CA2761253A1 - Combinations of therapeutic agents for treating melanoma - Google Patents

Abstract

The present invention shows that MDM4, a negative regulator of p53, is up-regulated in -60% of human cutaneous melanomas. Accordingly, treatment of metastatic melanoma cells with a specific inhibitor of the MDM4-p53 interaction decreases cell viability irrespective of their sensitivity to B-RAF-inhibitors and greatly synergizes with BRAF
inhibitors in cell killing. In addition, MDM4 targeting also greatly sensitizes metastatic melanoma cells to conventional chemotherapeutics.

Description

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Combinations of therapeutic agents for treating melanoma Field of the invention The present invention relates to the field of oncology, more particularly to the field of melanoma. The invention relates to a method of treating melanoma, particularly advanced cutaneous melanoma, with a combination of pharmaceutical agents comprising an inhibitor and one or more chemotherapeutic agents such as for examplealkylating agents (i.e. Dacarbazine (DIG) or melphalan), alkylating-like agents (i.e. cisplatin or carboplatin) or mitotic inhibitors (taxanesdocetaxel or paclitaxel) and B-RAE and MEK
inhibitors. The invention further relates to pharmaceutical formulations of an MDM4 inhibitor and a pharmaceutical formulation of one or more chemotherapeutic agents such as for example alkylating agents (i.e. Dacarbazine (DITC) or melphalan), alkylating-like agents (i.e. cisplatin or carboplatin) or mitotic inhibitors (taxanesdocetaxel or paclitaxel) and B-RAE and MEK
inhibitors.
Introduction to the invention Cutaneous malignant melanoma is the leading cause of skin cancer-related deaths. Its incidence has increased worldwide faster than any other cancer, with 5-year survival rates for patients with distant metastatic disease being less than 20%
(http://seercancergovicsr/1975_2007). Improvement of clinical outcomes for this aggressive, chemo- and radio-resistant, disease remains a major clinical challenge.
Significant progress in our understanding of the etiologies and genetic underpinnings of melanoma has nevertheless been made1'2. These advances have recently led to promising results in trials of targeted therapies for this diseases. The Ras/Raf/MEK/ERK pathway has been identified as the main regulator of cell proliferation in melanoma, with ERK being hyperactivated in up to 90% of human melanomas4. Activating NRAS mutations are a common route to activating this pathway; mutations affecting codon 61 being the most prevalent (NRASQ61K)5s. BRAF, one of the three human RAF genes, is also frequently mutated in melanomas', with the most common mutation being a glutamic acid for valine substitution at position 600 (V600E)7.
BRAFvsmE stimulates constitutive ERK signaling, leading to melanocyte hyper-proliferations.
Early clinical experience with the novel class I RAF-selective inhibitor, PLX4032, demonstrated an unprecedented 80% anti-tumor response rate among patients with BRA FV600E positive melanomas; unfortunately, patients acquire drug resistance within a few months of an initial responses. Because of its ability to acquire drug resistance, its chemoresistance and because melanoma is a highly dynamic and genetically heterogeneous tumor, novel treatment strategies and combination therapies are urgently needed. Restoration of the wild-type p53 tumor suppressor function has emerged as an attractive anti-cancer strategy for many tumor type510-12. Whether this approach can be therapeutically beneficial in malignant melanoma remains unknown. p53 pathway inactivation, which mainly arises as a consequence of inactivating mutations or allelic loss of the p53 gene itself, is the most common molecular defect in human cancers13.
Intriguingly, the p53 locus is intact in over 95% of melanoma cases14, raising questions as to the pathogenic relevance of p53 in the etiology of melanoma tumor formation. At the same time, there is an increasing body of evidence supporting a relevant role for p53 in melanoma development. Loss of p53 cooperates with melanocyte-specific overexpression of activated HRASv120 and BRAFv600E in promoting melanomagenesis in mice15'16and oncogenic NRAS
cooperates with p53 loss to generate melanomas in zebrafish17. Cancers that retain expression of wild-type p53 often find alternative ways to subvert p53 function, through either deregulation of upstream modulators and/or inactivation of downstream effectors18.
MDM2, which encodes an E3 ubiquitin ligase that control p53 levels and function19,is amplified in human melanomas but only in 3%-5% of documented cases20. The (CDKN2A) locus is often deleted or inactivated in heritable and sporadic melanoma'. This locus encodes two distinct tumor suppressors, p16INK44' (referred hereafter as INK4A) and p 14ARF (referred hereafter as ARF). INK4A positively regulates the pRB tumor suppressor and ARF is a potent MDM2 antagonist. Thus, decreased ARF expression or its complete loss could, in part, compromise p53 function in melanoma21. However, p53-independent functions of ARF have been described22and whether ARF restricts melanoma progression in a p53-dependent manner is still a matter of debate23. Overall, although several oncogenic events that compromise p53 function have been described in melanoma the number and the frequency of these events accounts for only a small proportion of melanoma cases, implying that additional, unidentified, mechanisms exist.Unveiling such mechanisms may lead to the development of novel targeted therapeutic strategies allowing re-activation of p53 tumor killing activities.
Summary of the invention In the present invention we provide evidence that the p53 pathway is inactivated in the majority of cutaneous melanomas as a result of deregulated expression of MDM4 (also known as HDMX or MDMX), a key negative regulator of p5324,25. In the present invention we further demonstrate that targeting the MDM4-p53 interaction inhibits the growth of melanoma cells in vitro and in vivo and significantly sensitizes them to conventional chemotherapeutics.

2 Also surprisingly, we show that MDM4 inhibitors affect the growth of melanoma cells that have acquired resistance to BRAF inhibitors and also synergize with BRAF
inhibitors in killing BRAF-mutant cells. Together our results identify the MDM4-p53 interaction as a key therapeutic target for melanoma treatment and a promising new candidate for combined therapy for this aggressive tumor type. Accordingly, it has been surprisingly found thatMDM4-inhibitors are synergistic (act synergistically) when used in combination with chemotherapeutic agents. Importantly, compelling evidence ¨including mouse genetic experiments- indicates that MDM4 targeting will not affect the viability of normal (non-cancer) cells. Therapeutic effects of combinations of chemotherapeutic agents with an inhibitoris therefore expected to result in lower toxicity of the chemotherapeutic agents as their efficacy is greatly enhanced at lower doses when used in combination with MDM4 inhibitors.
In a first aspect the invention provides a method for preventing the progression or treatment of patients with malignant melanoma, which comprises administering pharmaceutically effective amounts of a combination of i) an MDM4-inhibitor and ii) one or more chemotherapeutic agents selected from a microtubule active agent, an alkylating agent, an anti-neoplastic anti-metabolite, a platin compound, a Rafand MEK kinase inhibitor, a topoisomerase I inhibitor, a topoisomerase II inhibitor, a VEGF inhibitor, a tyrosine kinase inhibitor, an EGFR kinase inhibitor, an mTOR kinase inhibitor, an insulin-like growth factor I
inhibitor, a HDAC inhibitor, a proteasome inhibitor, and ionizing radiation for simultaneous, concurrent, separate or sequential use in for preventing or treating melanoma.
In another aspect the invention provides for a method for preventing or treating of melanoma according to claim 1 wherein said one or more chemotherapeutic agents are selected from camptothecin derivatives, paclitaxel, docetaxel, epothilone B, 5-FU, gemcitabine, oxaliplatin, cisplatinum, carboplatin, melphalan, dacarbazine, temozolomide, doxorubicin, imatinib, erlotinib, bevacizumab, cetuximab and Rafor MEK kinase inhibitor. In a particular aspect melanoma is cutaneous melanoma. In another particular aspect melanoma is metastatic melanoma. In another particular aspect said melanoma has a B-RAF activating mutation. In yet another particular aspect said melanoma comprises a B-Rafactivating mutation but has acquired resistance to a B-Raf kinase inhibitor. In yet another embodiment a melanoma has a B-Rafactivating mutation and has an enhanced protein expression of MDM4. In another aspect a Raf kinase inhibitor is a B-Raf kinase inhibitor.
In another aspect the invention provides for a pharmaceutical composition comprising i) an MDM4-inhibitor and ii) one or more chemotherapeutic agents selected from a microtubule active agent, an alkylating agent, an anti-neoplastic anti-metabolite, a platin compound, a

3 Raf kinase inhibitor, a topoisomerase I inhibitor, a topoisomerase II
inhibitor, a VEGF
inhibitor, a tyrosine kinase inhibitor, an EGFR kinase inhibitor, an mTOR
kinase inhibitor, an insulin-like growth factor I inhibitor, a HDAC inhibitor, a proteasome inhibitor.
In yet another aspect the invention provides for a pharmaceutical composition comprising as one or more chemotherapeutic agents selected from camptothecin derivatives, paclitaxel, docetaxel, epothilone B, 5-FU, gemcitabine, oxaliplatin, cisplatinum, carboplatin, melphalam, dacarbazine, temozolomide, doxorubicin, imatinib, erlotinib, bevacizumab, cetuximab and a Raf kinase inhibitor.
In yet another aspect the invention provides for a pharmaceutical composition comprising as one or more chemotherapeutic agents selected from oxaliplatin, cisplatinum, carboplatin, melphalam, dacarbazine, temozolomide and a RAF kinase inhibitor.
In yet another aspect the invention provides for pharmaceutical composition comprising an MDM4 inhibitor and aB-RAF kinase inhibitor. It is submitted that in the present invention the annotation "B-RAF" is equivalent with the annotation "BRAE".
In another aspect the invention provides a method for preventing or treating of melanoma, which comprises administering pharmaceutically effective amounts of an MDM4-inhibitor for use in for preventing or treating melanoma. In specific aspects melanoma is advanced cutaneous melanoma, or metastatic melanoma, or the melanoma has a B-Rafactivating mutation, or the melanoma has a B-Rafactivating mutation and has acquired resistance to B-RAF inhibitors.
In another aspect the invention provides for a method for testing the eligibility of a patient suffering from melanoma for treatment with an MDM4 inhibitor comprising determining the protein expression levels of MDM4 and MDM2 in a tumor sample derived from said patient and wherein an enhanced MDM4 protein expression (as compared to the MDM2 protein expression) selects the patient as eligible for treatment.
In another aspect the invention provides for a method for testing the eligibility of a patient suffering from melanoma for treatment with a pharmaceutical composition described herein before comprising determining the protein expression level of MDM4 and MDM2 and the B-RAF status in a tumor sample derived from said patient and wherein an enhanced protein expression (as compared to the MDM2 protein expression) and the presence of a B-RAF activating mutation selects the patient as eligible for treatment.

