WO2014012902A2 - Melanosome transport inhibition for the treatment of melanoma - Google Patents

Abstract

This invention relates to methods of increased the sensitivity of cancers, such as melanoma, to cytotoxic anti-cancer compounds, such as methotrexate, by inhibiting melanosome trafficking in the cancer cells, for example using Akt2 signalling inhibitors, such as 7-hydroxystraurosporine. Methods of treatment and compounds for use therein are provided.

Description

Melanosome Transport Inhibition for the Treatment of Melanoma

Field of Invention

This invention relates to compositions and methods for the treatment of melanoma and other cancer conditions.

Background of Invention

Malignant melanoma is a deadly disease in which standard treatment options have remained remarkably static over the past 30 years (Sullivan & Atkins, 2009) . At present, the incidence of melanoma continues to increase despite public health initiatives that have promoted protection against the sun. Thus, during the past ten years, the incidence and annual mortality of melanoma has increased more rapidly than any other cancer and according to an American Cancer Society estimate, there will have been approximately 68,720 new cases of invasive melanoma diagnosed in 2009 in the United States, which resulted in approximately 8,650 deaths (American Cancer Society, 2009) . Unfortunately, the increase in incidence has not been paralleled by the development of new therapeutic agents with a significant impact on survival. Although many patients with melanoma localized to the skin are cured by surgical excision, increased time to diagnosis is associated with higher stage of disease, and those with regional lymphatic or metastatic disease respond poorly to conventional radiation and chemotherapy with 5-year survival rates ranging from 10 to 50% (Tawbi & Buch, 2010) . Currently, limited therapeutic options exist for patients with metastatic melanomas, and all standard combinations currently used in metastasis therapy have low efficacy and poor response rates. For instance, the only approved chemotherapy for metastatic melanoma, dacarbacine, has a response rate of about 10% and a median survival of 8-9 months. The other approved agent for advanced melanoma is high dose interleukin-2 , which can induce dramatic complete and durable responses (Ascierto et al., 2010) . However, only one patient in twenty derives lasting benefit. These data indicate the needed for alternative therapies for this disease and recent results indicated that combined therapies could became an attractive strategy to fight melanoma. One example of the complications involved in melanoma chemotherapy is the limited effectiveness of antifolates. Although methotrexate (MTX) , the most frequently used antifolate, is an efficient drug for several types of cancer, it is not active against melanoma (Kufe et al., 1980) . Undoubtedly, unravelling the mechanism of the resistance of melanomas to this drug could help to improve current therapeutic approaches. Summary of Invention

The present invention relates to the finding that the sensitivity of melanoma to cytotoxic anti-cancer compounds, such as methotrexate (MTX) , may be increased by inhibiting melanosome transport systems, for example by inhibition of the Akt signalling pathway.

An aspect of the invention provides a method of treatment of melanoma comprising;

administering a cytotoxic anti-cancer compound and a

melanosome transport inhibitor to an individual in need thereof.

Other aspects of the invention provide a melanosome transport inhibitor for use in the treatment of melanoma in combination with cytotoxic anti-cancer compound and the use of a melanosome transpor inhibitor in the manufacture of a medicament for use in the

treatment of melanoma in combination with a cytotoxic anti-cancer compound .

Other aspects of the invention provide a cytotoxic anti-cancer compound for use in the treatment of melanoma in combination with melanosome transport inhibitor and the use of a cytotoxic anti- cancer compound in the manufacture of a medicament for use in the treatment of melanoma in combination with a melanosome transport inhibitor .

Other aspects of the invention provide a combination of a melanosome transport inhibitor and a cytotoxic anti-cancer compound for use in the treatment of melanoma and the use of a combination of a

melanosome transport inhibitor and a cytotoxic anti-cancer compound in the manufacture of a medicament for use in the treatment of melanoma .

Other aspects of the invention provide a pharmaceutical formulation comprising a melanosome transport inhibitor and a cytotoxic anticancer compound, optionally for use in the treatment of melanoma.

Preferred melanosome transport inhibitors may include Akt signalling pathway inhibitors, for example Akt2 signalling pathway inhibitors, such as 7-hydroxystraurosporine (UCN-01) and Akt Inhibitor VIII (1, 3-dihydro-l- (1- ( (4- ( 6-phenyl-lH-imidazo [4, 5-g] quinoxalin-7-yl) phenyl) methyl) -4-piperidinyl) -2H-benzimidazol-2-one . Preferred cytotoxic anticancer compounds include DNA crosslinking agents, for example anthracyclines , such as doxorubicin, adriamycin; Pt containing compounds, such as cisplatin, carboplatin, or

oxaliplatin; and antifolate compounds, such as methotrexate (MTX) , aminopterine (AMT) , trimetrexate (TMQ) fluorouracil , lometrexol

(LMTX) , pemetrexed, raltitrexed or prelatrexate, alkylating agents, such as dacarbazine, BRAF inhibitors such as vemurafenib or

dabrafenib, and microtubule-targeting drugs, such as docetaxel and vincri stine .

