effect of drug withdrawal in A375DR and mel888DR cells was limited, with cells surviving and continue to proliferate in the absence of drug.

PMA, a PKC activator activates the MAPK pathway in BRAF-mutant melanoma

The detrimental effect of drug withdrawal of the combination of BRAF and MEK inhibitors in the double resistant cell lines is accompanied by a strong increase in the levels of phospho- ERK.

We hypothesized that (hyper)-stimulation of the MAPK pathway upon drug withdrawal and may selectively affect MAPK-pathway inhibitor resistant cancers, including MAPK-pathway inhibitor resistant BRAF-mutant melanoma cells. The MAPK kinase pathway can be activated by exogenous stimulation of PKC. As a potent activator of PKC we first decided to use PMA (phorbol 12-myristate 13-acetate, also known as TPA). PMA is a diacylglycerol mimic that activates several protein kinase C (PKC) isoforms. Next, PKC activates Ras and Raf, leading to hyper-activation of MAPK pathway signaling. We assessed activation of Ras, c-Raf and ERK in A375 and mel888 cells upon stimulation of PKC.

Treatment with 10 nM PMA led to rapid activation of Ras and Raf, with peak activity around 1 hour after drug addition (Fig. 2A). Phospho-ERK was elevated after 1 h in mel888 but not in A375.

Given the efficient activation of c-Raf and to a lesser extend Ras, we expected PMA to cause resistance to MAPK pathway inhibition, for example BRAF inhibition. Indeed, both A375 and mel888 treated with 2 μΜ PLX4032 were rescued by PMA, while PMA treatment itself had very mild effects on proliferation of treatment naive cells (Fig. 2B). Despite the lack of activation of pERK in A375 with PMA, we did observe a partial rescue of the decrease in phospho-ERK levels upon treatment with BRAF inhibitor and PMA. The attenuation of the drop in MAPK signaling was sufficient to allow continued cell proliferation and prevent apoptosis.

Resistant BRAF-mutant melanomas are sensitive to MAPK hyper-activation by PKC activators

We next determined whether resistant BRAF-mutant melanoma lines are more sensitive to MAPK pathway hyper-activation than their corresponding sensitive parental lines.

In long-term colony formation assays, PMA had little effect on growth of parental lines, but intermediate to strong effect on growth of resistant lines after withdrawal of the BRAF inhibitor PLX4032 (Fig. 3A). Although PMA activates MAPK signaling through activation of PKC, we only observed highly elevated phospho-ERK levels in resistant mel888 in contrast to a very modest increase in resistant A375 (Fig. 3B). To ascertain that the detrimental effects of PMA were indeed caused by MAPK activation, we titrated in a MEK inhibitor with the PMA treatment. In all resistant cell lines, the detrimental effect of PMA could be rescued by MEK inhibition in a dose-dependent fashion. As an additional testimony to the MAPK activating capabilities of PMA, its addition leads to strongly decreased sensitivity to MEK inhibitor in both parental and resistant lines. The effect of PMA could be completely rescued by addition of a PKC inhibitor (GF109203X), proving that PKC activation was causal to the function of PMA (see, e.g. Figure 9). In other words, the effect is caused by PKC activation. PMA is well known for its tumor-promoting effects although it is unclear whether activation of PKCs or inactivation after prolonged exposure to PMA is associated with its tumorigenic capacities. Interestingly, small molecule activators of PKC exist that do not share the tumorigenic properties of PMA. An example of such a molecule is Prostratin. Importantly, we obtained similar inhibition of proliferation of BRAF inhibitor resistant BRAF mutant A375 melanoma cells using Prostratin (Fig. 4). The inhibitory effect of MAPK pathway hyper-activation on proliferation occurs after the development of resistance to MAPK pathway inhibition, here BRAF inhibition in BRAF mutant melanoma and after withdrawal of the MAPK pathway inhibitor(s).

We next tested whether the interval between drug withdrawal (i.e. discontinuing the treatment with the MAPK pathway inhibitor) and activation of PKC, for example by PMA treatment, is relevant. Since phospho-ERK elevation and c-Raf activation is observed upon withdrawal of BRAF and MEK inhibitors, we predicted highest efficacy for PMA when administered directly after drug withdrawal. However, we observed that an interval of 6 or even 12 days between drug withdrawal and PMA treatment did not decrease sensitivity of A375R and mel888R to PMA (Fig 5A). Clinically this is relevant, because it allows for more flexible timing of drug administration after cessation/discontinuation of the MAPK pathway inhibitor, e.g. BRAF or BRAF/ MEK inhibitor regime.

Oncogenic Ras activation is synthetic lethal with PMA in BRAF-mutant melanoma

Recent studies have demonstrated that a large amount of resistant BRAF-mutant melanomas have acquired mutations that cause activation of Ras. Examination of Ras in our panel of resistant and parental pairs indicated that two out of four resistant lines displayed elevated GTP-bound Ras, the active form of Ras. To test whether the presence of oncogenic Ras is sufficient to sensitize cells to PMA, we created A375 cells with an inducible oncogenic RasV12. Addition of tamoxifen (100 nM 40HT) to the culture medium stabilizes the ER- RasV12 fusion protein and restores phospho-ERK levels to that of untreated cells upon PLX4032 treatment. Without induction, the cells are sensitive to PLX4032 and insensitive to PMA, but upon induction of RasV12 the cells became resistant to PLX4032 and gained sensitivity to PMA. The levels of phospho-ERK are not increased by the expression of mutant RAS in the untreated setting, suggesting that the BRAF V600E mutation already activates the MAPK pathway to its maximum output. In agreement with this observation, the expression of oncogenic Ras has little or no effect on proliferation of these cells.