4 The present invention as claimed relates to:
- use of pharmaceutically effective amounts of a combination of i) an MDM4-inhibitor and ii) one or more chemotherapeutic agents selected from a microtubule active agent, an alkylating agent, an anti-neoplastic anti-metabolite, a platin compound, a Raf or MEK kinase inhibitor, a topoisomerase I inhibitor, a topoisomerase II
inhibitor, a VEGF inhibitor, a tyrosine kinase inhibitor, an EGFR kinase inhibitor, an mTOR
kinase inhibitor, an insulin-like growth factor I inhibitor, a HDAC inhibitor, a proteasome inhibitor, and ionizing radiation for simultaneous, concurrent, separate or sequential administration in preventing or treating melanoma having an activating B-RAF mutation;
- a pharmaceutical composition comprising i) an MDM4-inhibitor and ii) one or more chemotherapeutic agents selected from a microtubule active agent, an alkylating agent, an anti-neoplastic anti-metabolite, a platin compound, a Raf kinase inhibitor, a MEK kinase inhibitor, a topoisomerase I inhibitor, a topoisomerase II
inhibitor, a VEGF inhibitor, a tyrosine kinase inhibitor, an EGFR kinase inhibitor, an mTOR

kinase inhibitor, an insulin-like growth factor I inhibitor, a HDAC inhibitor, a proteasome inhibitor for simultaneous, concurrent, separate or sequential use in preventing or treating melanoma having an activating B-RAF mutation;
- use of a pharmaceutically effective amount of an MDM4-inhibitor for preventing or treating melanoma having an activating B-RAF mutation; and - a method for testing the eligibility of a patient suffering from melanoma having an activating B-RAF mutation, for treatment with an MDM4 inhibitor comprising determining the protein expression level of MDM4 in a tumor sample of said patient and wherein an enhanced MDM4 protein expression selects the patient as eligible for treatment.
4a Figures Figure 1: MDM4 is frequently overexpressed in human melanoma.
Protein levels were assessed by Western blotting analysis in total lysates from human melanoma samples and cell lines. (A) Protein expression of MDM4, MDM2 and p53 in melanoma samples from human patients. The samples are divided into four categories each containing 10 samples (primary, non-invasive lesions, regional dermal metastases, nodal metastatic lesions and distant metastatic lesions). Expression was evaluated in congenital melanocytic nevi (ON). (B) Expression levels of MDM4, MDM2, p53 and p21 were also determined in patient-derived short-term melanoma cell lines, in normal melanocytes (n.
melan) and (C) in long-term culture human melanoma cell lines. MCF7, U20S, were used as reference controls; Vinculin (Vinc.) and Tubulin as loading controls.
Figure 2: Inhibition of the MDM4-p53 interaction restores p53 activity in melanoma (A) Viability of MM031 (left) and MM011 (right) melanoma cell lines treated with 0,5-20 mM
SAH-p53-8, Nutlin-3, or an equimolar combination for 24h. The cells were exposed to CellTiter-Glo reagent and viability was assessed by ATP induced chemiluminescence. Data are mean SD for experiments performed in at least triplicate. (B) Dose-effect synergy analyses of MM031 (left) and MM011 (right) and melanoma cells treated with 0,5-20 pM
SAH-p53-8, Nutlin-3, or an equimolar combination. The EC50 values for each of the treatments are indicated. (C) RT-qPCR mRNA expression analysis of selected p53 target genes in melanoma cell lines 24h post SAH-p53-8 treatment (ECK of SAH used per each cell line); SAH-p53-8F19A was used as a point mutant control and DMSO as a vehicle control.
The data represent the mean ( SD) from three technical triplicates. The values are normalized to the level of mRNA expression in vehicle-treated cells. (D) SAH-p53-8 overcomes MDM4-mediated p53 suppression and blocks tumor growth in vivo.
(Left) Cohorts of MM031 xenograft mice were treated with vehicle (5% DMS0 in D5W) or mg/kg of SAH-p53-8 by intravenous injection daily for 5 consecutive days and tumor volume was monitored by caliper measurement daily for a period of 12 days. Data represent the mean ( SD) of 7 different biological replicates (P<0,005 on day 12). (Right) External views of representative tumor-bearing mice on day 12.
Figure 3: Therapeutic potential of targeting the MDM4-p53 pathway.
(A) and (B) Targeting the MDM4-p53 interaction sensitizes metastatic melanoma cells to chemotherapy. MM031 (A) and MM011 (B) melanoma cells were treated with 6,25-50 p,M
Cisplatin or 25-200 j.i.M Melpha Ian with or without E050 dose of SAH-p53-8 or with or without corresponding dose of nutlin-3. (B) Dose effect synergy analyses of MM011 melanoma cells treated with 6,25-50 pM cisplatin with or without 0,5-20 pM SAH-p53-8 or nutlin-3. Cell viability was measured at 24 hr by CellTiter-Glo assay. Data represent the mean ( SD) of at least three biological replicates. (C) Targeting the MDM4-p53 pathway sensitizes melanoma cells to a BRAFv600E-inhibitor. BRAF-resistant and parental cell lines were exposed to increasing doses of SAH-p53-8 (10-40 M). Cell viability was measured by CellTiter-Glo assay at 24 hr post SAH-p53-8 treatment. Data represent the mean ( SD) of at least three biological replicates. (D) Dose-effect synergy analyses of melanoma cells treated with 5 p,M
PLX4032 and an EC50 dose of SAH-p53-8 (18 M). Data represent the mean ( SD) of at least three biological replicates.
Detailed description of the invention In most tumor types, p53 is silenced mainly by missense mutations or deletions of the p53 gene itself. In melanoma, however, such genomic events are rare, making the relevance of the p53 pathway in melanomagenesisirrelevant. In the present invention we provide evidence that increased MDM4 protein expression contributes to p53 inactivation during melanomagenesis. Indeed, we demonstrate that the MDM4 protein is overexpressed in about 60% of cutaneous melanoma cases.ln addition to being very frequent, MDM4 up-regulation appears to be an early oncogenic event as high MDM4 levels are observed as early as in primary, non-invasive, melanoma lesions. Notably, MDM4 overexpression is only detected at the protein level explaining why this event has been missed by transcriptomic analyses previously performed on melanoma samples. We show that most metastatic melanoma cells depend on high MDM4 protein expression to keep p53 pro-apoptotic activities in check and to survive. Restoration of p53 function has been extensively pursued as a novel therapeutic approach to treat cancers that, like most melanomas, retain wild-type p53. Indeed, the therapeutic benefits of such an approach have been demonstrated in several preclinical mouse cancer mode1s45-47. Numerous efforts have focused on blocking MDM2 as a strategy for reactivating p53 in tumors31.45-51. However, several caveats to this approach have recently been uncovered52. One major limitation of anti-MDM2-based therapy is that tumor cells that overexpress MDM4 and low levels of MDM2, only poorly respond to MDM2 inhibition12'40. Here, we show that most human melanoma cell lines express high MDM4 and low MDM2 levels and that representative cell lines with such MDM4/MDM2 ratio respond very poorly to nutlin-3 alone. In the present invention it was surprisingly found that human melanoma cell lines are extremely sensitive to MDM4 inhibitors. Of note, melanomas that express high levels of both MDM4 and MDM2 were found to be extremely rare. Taken together our data show that while the majority of melanoma patients would respond poorly to MDM2 inhibition they would greatly benefit from pharmacological disruption of the MDM4-p53 interaction. The prognosis of patients with metastatic melanoma (MM) remains very poor, largely reflecting the failure of conventional chemotherapy regimens to impact on advanced disease53. Strategies that increase the sensitivity of melanoma cells to chemotherapeutics are therefore expected to decrease their toxicity and eventually improve their potency. The invention further shows in the examplesthat a stapled peptide inhibiting the MDM4/p53 interaction(designated further in the examples as SAH-p53-8) greatly potentiates the cytotoxic effects of two chemotherapeutic agents currently used in the clinic.
MDM4 inhibitors can therefore be used to enhance the effectiveness of conventional therapeutic agents in the treatment of melanoma, such as cutaneous melanoma, such as metastatic melanoma.
Yet another aspect is that the identification of activating mutations in BRAF, in 50-70% of malignant melanoma biopsies, raises the hope for targeted therapy as an attractive alternative to conventional chemotherapy in melanoma treatment. Indeed, encouraging results from a clinical trial with a small molecule BRAF inhibitor were recently reported54.
However, chronic treatment with BRAF inhibitors was shown to be invariably associated with the development of drug resistance42. Strategies for overcoming intrinsic and acquired drug resistance to small molecule BRAF inhibitors are thus urgently needed. The present invention convincingly shows that MDM4 overexpression occurs equally frequently in tumors harboring mutations in BRAF and NRAS, and that MDM4 inhibition equally affects the growth of both NRAS and BRAF melanoma cells. Importantly, we also show here that MDM4 inhibition is equally effective at inhibiting growth of BRAF-mutant cells that have acquired resistance to BRAF inhibitors. It is further also shown that combined treatment with MDM4 and BRAF inhibitors synergize in the killing of melanoma cells that are sensitive to the BRAF
inhibitors. Thus the invention discloses that MDM4 targeting also offers a therapeutic avenue in cases where BRAF inhibitors are no longer effective (i.e. melanoma cells having B-RAF
activating mutations which have become resistant to B-RAF inhibitors) and even against tumors harboring NRAS mutations against which no specific inhibitors exist.
Thus in a first embodiment the present invention provides a method for preventing or treating of melanoma, which comprises administering pharmaceutically effective amounts of an MDM4-inhibitor for use in for preventing or treating melanoma.
In yet another embodiment said melanoma is advanced cutaneous melanoma. In yet another embodiment said melanoma is metastatic melanoma.