In some preferred embodiments, the cytotoxic anticancer compound is methotrexate (MTX) and the melanosome transport inhibitor is 7- hydroxystraurosporine; the cytotoxic anticancer compound is

doxorubicin and the melanosome transport inhibitor is 7- hydroxystraurosporine; or the cytotoxic anticancer compound is methotrexate (MTX) and the melanosome transport inhibitor is Akt Inhibitor VIII ( 1 , 3-Dihydro-l- ( 1- (( 4- ( 6-phenyl-lH-imidazo [ 4 , 5- g] quinoxalin-7-yl) phenyl) methyl) -4-piperidinyl ) -2H-benzimidazol-2- one .

In other preferred embodiments, the cytotoxic anticancer compound is dacarbazine and the melanosome transport inhibitor is 7- hydroxystraurosporine; the cytotoxic anticancer compound is

vemurafenib and the melanosome transport inhibitor is 7- hydroxystraurosporine; the cytotoxic anticancer compound is

dacarbazine and the melanosome transport inhibitor is Akt Inhibitor VI II ( 1 , 3-Dihydro-l- ( 1- ( ( 4- ( 6-phenyl-lH-imidazo [4, 5-g] quinoxalin-7- yl) phenyl) methyl) -4-piperidinyl) -2H-benzimidazol-2-one; or the cytotoxic anticancer compound is vemurafenib and the melanosome transport inhibitor is Akt Inhibitor VIII ( 1 , 3-Dihydro-l- ( 1- (( 4- ( 6- phenyl-lH-imidazo [4, 5-g] quinoxalin-7-yl) phenyl ) methyl ) -4- piperidinyl ) -2H-benzimidazol-2-one ;

Brief Description of Figures

Figure 1 shows confocal microscopy assays for the localization of myosin Va (MyoVa) in SK-MEL-28 melanoma cells and the effect of ΙμΜ MTX. Co-localization with HMB45, melanophilin and Rab27a is shown.

Figure 2 shows time-courses for the fluorescence intensity

(arbitrary units) of MTX-FITC-containing vesicles inside SK-MEL-28 cells_treated with 20 μΜ MTX-FITC (1 h) . After extensive washing, the cells were imaged at 1 frame/min for 1 h in the presence of 20 μΜ unlabelled MTX by FLIM. Fluorescent MTX-FITC-containing exosomes were visible during the recording time.

Figure 3 shows a Pearson's coefficient of confocal images from immunohistochemistry (IHC) to estimate the degree of co-localization of the different melanosome markers with MyoVa. The Pearson^'s overlap coefficients are represented as the average of ten

individual cells. *P < 0.05 with respect to untreated control cells. The immunohistochemistry (IHC) analyzed the localization of MyoVa in untreated SK-MEL-28 cells and in cells treated for 10 h with 1 μΜ MTX.

Figure 4 shows that MyoVa silencing modifies MTX-FITC distribution in SKMEL-28 melanoma cells. Cells were imaged after 1 h treatment with 10 μΜ MTX-FITC.

Figure 5 shows that MyoVa siRNA sensitizes SK-MEL-28 to MTX-induced toxicity. Apoptosis was determined after 72 h treatments (*P < 0.05, respect to siCN-treated cells) (Upper panel) . The effective

silencing of MyoVa was tested by western blot relative to β-actin expression (Lower panel) .

Figure 6 shows the effect of MyoVa silencing on melanosome fraction exportation (A) and on the susceptibility of Sk-MEL-28 melanoma cells to MTX. siContro1 and siMyoVa-trans feeted cells were treated with increasing doses of MTX for 48 h (*P < 0.05) . The effective silencing of MyoVa was tested by western blot

Figure 7A shows relative MyoVa mRNA expression in SK-MEL-28 and B16/F10 melanoma cells after their exposure to 1 μΜ MTX as

determined by real time PGR. The estimated levels of mRNA relative to levels of 13-actin mRNA in MTX-treated cells were calculated and then compared to their expression levels in untreated cells (1- fold) . The changes observed after MTX treatment were not

statistically significant. Figure 3B shows the time-dependent effect of MTX treatment (1 μΜ) on the expression of MyoVa as assayed by western blot. The graph on the bottom shows the results of the densitometry quantification for the expression of MyoVa in melanoma cells after MTX treatment. Protein levels were normalized to --actin protein levels and to their respective untreated controls (1-fold) . Figure 3C shows that MTX induces phosphorylation of MyoVa at Serl650 in melanoma cells. MALDI-TOF mass spectra of tryptic digests of immuno-precipitated MyoVa are shown. Peptides were analysed in untreated SK-MEL-28 cells (control) or treated for 10 h with 1 μΜ MTX, The characteristics of peptides involved in post-translational modifications of MyoVa (phosphorylation = P) , as well as their measured and theoretical m/ z are shown in Table 1. The relative intensity of unphosphorylated (m/z 1841.91) and phosphorylated (m/z 1921.88) peptides in MyoVa-trypsin digested samples are also shown in Table 1. Peptides were analyzed in untreated SK-MEL-28 cells (CN) or treated for 10 h with 1 μΜ MTX (*P < 0.05) . Intensities were normalized with respect to an internal matrix control.