We generated four additional BRAF-mutant melanoma cell lines with ER-RasV12, Expression of oncogenic Ras intensified and prolonged MAPK signaling as determined by phospho-c-Raf and phospho-ERK levels (data not shown). Similar to the A375 cells, all four cell lines showed decreased sensitivity to PLX4032 but increased sensitivity to PMA upon induction of oncogenic Ras (data not shown).

Several resistance mechanisms, apart from RAS mutation, have been described that can activate Ras in melanoma including NF1 loss and RTK activation. We wanted to examine whether those mechanisms could also lead to the observed PMA sensitivity in BRAF mutant melanoma cells, similar to Ras activation itself. In A375, ectopic expression of the EGF receptor (Fig. 5B) gave rise to a moderate sensitivity to PMA. However, if these EGFR expressing cells were supplemented with EGF, they became extremely sensitive to PMA, suggesting that the coordinate MAPK activation by EGFR signaling and PMA leads to a synergistic detrimental effect.

We mimicked another resistance mechanism, NF1 loss, by short hairpin knockdown. A375 cells expressing a hairpin targeting NF1 but not a control vector became resistant to BRAF inhibitor and sensitive to PMA. Collectively, these data indicate that resistant cancers, e.g. resistant BRAF-mutant melanomas, with activated Ras signaling acquire sensitivity to PKC activators like PMA or Prostratin.

Double-resistant BRAF-mutant melanoma are hypersensitive to PMA

Currently, several proposed treatments of various cancers involve the use of combination of inhibitors, for example combination of MAPK pathway inhibitors or combinations with other drugs and including MAPK pathway inhibitors. For example, BRAF mutant melanoma is treated with a combination of BRAF and MEK inhibitors. We therefore asked whether double- resistant melanomas also respond to MAPK pathway hyper-activation through PKC activation. Given their increased sensitivity to drug withdrawal compared to single-resistant lines, we hypothesized that the double-resistant melanoma cells will also become more sensitive to PMA. Indeed, both the A375DR and mel888DR cells were very sensitive to PMA (Fig. 6). Although mel888DR already had a strong response to withdrawal of both MEK and BRAF inhibitor, the treated with PMA caused a strong reduction in colony outgrowth. As expected, the effect of PMA can be rescued by the addition of MAPK inhibitors.

Even though PMA is much more effective at preventing colony outgrowth than a drug holiday, we did not see a difference in the levels of phospho-ERK when comparing drug withdrawal and PMA treatment in both double-resistant lines. However, we did observe an increase in phosho-c-Raf indicative of additional MAPK pathway activation upon PMA treatment. This demonstrates the limitation of phospho-ERK as an indication of treatment efficacy of MAPK activation.

One major hurdle in the application of molecularly targeted inhibition is the appearance of drug tolerant persisters after prolonged exposure to drugs (Sharma et al. 2010 Cell 141 (1):69- 80)). We therefore asked whether prolonged PMA treatment of double-resistant cells would lead to the appearance of persisters. We were able to obtain drug tolerant persister cells from A375DR. Remarkably, the A375 PMA persisters were re-sensitized to BRAF and MEK combination inhibition. This suggests the possibility of sequential treatment with mitogen- activated protein kinase pathway inhibitors, followed by treatment with protein kinase C activators, and, after treatment with the protein kinase C activator is discontinued, further treatment with mitogen-activated protein kinase pathway inhibitors.

Finally, Figure 7 shows the effect of Prostratin on BRAF inhibitor or BRAF/MEK inhibitor resistant mel888 cells compared to the parental cells. Mell888R (resistant to BRAF inhibitor) and mel888DR cells (resistant to the combination of BRAF and MEK inhibitors) are sensitive to Prostratin and PMA after PLX4032 withdrawal. In the third and fourth column of the bottom panel the sensitivity to MEK inhibitor, or the rescue of the MEK effect with Prostratin in the Parental and single and double resistant mel888 cells is shown. The rescue of the reduction in colony growth after drug withdrawal with the MEK inhibitor (PD) again demonstrates the role of MAPK pathway activation in the detrimental effect of drug withdrawal. The effect of drug withdrawal can be exacerbated with the activation of PKC, for example with Prostratin or PMA.

Finally it was found from an experiment where PMA (or Prostratin) was kept with the cells only for a short time, after which outgrowth was analyzed after 10 days. Here, it was observed that a PMA or Prostratin exposure of 1 to 3 days is sufficient to achieve the maximum kill of the resistant cells (figure 8). Having now fully described this invention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation.

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

All references cited herein, including journal articles or abstracts, published or corresponding patent applications, patents, or any other references, are entirely incorporated by reference herein, including all data, tables, figures, and text presented in the cited references. Additionally, the entire contents of the references cited within the references cited herein are also entirely incorporated by references. Reference to known method steps, conventional methods steps, known methods or conventional methods is not in any way an admission that any aspect, description or embodiment of the present invention is disclosed, taught or suggested in the relevant art. The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art (including the contents of the references cited herein) , readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance presented herein, in combination with the knowledge of one of ordinary skill in the art.