In yet another embodiment said melanoma has a mutation which activates the activity of B-RAE.
In yet another embodiment the melanoma, or subcutaneous melanoma, with an activating B-RAF mutation has acquired resistance to a B-RAF inhibitor.
In a specific embodiment the MDM4-inhibitor is a stapled peptide such as disclosed in W02008095063.
In yet another embodiment the invention provides a method for preventing or treating of melanoma, which comprises administering pharmaceutically effective amounts of a combination of i) an MDM4-inhibitor and ii) one or more chemotherapeutic agents selected from a microtubule active agent, an alkylating agent, an anti-neoplastic anti-metabolite, a platin compound, a Rafor MEK kinase inhibitor, a topoisomerase I inhibitor, a topoisomerase II inhibitor, a VEGF inhibitor, a tyrosine kinase inhibitor, an EGFR kinase inhibitor, an mTOR
kinase inhibitor, an insulin-like growth factor I inhibitor, a HDAC inhibitor, a proteasome inhibitor, and ionizing radiation for simultaneous, concurrent, separate or sequential use in for preventing or treating melanoma.
In another embodiment the invention provides a method for preventing or treating of melanoma which comprises administering pharmaceutically effective amounts of a combination of i) an MDM4-inhibitor and ii) wherein one or more chemotherapeutic agents are selected from camptothecin derivatives, melphalan, temozolomide,dacarbazine,paclitaxel, docetaxel, epothilone B, 5-FU, gemcitabine, oxaliplatin, cisplatinum, carboplatin, doxorubicin, imatinib, erlotinib, bevacizumab, cetuximab and a Raf kinase inhibitor.
In yet another embodiment a Raf kinase inhibitor is a B-RAF kinase inhibitor.
In yet another particular embodiment an MDM4-inhibitor is a stapled peptide inhibitor such as disclosed in W02008095063.
In yet another embodiment in the method for treating or preventing melanoma, the melanoma is an advanced cutaneous melanoma.
In yet another embodiment in the method for treating or preventing melanoma, the melanoma is a metastatic melanoma.
In yet another embodiment in the method for treating or preventing melanoma, the melanoma has a B-RAF activating mutation.