Figure 8 shows the participation of Akt and protein phosphatase 2A (PP2A) in the MTX-induced phosphorylation of MyoVa. Figure 8A shows, in the left panels, fluorescent western blots show the simultaneous detection of unphosphorylated and phosphorylated Akt present in SK- MEL-28 control cells and cells treated with 1 μΜ MTX for the indicated times. The right panels of Figure 8A show the time dependent effect of MTX (1 μΜ) on the methylation status of the catalytic C subunit of PP2A (Leu309) in SK-MEL-28 cells as assayed by immunoblotting with anti-PP2A-C and methyl-specific anti-PP2A-C antibodies. Figure 8B shows the immunohistochemistry was used to analyze the localization of Aktl/2 and PP2A in control SK-MEL-28 cells and cells treated for 30 min with 1 μΜ MTX. Cells were stained with anti-Aktl/2 (red) and anti-PP2A-C (green) antibodies. Merged images are shown.

Figure 9 shows the effects of 1 μΜ MTX on Akt phosphorylation in melanoma cells. Figure 10 shows the effect of IAKT (10 h) on the MTX-induced phosphorylation of MyoVa (*P < 0.05) .

Figure 11 shows the density gradients of enriched melanosomal fractions obtained from untreated SK-MEL-28 cells and cells treated (5 h) with 10μΜ MTX and/or 10μΜ IAKT (Left panel) and electron micrographs of the treated cells (Right panel) .

Figure 12 shows histograms representing the effects of MTX/IAKT treatment (2 days) on SK-MEL-28 (*P < 0.05, respect to IAKT-treated cells) . Increasing IAKT concentrations were analyzed in the absence or presence of 1 μΜ MTX. The images show the effects of MTX and/or IAKT treatments on cell morphology. Figure 13 shows apoptosis in siControl (siCN)-and siAkt- transfected cells treated with 1 μΜ MTX (48 h) (*P < 0.05) . Akt silencing was examined by western blot. Figure 14 shows that MTX induces demethylation of the catalytic PP2A C subunit and inhibits PP2A activity in melanoma cells. The

histograms represent the time (at 1 μΜ MTX) and dose (at 60 min) effects of MTX on PP2A activity. *P < 0.05 compared with untreated controls .

Figure 15 shows Pearson' s coefficients of confocal images of SK-MEL- 28 melanoma cells treated during 24 h with MTX or cantharidin (both at 1 μΜ) to estimate the degree of co-localization of MyoVa with HMB45 (histograms) . The treatment promoted the co-localization of MyoVa with the melanosome stage II marker, HMB45. The Pearson^'s overlap coefficients are represented as the average of ten

individual cells. *P< 0.05 with respect to untreated control cells.

Figure 16 shows a western blot demonstrating the effects of UCN-01 on MTX-induced Akt2 phosphorylation. SK-MEL-28 cells were treated (1 h) with 1 M MTX or 1 M MTX plus 50 nM UCN-01.

Figure 17A shows UCN-01 inhibits MTX-induced phosphorylation of MyoVa at Serl 650. The relative intensity of unphosphorylated (m/z 1841.91) and phosphorylated (m/z 1921.88) peptides in MyoVa-trypsin digested samples are shown. Peptides were analyzed in lOh MTX- treated SK-MEL-28 cells (MTX) or treated for 10 h with a combination of 1 μΜ MTX and 50 nM UCN-01 (P < 0.05) . Intensities were normalized with respect to an internal matrix control. Figure 17B shows localization of MyoVa in SK-MEL-28 cells after 10 h treatment with 1 μΜ MTX or with the combination MTX/UCN-01 (10 μΜ/50 nM,

respectively) . The pictures show representative experiments repeated three times with similar results . Figure 18 shows the effect of UCN-01 on the MTX-induced

phosphorylation of MyoVa.

Figure 19 shows the sub-second frame-rate confocal microscopy examining the movements of individual MTX-FITC-containing vesicles. SK-MEL-28 cells were treated with 20 μΜ MTX-FITC or 20 μΜ MTX-FITC plus 50 nM UCN-01 for 1 h. After extensive washing, the cells were imaged at 2 frames/s for 60s in the presence of unlabelled MTX or MTX/UCN-01. Images at time zero (Left) and 60 s (Middle) .

Figure 20 shows time-courses of the fluorescence intensity of MTX- FITC-containing vesicles inside SK-MEL-28 cells treated with 20 μΜ MTX-FITC and 50 nM UCN-01 (lh) and analysed by FLIM. After extensive washing, the cells were imaged at 1 frame/min for 1 h in the presence of 20 μΜ unlabelled MTX and 50 nM UCN-01. Figure 21 shows subcellular localization of FITC-MTX in SK-MEL-28 melanoma cells after 5 h of drug exposure. Cells were treated with FITC-MTX 20 μΜ in the absence (left panels} or the presence (right panels) of 50 nM UCN-01. Figure 22 shows the determination of intracellular HRP activity in melanoma cells treated with HRP-MTX in the absence or the presence of 50 nM UCN-01. The right panels show the relative MTX levels in melanoma cells, based on HRP activity, assuming a factor of 1 for the levels of MTX in cells treated exclusively with 1 μΜ.

Figure 23 shows a combined treatment with MTX and UCN-01 inhibits melanoma growth and induces apoptosis in cultures of human and mouse cells. The dose-dependent effects (left panel) after 3 days of treatment with UCN-01 were analysed using increasing concentrations of UCN-01 in the absence or the presence of 1 μΜ MTX. Right panel, effect of p53 status on the induction of apoptosis by MTX and/or UCN-01 in melanoma cells after 3 days of treatment. The effect of p53 silencing in G361 cells on the induction of apoptosis during MTX and/or UCN-01 treatment was evaluated.

Figure 24 shows that MTX/UCN-01 treatment induces E2Fl-mediated apoptosis in melanoma. MTX (1 μΜ) and/or UCN-01 (50 nM) were used. In a synergy test for melanoma cell lines, MTX and UCN-01 were combined in 6 ^χ 6 matrices where the concentration of one drug was increased along each axis. Apoptosis (4 days post-treatment) was obtained in triplicate in two-independent experiments. Differences in apoptosis in MTX/UCN-01-treated cells were significant with respect to individual treatments for each drug concentration (P < 0.05) . MTX/UCN-01 combination showed clear synergistic behaviour.

Figure 25 shows dNTP quantification in melanoma cells. The data (left panel) were used to determine the total amount of each dNTP at each time point. The right panel represents dTTP levels in melanoma cells subjected to the indicated treatments (24 h) . *P < 0.05 with respect to untreated controls; **P = 0.001; ***Not statistically significant with respect to the untreated controls.

Figure 26 shows SK-MEL-28 cells treated with MTX (ΙμΜ) and/or UCN-01 (50nM) for 10 h and examined for yH2AX nuclear foci (middle) . Nuclei were counterstained with DAPI (left) . Figure 27 shows western blot analysis of E2Fl-proapoptotic related proteins. SK-MEL-28 were treated with MTX (ΙμΜ) and UCN-01 (50nM) at indicated times.

Figure 28 shows qRT-PCR of TAp73 and Apafl mRNA (Left panel) . SK- MEL-28 cells were treated with the indicated treatments (24 h) . mRNA levels are presented relative to beta-actin mRNA and compared with their expression levels in untreated cells (1-fold) . Right panel shows E2F1 occupancy on the TAp73 promoter of SKMEL-28 subjected to the indicated treatments (24 h) .

Figure 29 shows that MTX and UCN-01 combination therapy is effective in vivo. Treatment: 1 and 0.5 mg/kg/day for MTX and UCN-01, respectively. Left upper panel shows tumour area in C57BL/6 mice subcutaneously injected with B16/F10 cells. Means are representative of three independent experiments. Differences after 21 days of

MTX/UCN-01 treatments were statistically significant (P < 0.002) with respect to control mice or those subjected to individual MTX and UCN-01 treatments. Left-lower panel shows representative luciferase imaging of the control and MTX/UCN-01-treated mice 12 days after the intrasplenic injection of tumor cells. NS, not significant. Right panel shows the effect of MTX/UCN-01 on B16/F10 primary splenic tumors. Xenograft tumors treated (14-days) with DMSO (vehicle) or MTX/UCN-01. Vehicle-treated tumours showed normal splenic tissue (S) and tumour areas (T) but no discernible necrosis (N) . MTX/UCN-01-treated tumours showed haemorrhagic (H) necrosis. Bar = 40 μιτι

Figure 30 shows the results of bioluminescent liver imaging 14 days after the intrasplenic injection of B16-F10-luc2 cells from

untreated and MTX/UCN-01-treated mice (see table 2) . Figure 31 shows histograms representing the copies of tyrosinase mRNA for every 1 χ 103 copies of β-actin ± SD. *P = 0.001 between MTX/UCN-01 treated mice and untreated controls (vehicle) . Livers form non-melanoma cell inoculated mice (NT) were used as a control.

Figure 32 shows a proposed mechanism for MTX-induced melanosome transport in melanoma. PP2A is a trimeric serine/threonine

phosphatase that contains the regulatory subunit B, which is recruited by a C-A dimer composed of the catalytic subunit C (PP2AC) and structural subunit A. Recruitment occurs when C is carboxyl- methylated on the terminal Leu309, resulting in the assembly of the active PP2A trimer. Reversible PP2A methylation is catalysed by two conserved PP2A-specific enzymes: leucine carboxyl methyltransferase

(LCMT-1) and PP2A methylesterase (PME-1) . Because LCMT1 is a specific SAM dependent methyltransferase, the treatment of melanoma cells with MTX may result in the inhibition of the PP2A assembly. The left branch, corresponding with KIF3 activation, is hypothetical and inspired by the insulin-stimulated GLUT4 vesicle translocation process. If an atypical PKC does activate KIF3 in melanoma, UCN-01 treatment would result in the disruption of MTX-stimulated

microtubule and actin-dependent melanosome transport because PDKl is positioned upstream of the Akt and PKC pathways [Hodgkinson et al

(2002)]. Arrows represent MTX activated pathways and indicate the site of action for the assayed drugs UCN-01 and IAKT .