In yet another particular ,embodiment in the method for treating or preventing melanoma, the melanoma has a B-RAFv6wE mutation as a B-RAF activating mutation.
A "B-RAF activating mutation" is a mutation which makes the B-RAF kinase constitutively active. An example of a B-RAF activating mutation is the B-RAFv6mE mutation.
In yet another particular embodiment in the method for treating or preventing melanoma, the melanoma which has a B-RAF activating mutation has acquired a resistance to a B-RAF
inhibitor.The latter means that the melanoma does not respond anymore (i.e. as measured by the lack of induction of apoptosis or lack of growth arrest when a B-RAF
inhibitor is applied to such a B-RAF inhibitor resistant melanoma) to the B-RAF inhibitor treatment.
In yet another particular embodiment in the method for treating or preventing, melanoma, the melanoma which has a 8-RAF activating mutation has acquired a resistance to a inhibitor and has additionally an enhanced protein expression ratio level between MDM4 and MDM2.
An MDM4-inhibitor, in the context of the present invention, is a molecule which inhibits (or disrupts, or antagonizes) the interaction between p53 and MDM4. In certain instances an MDM4-inhibitor inhibits the interaction between p53 and MDM4 and additionally also inhibits the interaction between p53 and MDM2. Inhibitors inhibiting MDM2/p53 and MDM4/p53 are known in the art as dual-specificity inhibitors. Since MDM4 is considered to be a negative regulator of p53, the effect of a molecule which inhibits the MDM4/p53 interaction is an activation of p53 which results in the initiation of cell death in a cell wherein enhanced M DM4 protein expression occurs. Preferred examples of small molecules that antagonize the MDM4/p53 interaction are an imidazole, a beta-lactam, a tetrahydroquinoline, a aminomethyl phenol or a 1-(alkylsulfonyI)-4,5-dihydro-1H-imidazole. Variants of such molecules have been disclosed for example in W02008119741, W0201123677, W0201176786 and W02008130614. Next to small compounds, peptides have also been described which inhibit the interaction between MDM4 and p53 (or inhibit the interaction between MDM4/p53 and MDM2/p53). Examples of such peptides are disclosed in W02008106507, W02009149339 and W02011005219. in a preferred embodiment the MDM4/p53 inhibitor is a stapled peptide such as the peptides disclosed in W02008095063, in addition to the stapled peptide used in the examples.
The term "chemotherapeutic agents" is a broad one covering many chemotherapeutic agents having different mechanisms of action. Combinations of chemotherapeutic agents withMDM4-inhibitors result in synergistic effects and in improvements in melanoma cancer therapy. Generally, chemotherapeutic agents are classified according to the mechanism of action. Many of the available agents are anti-metabolites of development pathways of various tumors, or react with the DNA of the tumor cells.
By the term "chemotherapeutic agent" is meant chemotherapeutic agents selected from the list consisting of a topoisomerase I inhibitor or a derivative thereof, a microtubule active agent, an insulin-like growth factor I inhibitor, a protein tyrosine kinase inhibitor, a VEGF
inhibitor, an mTOR kinase inhibitor, an EGFR kinase inhibitor, an alkylating agent, an anti-neoplastic anti-metabolite, a platin compound, a topoisomerase II inhibitor, proteasome inhibitors, HDAC inhibitors, RAF kinase inhibitors and tumor cell damaging approaches, such as ionizing radiation.The term "microtubule active agent", as used herein, relates to microtubule stabilizing, microtubule destabilizing agents and microtublin polymerization inhibitors including, but not limited to taxanes, e.g. paclitaxel and docetaxel; vinca alkaloids, e.g. vinblastine, especially vinblastine sulfate; vincristine, especially vincristine sulfate and vinorelbine; discodermolides; colchicine and epothilone sand derivatives thereof, e.g.epothilone B or a derivative thereof. Paclitaxel is marketed as taxol;
docetaxel as taxotere; vinblastine sulfate as vinblastin and vincristine sulfate as farmistin. The term "alkylating agent", as used herein, includes, but is not limited to,Dacarbazine (DTIC) cyclophosphamide, ifosfamide, melphalan or nitrosourea (BCNU or Gliadel), or temozolomide (temodar). Cyclophosphamide can be administered, e.g. in the form as it is marketed, e.g. under the trademark cyclostin; and ifosfamide as holoxan. The term "anti-neoplastic anti-metabolite" includes, but is not limited to, 5-fluorouracil (5-FU); capecitabine;
gemcitabine; DNA de-methylating agents, such as 5-azacytidine and decitabine;
methotrexate; edatrexate; and folic acid antagonists.Capecitabine can be administered, e.g., in the form as it is marketed, e.g., under the trademark xeloda; and gemcitabine as gemzar.The term "platin compound", as used herein, includes, but is not limited to, carboplatin, cisplatin, cisplatinum, oxaliplatin, satraplatin and platinum agents, such as zd0473. Carboplatin can be administered, e.g., in the form as it is marketed, e.g., carboplat;
and oxaliplatin as eloxatin. Please note that in the field of oncology the platin compound are often designated as alkylating-like compounds. The term "topoisomerase I
inhibitors", as used herein, includes derivatives of the plant compound camptothecin.
Irinotecan (CPT-11) is a semi-synthetic derivative of camptothecin. Topotecan is another semi-synthetic analogue of camptothecin. There are other derivatives of camptothecin, as well as new formulations of the parent plant extract, that are in various stages of clinical trials.The term "topoisomerase II inhibitor", as used herein, includes, but is not limited to, the anthracyclines, such as doxorubicin, including liposomal formulation, e.g.
caelyx;daunorubicin, including liposomal formulation, e.g. daunosome; epirubicin; idarubicin and nemorubicin;
the anthraquinonesmitoxantrone and losoxantrone; and the podophillotoxinesetoposide and teniposide. Etoposide is marketed as etopophos; teniposide asVM 26-bristo;
doxorubicin as adriblastin or adriamycin; epirubicin asfarmorubicin; idarubicin as zavedos;
and mitoxantrone as novantron.The term "a VEGF inhibitor" includes, but is not limited to, compounds compounds targeting, decreasing or inhibiting the activity of the vascular endothelial growth factor (VEGF) receptors, such as compounds which target, decrease or inhibit the activity of VEGF, especially compounds which inhibit the VEGF receptor, such as, but not limited to, 7/-/-pyrrolo[2,3-d]pyrimidine derivative;
BAY 43-9006; isolcholine compounds disclosed in WO 00/09495, such as (4-tert-butyl-pheny1)-94-pyridin-4-ylmethyl-isoquinolin-1-y1)-amine. The term "insulin-like growth factor I
inhibitor" relates to compounds targeting, decreasing or inhibiting the activity of the insulin-likegrowth factor receptor 1 (IGF-1 R), such as compounds which target, decrease orinhibit the activity of IGF-IR, especially compounds which inhibit the IGF-1 R
receptor.Compounds include, but are not limited to, the compounds disclosed in W002/092599 and derivatives thereof of 4-amino-5-phenyl-7-cyclobutyl-pyrrolo{2,3- pyrimidine derivatives.The term a "protein tyrosine kinase", as used herein, relates to compounds targeting, decreasing or inhibiting the activity of protein-tyrosine kinase, such as imatinibmesylate (gleevec),tyrphostinorpyrymidylaminobenzamide and derivatives thereof. A
tyrphostin is preferably a low molecular weight (Mr<1500) compound, or a pharmaceutically acceptable salt thereof, especially a compound selected from the benzylidenemalonitrile class or the S-arylbenzenemalonirile or bisubstratequinoline class of compounds, more especially any compound selected from the group consisting of Tyrphostin A23/RG-50810, AG 99, Tyrphostin AG 213, Tyrphostin AG 1748, Tyrphostin AG 490, Tyrphostin B44, Tyrphostin B44 (+) enantiomer, Tyrphostin AG 555, AG 494, Tyrphostin AG 556; AG957; and adaphostin (4-{[(2,5-dihydroxyphenyl)methyl]amino}-benzoic acid adamantyl ester; NSC
680410, adaphostin).The term "EGFR kinase inhibitor", as used herein, relates to compounds targeting, decreasing or inhibiting the activity of the epidermal growth factor family of receptor tyrosine kinases (EGFR, ErbB2, ErbB3, ErbB4 as homo- or heterodimers), such as compounds which target, decrease or inhibit the activity ofthe epidermal growth factor receptor family are especially compounds, proteins orantibodies which inhibit members of the EGF receptor tyrosine kinase family, e.g.EGF receptor, ErbB2, ErbB3 and ErbB4 or bind to EGF or EGF-related ligands, and are in particular those compounds, proteins or monoclonal antibodies generically and specifically disclosed in W097/02266, e.g., the compound of Example 39, or inEP0564409, W099/03854, EP0520722, EP0566226, EP0787722,EP0837063,U.S. Patent No.5,747,498, W098/10767, W097/30034,W097/49688, W097/38983 and WO 96/30347, e.g. compound known as 0P358774, W096/33980, e.g. compound ZD1839; and W095/03283, e.g. compound ZM105180, e.g.trastuzumab (HERCEPTIN), cetuximab, iressa,OSI-774, CI-1033, EKB-569, GW-2016, E1.1, E2.4, E2.5, E6.2. -E6.4, E2.11 , E6.3 or E7.6.3, and erlotinib and gefitinib.
Erlotinibcan be administered in the form as it is marketed, e.g., tarceva, and gefitinib as iressa, humanmonoclonal antibodies against the epidermal growth factor receptor including ABX- EGFR.The term "mTOR kinase inhibitor" refers to compounds which target, decrease or inhibit the activity/function ofserine/theroninemTOR kinase are especially compounds, proteins or antibodieswhich target/inhibit members of the mTOR kinase family, e.g. RAD, RAD001 , CCI- 779, ABT578, SAR543, rapamycin and derivatives/analogs thereof, AP23573 andAP23841 from Ariad, everolimus (certican) and sirolimus. The term "proteasome inhibitors", as used herein, includes compounds which target, decrease or inhibit the activity of the proteosome. Compounds which target, -decrease or inhibit the activity of the proteosome include, but are not limited to, PS-341; MLN 341, bortezomib or velcade. The term "HDAC inhibitor", as used herein, relates to relates to compounds which inhibit the histone deacetylase and which possess anti-proliferative activity.
This includes but is not limited to compounds disclosed in WO 02/22577. It further especially includes suberoylanilidehydroxamic acid (SAHA); [4-(2-amino-phenylcarbamoy1)-benzylj-carbamic acid pyridine-3-ylmethyl ester and derivatives thereof; butyric acid, pyroxamide, trichostatin A, oxamflatin, apicidin, depsipeptide, depudecin and trapoxin.The term "tumor cell damaging approaches" refers to approaches, such as ionizing radiation. The term "ionizing radiation", referred to above and hereinafter, means ionizing radiation that occurs as either electromagnetic rays, such as X-rays and gamma rays; or particles, such as alpha, beta and gamma particles. Ionizing radiation is provided in, but not limited to, radiation therapy and is known in the art. See Hellman, Cancer, 41h Edition, Vol. 1, Devita et al.,Eds., pp. 248-275 (1993).
A "RAF kinase inhibitor" refers to compounds (or "inhibitors" or "antagonists"
which are equivalent terms) which interfere with the abnormal activation of a RAF
kinase. The meaning of "abnormal activation of a RAF kinase" is further explained. The Ras/Raf/Mek/ERK
(mitogen-activated protein kinase) signaling pathway plays a critical role in transmitting proliferation signals generated by the cell surface receptors and cytoplasmic signaling elements to the nucleus. Constitutive activation of this pathway is involved in malignant transformation by several oncogenes. Activating mutations in RAS occur in approximately 15 % of cancers, and recent data has shown that the RAF kinase, B-RAF, is mutated in about 7 % of cancers (Wellbrock et al., Nature Rev. Mol. Cell Biol. 2004, 5:875-885).
In mammals, the RAF family of serine/threonine kinases comprises three members: A-RAF, B-RAF and C-RAF. However, activating mutations have so far been only identified in B-RAE
underlining the importance of this isoform. The most common cancer mutation in B-RAF
results in a valine to glutamic acid exchange at position 600 of the protein (mutant is designated as BRAFvw E), which dramatically enhances the activity of B-RAE. Thus, RAF
inhibitors, particularly B-RAF inhibitors, interfere with cells comprising B-RAF
mutations, in particular with cells having the B-RAFvemE mutation. A number of B-RAF inhibitors are described in the art and it is believed that these B-RAF inhibitors are suitable for use in the present invention.
The following references disclose 13-RAF inhibitors:
W02011117381, W02011119894, W02011097594, W02011097526, W02011085269, W02011090738, W02011025968, W02011025927, W02011023773, W02011028540, W02010111527, W02010104973, W02010100127, W02010078408, W02010065893, W02010032986, W02009115572, W02009115572, W02009108838, W02009108827, W02009111260, W02009100536, W02009059272, W02009039387, W02009021869, W02009006404, W02009006389, W02009006389, W02008140850, W02008079277, W02008055842, W02008034008, W02008115263, W02008030448, W02008028141, W02007123892, W02007115670, W02007090141 ,W020070760 92,W02007067444, W02007056625, W02007031428, W02007027855, W02006125101, W02006124874, W02006124780, W02006124780, W02006102079 , W02006108482, W02006105844, W02006084015, W02006076700, W02006050800, W02006040569, W02005112932, W02005075425, W02005049603, W02005037285, W02005037273 and W02005032548.
In the present invention the compounds used as active ingredients in the combinations disclosed herein can be prepared and administered as described in the cited documents.
The structure of the active agents identified by code numbers, generic or trade names may be taken from the actual edition of the standard compendium The Merck Index"
or from databases, e.g., Patents International, e.g., IMS World Publications, or the publications mentioned above and below.

It will be understood that references to the MDM4-inhibitors and the chemotherapeutic compounds are meant to also include the pharmaceutically acceptable salts of any of the active substances. If active substances comprised by the components have, e.g.
at least one basic center, they can form acid addition salts. Corresponding acid addition salts can also be formed having, if desired, an additionally present basic center. Active substances having an acid group, e.g. COOH, can form salts with bases. The active substances comprised in the components or a pharmaceutically acceptable salt thereof may also be used in form of a hydrate or include other solvents used for crystallization.
Combination treatment Thus, in a particular embodiment the present invention relates to a method for the prevention of treatment of melanoma, particularly advanced cutaneous melanoma, which comprises treating the patient concurrently or sequentially with pharmaceutically effective amounts of a combination of: (a)an MDM4-antagonist and(b) one or more chemotherapeutic agents.
In a yet further aspect, the present invention provides a pharmaceutical preparation comprising: (a) an MDM4-antagonist; and(b) one or more chemotherapeutic agents, together with a pharmaceutically acceptable carrier.
In preferred embodiment, the present invention provides a pharmaceutical preparation comprising: (a) an MDM4-antagonist; and(b) one or more chemotherapeutic agents selected from a microtubule active agent; an alkylating agent; an anti-neoplastic anti-metabolite; a platin compound; a topoisomerase I inhibitor, a topoisomerase II inhibitor; a VEGF inhibitor;
a tyrosine kinase inhibitor; an EGFR kinase inhibitor; an mTOR kinase inhibitor; an insulin-like growth factor I inhibitor; a Raf kinase inhibitor; a MEK kinase inhibitor, a proteasome inhibitor; a HDAC inhibitor; and ionizing radiation.
Any of the combination of components (a) and (b), the method of treating a human comprising administering these two components, a pharmaceutical composition comprising these two components for simultaneous, separate or sequential use, the use of the combination for the delay of progression or the treatment of melanoma, in particularly cutaneous melanoma, or metastatic melanoma, or for the manufacture of a pharmaceutical preparation for these purposes or a commercial product comprising such a combination of components (a) and (b), all as mentioned or defined above, will be referred to subsequently also as "combination of the invention" (so that this term refers to each of these embodiments which thus can replace this term where appropriate).