Detailed Description of Invention

In various aspects, this invention relates to the treatment of cancer by administering a cytotoxic anti-cancer compound and a melanosome transport inhibitor to an individual in need thereof.

The cancer may be resi stant to the cytotoxic anti-cancer compound i.e. cells of the caneer suffer reduced cell death compared to non resistant cancer cells in the presence of the same concentration o compound .

In some embodiments, cancer cells in the individual may sequester and/or export the cytotoxic anti-cancer compound before the

intracellular concentration becomes cytotoxic, for example using myosin Va (MyoVa) mediated transport mechanisms, such as MyoVa- mediated melanosome transport. Myosin Va (MyoVa) is a dimeric molecular motor that moves processively on actin by converting the energy released by ATP hydrolysis into mechanical force or movement (Mehta et al . , 1999; Veigel et al . , 2002; Lambert et al 1988b) . MyoVa is recruited to the melanosome membrane of cells by melanophilin (Wu et al . , 2001, 2002a; Fukuda et al . , 2002; Hume et al . , 2002; Strom et al . , 2002; Westbroek et al., 2003) . Human MyoVa (Gene ID4644) has the

reference sequence NP 000250.3 GI : 215982791. The sequences of other MyoVa isoforms in humans and other species are available on public databases .

In preferred embodiments, the cancer is melanoma.

Melanoma is a malignant neoplasm of melanocytes in the skin.

Melanoma which may be treated as described herein may include primary melanoma, for example, superficial spreading melanoma, nodular melanoma, acral lentiginous melanoma or lentigo maligna (melanoma) ; and metastatic melanoma, for example melanoma displaying local or distant metastases. The melanoma may be at any stage. For example, the melanoma may be stage 0, I, II, III or IV melanoma as described in Balch C et al (2001) . J Clin Oncol 19 (16) : 3635-48.

In some embodiments, melanoma cells in the individual may have activated MAPK signalling and may for example have mutations in BRAF (v-raf murine sarcoma viral oncogene homolog Bl; Gene ID673; Ref sequence NP_004324.2 GI : 33188459) .

In addition to melanoma, the compounds and combinations described may also be useful in the treatment of other forms of cancer, for example bladder cancer, breast cancer, uterus cancer, ovary cancer, prostate cancer, lung cancer, colorectal cancer, cervical cancer, liver cancer, head and neck cancer, oesophageal cancer, pancreas cancer, renal cancer, stomach cancer, cerebral cancer or non- melanoma skin cancer.

In some embodiments, the cancer may be a metastatic cancer.

In some preferred embodiments, the melanoma or other cancer may be characterised by the expression of one or more MyoVa exon F isoforms (i.e. MyoVa isoforms encoded by sp ice variants which contain exon F) . A cytotoxic anti-cancer compound is a compound which induces apoptosis or cell-death in cancer cells, but has a reduced effect or no effect on non-cancer cells at the same concentration.

A large number of cytotoxic anti-cancer compounds are known in the art. Suitable cytotoxic anti-cancer compounds may activate or stimulate cellular export systems, such as MyoVa and/or

MyoVa-mediated melanosome transport, in cancer cells. For example, the cytotoxic anti-cancer compound may stimulate or activate the phosphorylation of Ser¹⁶⁵⁰ and/or Ser¹⁸¹² of MyoVa .

In some embodiments, the cytotoxic anticancer compound may activate the Akt2 signalling pathway, for example by stimulating the

phosphorylation of Thr³⁰⁸ and/or Ser⁴⁷³ of Akt2, for example by increasing the methylation or inhibiting the demethylation of PP2A.

Suitable cytotoxic anti-cancer compounds may include antifolate compounds .

Antifolate compounds possess anti-folate activity and impair folic acid metabolism in a cell. An antifolate compound may inhibit the activity of dihydrofolate reductase (DHFR; 5,6,7,? -tetrahydrofolate

NADP+ oxidoreductase , EC 1 .5.1.3) .

Antifolate compounds inhibit the production of purine and pyrimidine precursors and are generally cytotoxic during the S-phase of the cell cycle, when DNA replication occurs. Because antifolate

compounds target nucleic acid synthesis, cells which are rapidly dividing, such as cancer cells, are generally more sensitive to antifolate compounds than other cell types.

Antifolate compounds are well-known in the art for the treatment cancer and other conditions and include methotrexate (MTX) ,

aminopterine (AMT) , trimetrexate (TMQ) fluorouracil , lometrexol (LMTX) , pemetrexed, raltitrexed and prelatrexate .

Suitable assays to determine the inhibition of dihydrofolate reductase (DHFR) by a compound are well-known in the art.

In some preferred embodiments, the antifolate compound is

methotrexate (MTX) . Other suitable cytotoxic anti-cancer compounds may include DNA cross-linking and/or DNA intercalating agents, including Pt

containing compounds, such as cisplatin, carboplatin, and

oxaliplatin; adriamycin and anthracylines , such as doxorubicin, microtubule-targeting drugs, such as docetaxel and vincristine, alkylating agents, such as dacarbazine (4- [ (IE) -3, 3-Dimethyl-l- triazen-l-yl] -lH-imidazole-5-carboxamide) , and BRAF inhibitors such as vemurafenib (N-(3-{ [ 5- ( 4-Chlorophenyl ) -lH-pyrrolo [ 2 , 3-b] pyridin- 3-yl ] carbonyl } -2 , 4-difluorophenyl ) -1 -propanesulfonamide) or

dabrafenib (N-{3- [5- (2-aminopyrimidin-4-yl ) -2- tert-butyl-1 , 3- thiazol-4-yl] -2- fluorophenyl } -2 , 6-difluorobenzenesulfonamide ) .