Simultaneous administration may, e.g. take place in the form of one fixed combination with two or more active ingredients, or by simultaneously administering two or more active ingredients that are formulated independently. Sequential use (administration) preferably means administration of one (or more) components of a combination at one time point, other components at a different time point, that is, in a chronically staggered manner, preferably such that the combination shows more efficiency than the single compounds administered independently (especially showing synergism). Separate use (administration) preferably means administration of the components of the combination independently of each other at different time points.Also combinations of two or more of sequential, separate and sinnultaneousadministration are possible, preferably such that the combination component-drugs show a joint therapeutic effect that exceeds the effect found when the combination component-drugs are used independently at time intervals so large that no mutual effect on their therapeutic efficiency can be found, a synergistic effect being especially preferred.
Determining a synergistic interaction between one or more components, the optimum range for the effect and absolute dose ranges of each component for the effect may be definitively measured by administration of the components over different w/w ratio ranges and doses to patients in need of treatment. For humans, the complexity and cost of carrying out clinical studies on patients renders impractical the use of this form of testing as a primary model for synergy. However, the observation of synergy in one species can be predictive of the effect in other species and animal models exist, as described herein, to measure a synergistic effect and the results of such studies can also be used to predict effective dose and plasma concentration ratio ranges and the absolute doses and plasma concentrations required in other species by the application of pharmacokinetic/pharmacodynamic methods.
Established correlations between tumor models and effects seen in humans suggest that synergy in animals may e.g. be demonstrated in the melanoma tumor model as described in the examples below.
The term "delay of progression", as used herein, means administration of the combination to patients being in a pre-stage or in an early phase of melanoma, of the first or subsequent manifestations; or a relapse of the melanoma to be treated in which patients, e.g. a pre-form of the corresponding disease is diagnosed; or which patients are in a condition, e.g. during a medical treatment.
"Jointly therapeutically active" or "joint therapeutic effect" means that the compounds may be given separately (in a chronically staggered manner, especially a sequence-specific manner) in such time intervals that they preferably, in the human, to be treated, still show a (preferably synergistic) interaction (joint therapeutic effect).
"Pharmaceutically effective" preferably relates to an amount that is therapeutically or in a broader sense also prophylactically effective against the progression of melanoma, particularly subcutaneous melanoma, particularly metastatic melanoma.
A pharmaceutical product of the invention, as used herein defines especially a "kit of parts"
in the sense that the components (a), which is the MDM4-inhibitor and (b), which includes one or more chemotherapeutic agents, as defined above, can be dosed independently or by use of different fixed combinations with distinguished amounts of the components (a) and (b), i.e. simultaneously or at different time points. Moreover, these terms comprise a commercial package comprising (especially combining) as active ingredients components (a) and (b), together with instructions for simultaneous, sequential, chronically staggered, in time-specific sequence, preferentially) or (less preferably) separate use thereof in the delay of progression or treatment of melanoma, particularly subcutaneous melanoma, particularly metastatic melanoma. The parts of the kit of parts can then, e.g. be administered simultaneously or chronologically staggered, that is at different time points and with equal or different time intervals for any part of the kit of parts. Very preferably, the time intervals are chosen such that the effect on the treated melanoma in thecombined use of the parts is larger than the effect which would be obtained by use of only any one of the combination partners (a) and (b) as can be determined according to standard methods. The ratio of the total amounts of the combination partner (a) to the combination partner (b) to be administered in the combined preparation can be varied, e.g. in order to cope with the needs of a patient sub-population to be treated or the needs of the single patient which different needs can be due to the particular disease, age, sex, body weight, etc. of the patients.
Preferably, there is at least one beneficial effect, e.g. a mutual enhancing of the effect of the combination partners (a) and (b), in particular, a more than additive effect, which hence could be achieved with lower doses of each of the combined drugs, respectively, than tolerable in the case of treatment with the individual drugs only without combination, producing additional advantageous effects, e.g. less side effects or a combined therapeutic effect in a non-effective dosage of one or both of the combination partners (components) (a) and (b), and very preferably a strong synergism of the combination partners (a) and (b).
In a particular embodiment the "combination of the invention" can also be applied in combination with other treatments, e.g. surgical intervention, hyperthermia and/or irradiation therapy.

In yet another embodiment the invention provides a pharmaceutical composition comprising i) an MDM4-inhibitor and ii) one or more chemotherapeutic agents selected from a microtubule active agent, an alkylating agent, an anti-neoplastic anti-metabolite, a platin compound, a Raf kinase inhibitor, a MEK kinase inhibitor, a topoisomerase I
inhibitor, a topoisomerase II inhibitor, a VEGF inhibitor, a tyrosine kinase inhibitor, an EGFR kinase inhibitor, an mTOR kinase inhibitor, an insulin-like growth factor! inhibitor, a HDAC inhibitor, a proteasome inhibitor.
In yet another embodiment the invention provides a pharmaceutical composition comprising I) an MDM4-inhibitor and ii) one or more chemotherapeutic agents selected from camptothecin derivatives, paclitaxel, docetaxel, melphalan, temozolomide, cacarbazine,epothilone B, 5-FU, gemcitabine, oxaliplatin, cisplatinum, carboplatin, melphalam, doxorubicin, imatinib, erlotinib, bevacizumab, cetuximab and a Raf kinase inhibitor.
In yet another embodiment the invention provides a pharmaceutical composition comprising i) an MDM4-inhibitor and ii) one or more chemotherapeutic agents selected from oxaliplatin, cisplatinum, carboplatin, melphalam, dacarbazine, temozolomide and a RAF
kinase inhibitor.
In yet another embodiment the invention provides a pharmaceutical composition comprising i) an MDM4-inhibitor and ii) a RAF kinase inhibitor.
The pharmaceutical compositions according to the present invention can be prepared by conventional means and are those suitable for enteral, such as oral or rectal, and parenteral administration to mammals including man, comprising a therapeutically effective amount of an MDM4 inhibitor and at least one chemotherapeutic agent alone or in combination with one or more pharmaceutically acceptable carriers, especially those suitable for enteral or parenteral application.
The pharmaceutical compositions comprise from about 0.00002% to about 100%, especially, e.g. in the case of infusion dilutions that are ready for use) of 0.0001-0.02%, ore.g., in case of injection or infusion concentrates or especially parenteral formulations, from about 0.1% to about 95%, preferably from about 1% to about 90%, more preferably from about 20% to about 60%, active ingredient (weight by weight, in each case). Pharmaceutical compositions according to the invention may be, e.g., in unit dose form, such as in the form of ampoules, vials, dragees, tablets, infusion bags or capsules.
The effective dosage of each of the combination partners employed in a formulation of the present invention may vary depending on the particular compound or pharmaceutical compositions employed, the mode of administration, and the stage of the melanoma being treated. A physician, clinician or veterinarian of ordinary skill can readily determine the effective amount of each of the active ingredients necessary to prevent, treat or inhibit the progress of the melanoma.
Pharmaceutical preparations for the combination therapy for enteral or parenteral administration are, e.g. those in unit dosage forms, such as sugar-coated tablets, capsules or suppositories; and furthermore ampoules. If not indicated otherwise, these formulations are prepared by conventional means, e.g., by means of conventional mixing, granulating, sugar-coating, dissolving or lyophilizing processes. It will be appreciated that the unit content of a combination partner contained in an individual dose of each dosage form need not in itself constitute an effective amount since the necessary effective amount can be reached by administration of a plurality of dosage units. One of skill in the art has the ability to determine appropriate pharmaceutically effective amounts of the combination components.
Preferably, the compounds or the pharmaceutically acceptable salts thereof, are administered as an oral pharmaceutical formulation in the form of a tablet, capsule or syrup;
or as parenteral injections if appropriate.
In preparing compositions for oral administration, any pharmaceutically acceptable media may be employed, such as water, glycols, oils, alcohols, flavoring agents, preservatives or coloring agents. Pharmaceutically acceptable carriers include starches, sugars, m icrocrystalline celluloses, diluents, granulating agents, lubricants, binders and disintegrating agents.
Solutions of the active ingredient, and also suspensions, and especially isotonic aqueous solutions or suspensions, are useful for parenteral administration of the active ingredient, it being possible, e.g., in the case of lyophilized compositions that comprise the active ingredient alone or together with a pharmaceutically acceptable carrier, e.g., mannitol, for such solutions or suspensions to be produced prior to use. The pharmaceutical compositions may be sterilized and/or may comprise excipients, e.g., preservatives, stabilizers, wetting and/or emulsifying agents, solubilizers, salts for regulating the osmotic pressure and/or buffers, and are prepared in a manner known per se, e.g., by means of conventional dissolving or lyophilizing processes. The solutions or suspensions may comprise viscosity-increasing substances, such as sodium carboxymethylcellu lose, carboxymethylcellu lose, dextran, polyvinylpyrrolidone or gelatin. Suspensions in oil comprise as the oil component the vegetable, synthetic or semisynthetic oils customary for injection purposes.