Cytotoxic anti-cancer compounds may possess intrinsic activity (drugs) or may be prodrugs of active compounds which may themselves exhibit little or no intrinsic activity.

Melanosome transport inhibitors inhibit, block or abolish

melanosome-mediated export from cancer cells, preferably

MyoVa-mediated melanosome transport.

A Melanosome transport inhibitor may target one or more of the Akt, PKD/PKCX, PI3K/PKCX and Chkl signalling pathways.

Melanosome transport inhibitors may include Akt signalling pathway inhibitors and PKCX signalling pathway inhibitors.

Other suitable melanosome transport inhibitors may be identified using standard techniques. For example, the export of the melanosome fraction may be determined in melanoma cells, for example using sucrose gradients and zonal centrifugation . Reduction, inhibition or abolition of melanosome export may be indicative of a melanosome transport inhibitor.

An Akt signalling pathway inhibitor reduces, blocks or inhibits signalling through the Akt pathway. This inhibits the activation of MyoVa, for example by inhibiting the Akt-mediated phosphorylation of Ser¹⁶⁵⁰ and/or Ser¹⁸¹² of MyoVa.

Akt (also known as protein kinase B PKB) is a serine/threonine kinase which is involved in a range of cellular processes. Akt may include Aktl (Gene ID 207; NP_005154.2 GI : 62241011) or Akt 2 (Gene ID 208; NP 001617.1 GI : 4502023 or NP 001229956.1 GI : 339895853) . Preferably, the Akt signalling pathway inhibitor is an Akt2

signalling pathway inhibitor.

An Akt signalling pathway inhibitor may inhibit Akt or another member of the pathway. For example, an Akt signalling pathway inhibitor may inhibit Akt or an activator or co-factor of Akt; or an Akt signalling pathway inhibitor may activate an inhibitor or repressor of Akt, such as protein phosphatase 2A (PP2A) . The Akt signalling pathway is well characterised in the art (see for example, Sato et al., 2002; Kondapaka et al . , 2004) and members of the pathway include Akt (also termed PKB) , phosphoinositide 3 kinase (PI3K) and PDK-1. A suitable Akt signalling pathway inhibitor may inhibit the

activation or nuclear translocation of Akt, for example by

inhibiting phosphorylation at Thr³⁰⁸ and/or Ser⁴⁷³ of Akt, preferably Akt2. For example, the Akt signalling pathway inhibitor may be a PDK1 inhibitor. In some embodiments, an Akt signalling pathway inhibitor may inhibit the inactivation of protein phosphatase 2A (PP2A) , for example by blocking demethylation of the catalytic subunit of PP2A .

Suitable assays for the identification of Akt signalling pathway inhibitors are well known in the art and described herein. For example, the export of the melanosome fraction may be determined in melanoma cells in the presence and absence of a candidate Akt signalling pathway inhibitor, for example using sucrose gradients and zonal centrifugation . Reduction, inhibition or abolition of melanosome export may be indicative of an Akt signalling pathway inhibitor. A suitable Akt signalling pathway inhibitor may for example, inhibit or reduce phosphorylation of Ser¹⁶⁵⁰ or Ser¹⁸¹² of MyoVa . Activation of Akt may be determined in melanoma cells by determining the phosphorylation of Akt in the presence and absence of a

candidate Akt signalling pathway inhibitor. Phosphorylation of Akt, for example at Thr308 and/or Ser473, may be determined by routine techniques, including western blot, confocal microscopy MALDI-TOFMS (Bellacosa A, et al . (2005) Adv Cancer Res. 2005; 94: 29-86) .

Reduction, inhibition or abolition of phosphorylation of Akt, for example at Thr³⁰⁸ and/or Ser^473, may be indicative of an Akt signalling pathway inhibitor.

In some embodiments, the Akt signalling pathway inhibitor may inhibit PDK1 , which mediates the activation of Akt2 by

phosphorylation of Thr³⁰⁸ and/or Ser⁴⁷³ residues.

PDK1 (also known as pyruvate dehydrogenase kinase isoform 1) is involved in a range of cellular processes and has the reference sequence Gene ID 5163; NP_002601.1 GI : 4505689.

Suitable Akt pathway inhibitors include 7-hydroxystraurosporine (UCN-01), triciribine, honokiol and perifosine (1, 1- dimethylpiperidinium-4-yl octadecyl phosphate) and Akt Inhibitor VI II ( 1 , 3-Dihydro-l- ( 1- ( ( 4- ( 6-phenyl-lH-imidazo [4, 5-g] quinoxalin-7- yl) phenyl) methyl) -4-piperidinyl) -2H-benzimidazol-2-one ,

In some preferred embodiments, the Akt pathway inhibitor is 7- hydroxystraurosporine (UCN-01) . 7-hydroxystraurosporine has been the subject of multiple preclinical trials and is well-known in the art. 7-hydroxystraurosporine is shown herein to display greater

efficiency than other Akt pathway inhibitors, for example when combined with MTX, and induced consistent apoptosis in melanoma cells at doses as low as 10 nM.