The isotonic agent may be selected from any of those known in the art, e.g.
mannitol, dextrose, glucose and sodium chloride. The infusion formulation may be diluted with the aqueous medium. The amount of aqueous medium employed as a diluent is chosen according to the desired concentration of active ingredient in the infusion solution. Infusion solutions may contain other excipients commonly employed in formulations to be administered intravenously, such as antioxidants.
In a particular embodiment the invention offers a method for testing the eligibility of a patient suffering from melanoma, or cutaneous melanoma, or metastatic melanoma, for treatment with an MDM4 inhibitor comprising determining the protein expression level of MDM4 in a melanoma tumor sample derived from said patient and wherein an enhanced MDM4 protein expression compared to the MDM2 protein expression selects the patient as eligible for =
treatment. In other words, the invention provides for a companion diagnostic for testing the eligibility of a patient suffering from melanoma for treatment with an MDM4 inhibitor or a pharmaceutical composition of the invention comprising an MDM4 inhibitor.
In yet another embodiment the invention offers a method for testing the eligibility of a patient suffering from melanoma for treatment with a pharmaceutical composition of a combination between an MDM4-inhibitor and one or more chemotherapeutic agents as described before comprising determining the protein expression level of MDM4 and determining the B-RAE
status in a melanoma tumor sample derived from said patient and wherein an enhanced MDM4 protein expression compared to the MDM2 protein expression and the presence of a B-RAE activating mutation selects the patient as eligible for treatment. The term "B-RAF
status" means that the B-RAF gene is screened for the presence of B-RAF
activating mutations, in particular for the presence of the B-RAE mutation, B-RAFv600E.
Determination of the B-RAE status typically comprises FOR amplification of the B-RAF
nucleotide sequence derived from a tumor sample and determining the nucleotide sequence of the 8-RAE amplified nucleotide sequence. A "melanoma tumor sample derived from a patient"
typically means a tissue biopsy derived from a melanoma present in a patient.
The term "an enhanced MDM4 protein expression compared to the MDM2 protein expression"
refers to the ratio between the MDM4 protein expression level and the MDM2 protein expression level, which ratio is typically higher than 1, higher than 2, higher than 3, higher than 4 or even higher than 5. The absence of MDM4 protein expression means that the patient is not eligible for treatment with the pharmaceutical compositions of the invention.
One of the merits of the present invention is that it was found that the protein expression levels of MDM4 and MDM2 were mutually exclusive (an exception being only a low fraction of metastatic melanomas where the protein expression levels of MDM4 and MDM4 were found high; it is submitted that also the latter group is eligible for treatment with the pharmaceutical compositions of the invention). In a particular embodiment the protein expression level of MDM2 is absent or not detectable in the melanoma biopt. Indeed, we have found that in most cases of melanoma the protein expression level of MDM2 is non-detectable or very low.ln yet another embodiment the protein expression level of MDM4 in a suspected melanoma biopt is quantified as compared to the protein expression level of MDM4 in benign congenital melanocytic nevi. In these benign congenital melanocytic nevi we observed that the MDM4 protein expression level was very low. Hence, a high ratio of MDM4 protein expression level (biopt versus benign congenital melanocytic levi) which is preferably higher than 2, 3, 4, 5, 6, 7, 8, 9, 10 or more also means that the patient is eligible for treatment with the pharmaceutical compositions of the invention.
The determination of the protein expression level of MDM2 and the protein expression level of MDM4 can be carried out with a number of methods described in the art for determining the protein expression level, Non-limiting examples are antibody based methods for the detection and quantification of MDM4 and MDM2 such as western blot analysis, ELISA, immunoblotting, immunelectrophoresis, immunoprecipitation. Antibodies which can be used are monoclonal or polyclonal antibodies directed against MDM4 or MDM2. Yet another method to detect and quantify the ratio between MDM4 and MDM2 protein expression levels is the proximity ligation assay (see for example SOderberg, Ola et al (2006).
"Direct observation of individual endogenous protein complexes in situ by proximity ligation".Nature Methods3 (12): 995-1000.
The following Examples illustrate the invention described above; they are not, however, intended to limit the scope of the invention in any way. The beneficial effects of the pharmaceutical composition of the invention can also be determined by other test models known as such to the person skilled in the pertinent art.
Examples 1.MDM4 is overexpressed in 60% of human melanomas In most human melanoma cell lines p53 is wild-type and highly expressed, but transcriptionally inactive26. Since MDM4 overexpression stabilizes p53 by competing with MDM2 for p53 binding and inhibits p53 transcriptional activity24 we hypothesized that MDM4 overexpression might contribute to inactivation of p53 in melanomas. Although we have previously found MDM4 amplification in retinoblastoma and breast cancer27'28 we did not find evidence for an increased somatic copy-number of MDM4 in melanomas through in siiicoanalysis29.30. Meta-analysis of Oncomine microarray data (Oncomine 4.4 Research Edition; http://www.oncomine.org) failed to identify any significant or recurrent up-regulation of MDM4 mRNA expression in melanomas. We nevertheless assessed MDM4 levels in a panel of 40 primary skin melanomas: 10 primary, non-invasive, 10 regional dermal metastases, 10 nodal metastatic lesions and 10 distant metastatic lesions.
Consistent with the in silico data, the large majority of these samples (36/40) expressed MDM4 mRNA levels that were lower than in MCF-7 a cell line expressing relatively high MDM4 levels27. In contrast, MDM4 protein levels were considerably higher than in MCF-7 in ¨60%
of cases (Figure 1A). Whereas MDM4 protein expression was barely detectable in benign congenital melanocytic nevi (ON), high MDM4 protein levels were already evident in many of the primary, non-invasive, tumor samples (6/10) indicating that MDM4 protein up-regulation is an early event during melanomagenesis. In contrast, MDM2 protein levels ranged from non-detectable to low in the majority of cases. High MDM2 levels were only found in a few metastatic lesions: 1/10 regional dermal, 1/10 nodal and 4/10 distant metastatic lesions (Figure 1A). Overexpression of MDM2 and MDM4 was mutually exclusive, except in (samples 33 and 37) metastatic melanomas. p53 levels varied from sample to sample and were only very high in two samples (samples 23 and 24). As mutant p53 is often expressed at very high levels this observation raises the possibility that p53 is only mutated in these two particular samples (Figure 1A), an observation which is consistent with the previously reported low mutation rate of p53 in melanoma14=26.
MDM4 protein overexpression was further confirmed in a panel of human metastatic melanoma (MM) cell lines. MDM4 protein levels were very high in 8 (MM001, MM011, MM031, MM032, MM047, MM057, MM117, MM120) out of 17 patient-derived short-term cultures established from metastatic tumors and in all (4/4) commonly used cell lines harboring wild-type p53 (A375, WM9, Mel-501, Lu1205) (Figure 1B). Importantly, protein was not detectable in normal primary melanocyte cultures. In addition to the very high expressers, MDM4 protein levels were also higher than in normal melanocytes in 6 (MM029, MM034, MM061, MM074, MM087, MM118) of 17 cell lines; hence, overall protein levels were elevated in 14 out of the 17 lines compared to primary melanocytes.
Consistent with a posttranscriptional mechanism being responsible for MDM4 up-regulation, MDM4 mRNA levels were only significantly elevated in one of the 17 short-term cultured cell lines (MM120). As in the primary melanoma samples, MDM2 protein levels in the short-term cultures ranged from non-detectable to low in the majority of cases and were only high in 4 cases (MM011, MM034, MM061, MM117) and very high in two cases (MM001, MM120).
Notably, MDM2 protein expression levels were very high in all (4/4) well-established melanoma cell lines, a situation which could be a consequence of extensive in vitropassaging of these cells.
Finally, all the MM cell lines expressed relatively high levels of p53. To test whether the p53 tumor suppressor response was intact downstream of MDM4 we exposed most of the short-term cultured cell lines to the DNA-damaging agent doxorubicin (Doxo) and to high concentrations of the MDM2 antagonist nutlin-331. All but one (MM87) of the cell lines overexpressing MDM4 had an intact p53 pathway as evidenced by increased p53 protein levels and induction of expression of p21, an established p53-transcriptional target32. p53 stabilization and concomitant induction of p21 was also observed in response to these treatments in two of the commonly used cell lines analyzed (A375, and Lu1205).
BRAF and NRAS mutational status was determined in all primary tumors and cell lines described above. MDM4 overexpression was observed at comparable frequencies in tumors and cell lines harboring either wild-type or NRAS or BRAF mutations indicating that MDM4 overexpression is independent of the BRAF and NRAS mutational status.
Hence, MDM4 protein, but not mRNA, is frequently overexpressed at very high levels in human primary melanoma tumors and metastatic melanoma cell lines. Observing MDM4protein overexpression already in the non-invasive, non-metastatic, melanomas may indicate a significant role for this oncogene in early melanocyte transformation. Collectively, our results indicate that MDM4 protein up-regulation is an important driving oncogenic event and a key mechanism that contributes to p53 inactivation during melanomagenesis.
2.Inhibition of the MDM4-p53 interaction restores p53 activity in melanoma The reliance of melanoma on MDM4 for survival suggests that antagonizing its interaction with wild-type p53 should restore p53-driven pro-apoptotic activities in melanoma cells.
Recently, Bernal and co-workers described the design, synthesis and evaluation of stabilized alpha helical peptides (SAH) based on the transactivation domain of p5339. One of these compounds, SAH-p53-8, binds directly MDM4 within its p53-binding pocket with high affinity and is capable of disrupting p53-MDM4 complexes in vitro and in vivo40. In contrast to nutlin-3, this compound is capable of reactivating the p53 tumor suppressor functions and induce apoptosis in cells with high levels of wild-type p53 and .MDM449. To test whether direct inhibition of MDM4 is a viable therapeutic strategy for melanoma we treated the MM cell lines with SAH-p58-8, its biologically inactive point mutant analog SAH-p53-8H9A, the MDM2-specific inhibitor nutlin-3, or a 1:1 stoichiometric combination of both SAH-p53-8 and nutlin-3 (Figure 2A). As expected since the MM031 cells express low levels of MDM2 and high levels of MDM4, nutlin-3 had only a marginal cytotoxic effect on these cells. In sharp contrast, MM031 exhibited robust growth inhibition in response to SAH-p53-8 alone, and there was only a very modest increase in cytotoxicity upon combination with nutlin-3 (Figure 2B left panel; Combination Index CI=0.90).

Consistent with previous work40, the MM011 cells which express high levels of both MDM4 and MDM2 showed sensitivity to the single agents SAH-p53-8 and nutlin-3 (Figure 2A, right panel). Most notably, these two compounds synergize strongly with one another displaying enhanced cytotoxicity over the single treatment regimen (Figure 2B, right;
combination index CI=0.32). Similar effects were observed in the conventional melanoma cell line Lu1205, which expresses both MDM4 and MDM2 at high levels (data not shown). As further demonstration of specificity, the mutant SAH-p53-8F19A peptide did not induce any measurable cytotoxic effects in any of the melanoma cell lines. Together our data indicate that all human melanoma cell lines are highly sensitive to MDM4 inhibition alone or in combination with MDM2 inhibition. RT-qPCR analyses further confirmed the ability of SAH-p53-8, but not SAH-p53-8F19A, to induce expression of a series of established p53-target genes in all melanoma cell lines analyzed (Figure 2C).
To test the therapeutic potential of inhibiting the p53-MDM4 interaction on melanoma progression in vivo we compared the activity of vehicle and SAH-p53-8 in a MM031 murine xenograft model. MM031 xenografts were established by subcutaneous injections into the flanks of immunocompromised mice. When tumors reached an average volume of 200 mm3, cohorts were treated intravenously with vehicle or SAH-p53-8 daily for 5 consecutive days.
Whereas tumor growth rate was unaffected in vehicle-treated mice, treatment with SAH-p53-8 significantly suppressed tumor growth throughout the 10-day evaluation period (Figure 2D). As previously shown, histological examination of SAH-p53-8-treated mice showed no obvious toxicity of the compound to normal tissues43. Collectively, these data highlight the pharmacologic potential of existing MDM4 inhibitors for the treatment of melanoma.
3.Targetinq the MDM4-p53 pathway sensitizes melanoma to conventional chemotherapy The use of DNA-damaging agents such as cisplatin or melphalan in the clinic has yielded low response rates in melanoma, of which few are durable". Strategies that increase the sensitivity of melanoma cells to genotoxic agents are expected to decrease their toxicity and eventually improve their potency. Since the effectiveness of these agents relies on the reactivation of a genetically uncompromised p53 pathway, we hypothesized that combination treatment with p53-MDM4 inhibitors would enhance cytotoxicity caused by DNA-damaging agents. To test this possibility, we investigated the effects of cisplatin and melphalan alone or in combination with nutlin-3 or SAH-p53-8 on the growth of the MM011 and MM031 cell lines. Treatment of these cells with cisplatin or melphalan alone yielded variable growth inhibition effects (Figure 3A and 3B). Importantly, the cytotoxic effects of these alkylating agents were greatly potentiated by co-treatment with SAH-p53-8. Because they express low levels of MDM2 and consistent with our mechanistic hypothesis, MM031 cells responded more favorably to the combination of cisplatin or melphalan with SAH-p53-8 than co-treatment with nutlin-3 (Figure 3A). On the other hand, the high levels of MDM2 in MM011 cells render SAH-p53-8 as effective as nutlin-3 in synergizing with cisplatin (Figure 3B).
These data demonstrate that targeting the MDM4-p53 axis significantly sensitizes metastatic melanoma cells to DNA-damaging agents.
4.Targeting the MDM4-p53 axis kills BRAF inhibitor-resistant melanoma cells Chronic treatment with BRAF inhibitors is invariably associated with the development of drug resistance42. Overcoming BRAF inhibitor resistance is likely to require targeting of multiple signaling pathways. Since MDM4 overexpression was observed in tumors irrespective of BRAF mutational status, we investigated whether targeting the MDM4-p53 interaction could affect the growth of BRAF inhibitor-sensitive parental melanoma cell lines (451Lu and Me11617) as well as BRAF-resistant sub-lines (451Lu-R and Me11617-R) which were artificially derived by chronic exposure to a BRAF inhibitor43. Strikingly, treatment of both parental and BRAF-resistant cell lines with increasing doses of SAH-p53-8 led to decreased viability, indicating that cells that have acquired drug resistance to BRAF
inhibitor remain sensitive to MDM4-p53 targeting (Figure 3C). To investigate whether combined BRAF and MDM4 inhibition synergizes to induce cytotoxic effects we treated BRAF
inhibitor-sensitive lines with a BRAF-inhibitor, PLX4032, and SAH-p53-8 as single agents or in combination. As expected, exposure to PLX4032 alone was sufficient to induce a significant decrease in cell viability (Figure 3D). More significantly simultaneous treatment with PLX4032 and SAH-p53-8 greatly enhanced the effect when compared with each individual compound (Figure 3D).
The collective data show that co-targeting of MDM4 and mutant BRAF results in potent anti-tumor activity in melanomas harboring BRAF mutations, and may therefore offer a novel therapeutic avenue for limiting and overcoming the resistance to BRAF
inhibitors.
Additionally, as melanoma cells, that acquire resistance to BRAF inhibitors, remain addicted to high protein levels of MDM4, targeting the MDM4-p53 pathway is a valid therapeutic approach to treat relapsed patients.
Materials and Methods 1 .Reagents The stapled peptides SAH-p53-8 and SAH-p53-8 FisA were obtained from Dr.
Federico Bernal, National Cancer Institute, CCR, Metabolism Branch, Rockville, MD
20852. PLX4032 (also known as vemurafenib or RG7204 or R05185426) was purchased from Selleck Chemicals. Before application 10mg of PLX4032 was suspended in DMSO at a concentration of 2.5 mM.

2.Melanoma cell cultures The panel of 40 primary skin melanomas was kindly provided by the Institute Jules Bordet, Brussels, Belgium. The panel of human metastatic melanoma (MM) cell lines was kindly provided by the UMDNJ-Robert Wood Johnson Medical School, CINJ, NJ, US. Human melanoma cell lines MM001, MM011, MM031, MM117, were cultured in F10 medium with

5% fetal bovine serum (HyClone), 5% calf bovine serum (HyClone) and antibiotics. A375 and Lu1205 melanoma cell lines were cultured in respectively RPM' and DMEM
medium with 10% fetal bovine serum (Sigma) and antibiotics. 451Lu, 451Lu-R, Me11617 and Me11617-R cell lines were kindly provided by M. Herlyn (The Wistar Institute, Philadelphia, US), these cell lines were cultured in DMEM plus 5% fetal bovine serum (HyClone) and antibiotics. The resistant cells were maintained in 1pM BRAF inhibitor PLX4032, supplemented every 72 hr.
3.Cell Growth and Viability Cell growth was measured using the WST-1 assay (Roche). Cells were counted and seeded in triplicate in 96-well plates at a density of 3000 or 6000 cells per well, in a total volume of 100 pl medium. Cells were incubated with 10 pl WST-1 reagent for 2 h and absorbance (450 nm) was measured in a microplate reader (Victor; Perkin Elmer).
Alternatively, cell growth was measured using the CellTiter-Glo Assay (Promega). Cells were counted and seeded in triplicate in 96-well plates at a density of 15,000 cells per well, and treated with chemotherapeutics: cisplatin (Sigma) or melphalan (Sigma), SAH-p53-8 and SAH-p53-8F19A (both synthesized and characterized in the Bernal laboratory, NCI)40, BRAF
inhibitor (PLX4032, Roche) or nutlin-3 (Johnson & Johnson) for 24h after which the CellTiter-Glo reagent was added to the cells in a 1:1 ratio. Luminescence was measured in a microplate reader (Victor; Perkin Elmer).
4.Apoptosis Assays Apoptosis was measured using the Caspase-Glo 3/7 Assay (Promega). Cells were counted and seeded in triplicate in 96-well plates at a density of 12000 cells per well. Next day the Caspase-Glo 3/7 reagent was added to the cells in a 1:1 ratio. Luminescence was measured in a microplate reader (Victor; Perkin Elmer) after 1h shaking at RT.
5.Colony Formation Assays Cells were plated at a density between 2x103 and 32x103 cells per 6-well plate and cultured for 12 days. The cells were then washed with PBS 1X, fixed and stained 5 min with a 1%

crystal violet in 35% methanol solution.

6.Western blotting analysis Cells, adult tissue samples, or tumors were lysed in Giordano buffer [50 mMTris-HCl (pH