A PKCX signalling pathway inhibitor reduces, blocks or inhibits signalling through the PKCX/KIF3 pathway. This inhibits the drug induced export of melanocytes. PKCX is a serine/threonine kinase which is involved in a range of cellular processes. Human PKCX (Gene ID 5582) has the reference sequence NP_002730.1 GI : 13384594.

A PKCX signalling pathway inhibitor may inhibit PKCX or another member of the pathway. For example, a PKCX signalling pathway inhibitor may inhibit PKCX or an activator or co-factor of PKCX; or an PKCX signalling pathway inhibitor may activate an inhibitor or repressor of PKCX. The PKCX signalling pathway is well-characterised in the art and members of the pathway include PKCX and KIF3. Suitable PKCX pathway inhibitors include sodium aurothiomalate .

Methods of the invention may be useful in reducing the resistance of a cancer cell to a cytotoxic anti-cancer compound, such as an antifolate compound. For example, a method of increasing the sensitivity of a cancer cell to a cytotoxic anti-cancer compound, or reducing the export of a cytotoxic anti-cancer compound from a cancer cell, may comprise treating or contacting the cancer cell with a melanosome transport inhibitor, such as an Akt signalling pathway inhibitor as described herein.

The cancer cell may be treated or contacted in vitro or in vivo.

Another aspect of the invention provides a method of reducing the melanosome mediated export of a cytotoxic anti-cancer compound from a cancer cell comprising contacting the cell with a MyoVa melanosome transport inhibitor, as described above.

The cancer cell may be treated with the melanosome transport inhibitor before, simultaneous with or after treatment with the cytotoxic anti-cancer compound.

In some preferred embodiments, the cancer cell is a melanoma cell.

An individual suitable for treatment as described above may be a mammal, such as a rodent (e.g. a guinea pig, a hamster, a rat, a mouse), murine (e.g. a mouse), canine (e.g. a dog), feline (e.g. a cat), equine (e.g. a horse), a primate, simian (e.g. a monkey or ape), a monkey (e.g. marmoset, baboon), an ape (e.g. gorilla, chimpanzee, orang-utan, gibbon), or a human. In some preferred embodiments, the individual may be human or equine.

Combinations of cytotoxic anti-cancer compounds, such as MTX, and melanosome transport inhibitors, such as 7-hydroxystraurosporine as described herein, may be the sole therapeutic agents which are administered to the individual or they may be administered in combination with one or more additional active compounds .

In some embodiments, combinations of cytotoxic anti-cancer compounds and melanosome transport inhibitors may be administered along with compounds that inhibit the methionine cycle or disrupt the adenosine metabolism. Suitable compounds include S-adenosylmethionine (SAM) , S-adenosylhomocysteine (SAH) , 5 ' -methylthioadenosine , 5-azacytidine , 5-aza-2 ^' -deoxycytidinea and 3-deazaneplanocin and ornithine

decarboxylase inhibitors, such as difluoromethylornithine .

In some embodiments, combinations of cytotoxic anti-cancer compounds and melanosome transport inhibitors may be administered with inhibitors of the equilibrate nucleoside transporters and/or inhibitors of adenosine deaminase, such as dipyridamole, pyridamole propentofylline, p-nitrobenzylthioinosine, l-deaza-erythro-9- (2- hydroxy-3-nonyl ) adenine, 4-amino-2- (2-hydroxy-l-decyl) pyrazole [3,4- d] pyrimidine , or-deazaadenosine .

Methods of measuring the effect of a combination of compounds as described above on melanoma or other cancer cells are well-known in the art and are exemplified herein. For example, the effect of combinations of cytotoxic anti-cancer compounds and melanosome transport inhibitors; antifolate compounds and Akt pathway

inhibitors; or cytotoxic anti-cancer compounds and 7- hydroxystraurosporine on the cell death in melanoma or other cancer cells may be determined by contacting a population of melanoma or other cancer cells with the combination, preferably in the form of a pharmaceutically acceptable composition ( s ) , and determining the amount of cell death in the population. An increase in cell death in the cancer cell population treated with the combination, relative to untreated cancer cells or cancer cells treated with either one of the compounds individually, is indicative that the combination has a cytotoxic effect on the cancer cells. Suitable methods may be practised in vitro or in vivo.

The term "treatment" as used herein in the context of treating a cancer condition, such as melanoma, pertains generally to treatment and therapy, whether of a human or an animal (e.g. in veterinary applications), in which some desired therapeutic effect is achieved, for example, the inhibition or delay of the progress of the

condition, and includes a reduction in the rate of progress, a halt in the rate of progress, amelioration of the condition, and cure of the condition. Treatment as a prophylactic measure (i.e.

prophylaxis) is also included. For example, an individual

susceptible to or at risk of the occurrence or re-occurrence of melanoma may be treated as described herein. Such treatment may prevent or delay the occurrence or re-occurrence of melanoma in the individual . The compounds described herein may be administered in

therapeutically-effective amounts .