7,4); 250 mMNaCI; 0,1% Triton X-100; 5 mM EDTA] containing phosphatase and protease inhibitors (Sigma). The adult tissue samples were sonicated three times 10 sec. The protein concentration was determined by Bradford assay (OD 595 nm; Bio-Rad Protein Assay) and 40 pg of each sample were fractionated by SDS-PAGE (Invitrogen; NuPAGEO Novex 12% Bis-Tris Gel). The fractionated extracts were then transferred to a PVDF
membrane (iBlot Dry Blotting System). Membranes were blocked in Tris-buffered saline, 0.2% Tween-20 (TBST) containing 5% or 10% non-fat dry milk, and subsequently incubated with the appropriate primary antibody. Membranes were washed five times with TBST and incubated with either horseradish peroxidase -conjugated horse anti-mouse or goat anti-rabbit secondary antibody (Cell Signaling). After five washies with TBST, proteins were detected by enhanced chemiluminescense"ECL Western Blotting Detection Reagents" (Amersham Biosciences) or "SuperSignal West Femto Maximum Sensitivity Substrate" (Thermo Scientific) and visualized by exposure to X-ray film (Amersham Biosciences).
The following primary antibodies were used: rabbit anti-MDM4 (IHC-00108, Bethyl Laboratories), mouse anti-Mdm2 (mixture of 2A10 and 4B2 and SMP14, Santa Cruz Biotechnology), mouse anti-p53 (D0-1, Santa Cruz Biotechnology), mouse anti-p21 (F-5, Santa Cruz Biotechnology), mouse anti-b-Tubulin (Sigma-Aldrich), mouse anti-Vinculin (Sigma-Aldrich).
7.Quantitive real-time PCR (RT¨gPCR) RNA was isolated using the "RNeasyminikit" (Qiagen) according to the manufacturer's protocol. The RNA was quantified using a Nanodrop 1000 (Thermoscientific). 2 pg of total RNA of each sample of interest was reverse-transcribed using the "High-Capacity cDNA
Reverse Transcription Kit" (Applied Biosystems) to obtain cDNA which was further used as template for PCR amplification. Quantitative reverse transcriptase PCR (RT-qPCR) assays were performed using Fast SYBR Green 2x Master Mix, following the manufacturer's instructions (Applied Biosystems). For normalization the geometric mean of at least three reference genes was used. For mouse samples TaqMan probes were designed by Applied Biosystems (assays on demand). For human samples the primers used were as follows:
APAF-1 [Fwd, 5'- CCIGTIGTCTCTTCTTCCAGTGT-3', Rev, 5-AAAACAACTGGCCICTGTGG-31, BAX [Fwd, 5'- ATGTTTTCTGACGGCAACTTC-3', Rev, 5- ATCAGTTCCGGCACCTTG-3'], MDM4 [Fwd, 5'- AGGTGCGCAAGGTGAAATGT-3', Rev, 5- CCATATGCTGCTCCTGCTGAT-3'], MDM2 [Fwd, 5'- AGGAGATTTGTTTGGCGTGC-3', Rev, 5- TGAGTCCGATGATTCCTGCTG-3'], PUMA [Fwd, 5'- GACCTCAACGCACAGTA-3', Rev, 5- CTAATTGGGCTCCATCT-3'], p21 [Fwd, 5'- AGCAGAGGAAGACCATGTGGA-3', Rev, 5- AATCTGTCATGCTGGTCTGCC-3']. Following reference genes were used: GAPDH
[Fwd, 5'- TGCCATGTAGACCCCTTGAAG-3', Rev, 5- ATGGTACATGACAAGGTGCGG-3'], HMBS [Fwd, 5'- GGCAATGCGGCTGCAA-3', Rev, 5- GGGTACCCACGCGAATCAC-3'], RLP13a [Fwd, 5'- CCTGGAGGAGAAGAGGAAAGAGA-3', Rev, 5-TTGAGGACCTCTGTGTATTIGTCAA-3], TBP [Fwd, 5'- CGGCTGTTTAACTTCGCTTC-3', Rev, 5- CACACGCCAAGAAACAGTGA-3']. Gene expression levels and errors on the gene expression levels were calculated using qBasePLUS 1.0 analysis software69 .

8.Determining the BRAF and NRAS status To analyze the mutational status of BRAF and NRAS in the short-term melanoma cell lines, 200 ng of cDNA from each of the cell line was used as a template for a PCR
reaction. The subsequent primer sequences were used for genotyping: Primer BRAF [Fwd, 5'-AGCACCTACACCTCAGCAGTTACA-3', Rev, 5'-ACAGGTATCCTCGTCCCACCATAA-3'], Primer NRAS [Fwd, 5'-ACAAACTGGTGGIGGTTGGA-3', Rev, 5'-TGGCCATCCCATACAACCCT-3']
Subsequently, PCR reaction was purified using the QIAquick PCR Purification Kit, according to the manufacturers protocol. The purified PCR products were sequenced using following nested primers: Primer BRAF (Nested)5'-AGGGCATGGATTACTTACACGCCA-3', Primer NRAS (Nested) 5'-ACTCGCTTAATCTGCT000TGT-3'

9.Histoloqv and IHC
Tissues were fixed overnight in 4% paraformaldehyde, dehydrated, paraffin embedded, sectioned (6 um) and stained with hematoxylin and eosin (H&E). For immunohistochemistry (IHC), slides were bleached for 5h in 10% H202 solution, and stained with antibodies against S100 (rabbit, Z0311, 1:300; Dako). Detection was performed with the secondary goat anti-rabbit antibody (E0432, 1:500; Dako) combined with the incubation in an ABC
reagent (Vector). Sections were counterstained with hematoxylin.

10.Xenograft Experiments All animal experiments were performed in accordance with the guidelines of the University of Leuven Animal Care and Use ethical Committee. Mice were injected subcutaneously with human cell lines in sterile phosphate buffered saline [PBS 1X (pH 7,4)]. MM031 xenografts were established by injecting 107 cells, whereas Lu1205 and A375 xenografts were established by injecting 106 cells subcutaneously into the flanks of 8-weeks female Rj:NMRI-nu (nu/nu) mice (Jackson Labs). For the knock-down experiments, tumor growth was monitored with a caliper twice a week, and the volume was calculated using the following formula V= a * b2* 0.5, where a is the largest, and b the smallest diameter of the tumor. The SAH-p53-8 experiment was performed on MM031 cell line-derived tumors with an average volume of 200 mm3. Subsequently, cohorts (n = 7) were treated with vehicle (5%
DMSO in D5W) or SAH-p53-8 (10 mg/kg), once daily for 5 consecutive days by intravenous injection and tumor volume was monitored by caliper measurement daily for a period of 12 days.

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Claims (19)

CLAIMS:

1. Use of pharmaceutically effective amounts of a combination of i) an MDM4-inhibitor and ii) one or more chemotherapeutic agents selected from a microtubule active agent, an alkylating agent, an anti-neoplastic anti-metabolite, a platin compound, a Raf or MEK
kinase inhibitor, a topoisomerase I inhibitor, a topoisomerase II inhibitor, a VEGF inhibitor, a tyrosine kinase inhibitor, an EGFR kinase inhibitor, an mTOR kinase inhibitor, an insulin-like growth factor I
inhibitor, a HDAC inhibitor, a proteasome inhibitor, and ionizing radiation for simultaneous, concurrent, separate or sequential administration in preventing or treating melanoma having an activating B-RAF mutation.

2. The use according to claim 1 wherein said one or more chemotherapeutic agents are selected from camptothecin derivatives, paclitaxel, docetaxel, melphalan, dacarbazine, temozolomide, epothilone B, 5-FU, gemcitabine, oxaliplatin, cisplatinum, carboplatin, doxorubicin, imatinib, erlotinib, bevacizumab, cetuximab and a Raf kinase inhibitor.

3. The use according to claim 1 or 2 wherein said Raf kinase inhibitor is a B-RAF kinase inhibitor.

4. The use according to any one of claims 1 to 3 wherein said MDM4-inhibitor is a stapled peptide inhibitor.

5. The use according to any one of claims 1 to 4 wherein said melanoma is advanced cutaneous melanoma.

6. The use according to any one of claims 1 to 5 wherein said melanoma having an activating B-RAF mutation has acquired a resistance to a B-RAF inhibitor.

7. The use according to any one of claims 1 to 6 wherein said melanoma has additionally an enhanced protein expression level of MDM4.

8. A pharmaceutical composition comprising i) an MDM4-inhibitor and ii) one or more chemotherapeutic agents selected from a microtubule active agent, an alkylating agent, an anti-neoplastic anti-metabolite, a platin compound, a Raf kinase inhibitor, a MEK kinase inhibitor, a topoisomerase I inhibitor, a topoisomerase II inhibitor, a VEGF
inhibitor, a tyrosine kinase inhibitor, an EGFR kinase inhibitor, an mTOR kinase inhibitor, an insulin-like growth factor I inhibitor, a HDAC inhibitor, a proteasome inhibitor for simultaneous, concurrent, separate or sequential use in preventing or treating melanoma having an activating B-RAF
mutation.

9. The pharmaceutical composition according to claim 8 comprising one or more chemotherapeutic agents selected from camptothecin derivatives, paclitaxel, docetaxel, melphalan, dacarbazine, temozolomide, epothilone B, 5-FU, gemcitabine, oxaliplatin, cisplatinum, carboplatin, melphalam, doxorubicin, imatinib, erlotinib, bevacizumab, cetuximab and a Raf kinase inhibitor.

10. The pharmaceutical composition according to claim 8 comprising one or more chemotherapeutic agents selected from oxaliplatin, cisplatinum, carboplatin, melphalam, dacarbazine, temozolomide and a RAF kinase inhibitor.

11. The pharmaceutical composition according to claim 8 comprising as a chemotherapeutic agent a RAF kinase inhibitor.

12. Use of a pharmaceutically effective amount of an MDM4-inhibitor for preventing or treating melanoma having an activating B-RAF mutation.

13. The use according to claim 12 wherein said melanoma is advanced cutaneous melanoma.

14. The use according to claim 12 or 13 wherein said melanoma having an activating B-RAF
mutation has acquired resistance to a B-RAF inhibitor.

15. The use according to any one of claims 12 to 14 wherein said MDM4-inhibitor is a stapled peptide.

16. A method for testing the eligibility of a patient suffering from melanoma having an activating B-RAF mutation, for treatment with an MDM4 inhibitor comprising determining the protein expression level of MDM4 in a tumor sample of said patient and wherein an enhanced MDM4 protein expression selects the patient as eligible for treatment.

17. A method for testing the eligibility of a patient suffering from melanoma having an activating B-RAF mutation for treatment with the pharmaceutical composition according to claim 11 comprising determining the protein expression level of MDM4 and the B-RAF
status in a tumor sample of said patient and wherein an enhanced MDM4 protein expression and the presence of a B-RAF mutation selects the patient as eligible for treatment.

18. The use according to any one of claims 1 to 7, or 13 to 15, or the pharmaceutical composition according to any one of claims 8 to 11, or the method according to claim 16, wherein the MDM4-inhibitor is SAH-p53-8.

19. The use according to any one of claims 1 to 7, or 13 to 15, or the pharmaceutical composition according to any one of claims 8 to 11, wherein the Raf kinase inhibitor is PLX4032.