The term "therapeutically-effective amount" as used herein, pertains to that amount of an active compound, or a combination, material, composition or dosage form comprising an active compound, which is effective for producing some desired therapeutic effect,

commensurate with a reasonable benefit/risk ratio.

While it is possible for the compounds in a combination described above to be administered alone, it is preferable to present the compounds in the same or separate pharmaceutical compositions (e.g. formulations) comprising the compound(s) as defined above, together with one or more pharmaceutically acceptable carriers, adjuvants, excipients, diluents, fillers, buffers, stabilisers, preservatives, lubricants, or other materials well known to those skilled in the art. Optionally, other therapeutic or prophylactic agents may be included .

For example, methionine cycle inhibitors; adenosine metabolism inhibitors; equilibrate nucleoside transporters inhibitors and/or adenosine deaminase inhibitors as described above may be included in the pharmaceutical compositions.

Thus, the present invention further provides pharmaceutical

compositions, as defined above, and methods of making a

pharmaceutical composition comprising admixing a cytotoxic anticancer compound, such as an antifolate compound, and a melanosome transport inhibitor, for example an Akt pathway inhibitor, such as 7-hydroxystraurosporine, together with one or more pharmaceutically acceptable carriers, excipients, buffers, adjuvants, stabilisers, or other materials, as described herein.

The term "pharmaceutically acceptable" as used herein pertains to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of a subject (e.g. human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, excipients, etc. can be found in standard pharmaceutical texts, for example, Remington's Pharmaceutical

Sciences, 18th edition, Mack Publishing Company, Easton, Pa., 1990. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of

pharmacy. Such methods include the step of bringing into

association the active compound with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active compound with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.

Formulations may be in the form of liquids, solutions, suspensions, emulsions, elixirs, syrups, tablets, losenges, granules, powders, capsules, cachets, pills, ampoules, suppositories, pessaries, ointments, gels, pastes, creams, sprays, mists, foams, lotions, oils, boluses, electuaries, or aerosols. The active compound or pharmaceutical composition comprising the active compound may be administered to a subject by any convenient route of administration, whether systemically/ peripherally or at the site of desired action, including but not limited to, oral (e.g. by ingestion); topical (including e.g. transdermal, intranasal, ocular, buccal, and sublingual); pulmonary (e.g. by inhalation or insufflation therapy using, e.g. an aerosol, e.g. through mouth or nose); rectal; vaginal; parenteral, for example, by injection, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal,

intracapsular, subcapsular, intraorbital, intraperitoneal,

intratracheal, subcuticular, intraarticular, subarachnoid, and intrasternal ; by implant of a depot, for example, subcutaneously or intramuscularly . Formulations suitable for oral administration (e.g. by ingestion) may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active compound; as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion; as a bolus; as an electuary; or as a paste. A tablet may be made by conventional means, e.g., compression or moulding, optionally with one or more accessory ingredients.

Compressed tablets may be prepared by compressing in a suitable machine the active compound in a free-flowing form such as a powder or granules, optionally mixed with one or more binders (e.g.

povidone, gelatin, acacia, sorbitol, tragacanth, hydroxypropylmethyl cellulose); fillers or diluents (e.g. lactose, microcrystalline cellulose, calcium hydrogen phosphate); lubricants (e.g. magnesium stearate, talc, silica); disintegrants (e.g. sodium starch

glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose); surface-active or dispersing or wetting agents (e.g. sodium lauryl sulfate); and preservatives (e.g. methyl

p-hydroxybenzoate, propyl p-hydroxybenzoate, sorbic acid) . Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active compound therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile. Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach.

Formulations suitable for topical administration (e.g. transdermal, intranasal, ocular, buccal, and sublingual) may be formulated as an ointment, cream, suspension, lotion, powder, solution, past, gel, spray, aerosol, or oil. Alternatively, a formulation may comprise a patch or a dressing such as a bandage or adhesive plaster

impregnated with active compounds and optionally one or more excipients or diluents. table for topical

comprising the ac

ucrose and acacia

ctive compound in

glycerin, or sucrose and acacia; and mouthwashes comprising the active compound in a suitable liquid carrier.

Formulations suitable for topical administration to the eye also include eye drops wherein the active compound is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the active compound. Formulations suitable for nasal administration, wherein the carrier is a solid, include a coarse powder having a particle size, for example, in the range of about 20 to about 500 microns which is administered in the manner in which snuff is taken, i.e. by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. Suitable formulations wherein the carrier is a liquid for administration as, for example, nasal spray, nasal drops, or by aerosol administration by nebuliser, include aqueous or oily solutions of the active compound.

Formulations suitable for administration by inhalation include those presented as an aerosol spray from a pressurised pack, with the use of a suitable propellant, such as dichlorodifluoromethane,

trichlorofluoromethane , dichoro-tetrafluoroethane, carbon dioxide, or other suitable gases.

Formulations suitable for topical administration via the skin include ointments, creams, and emulsions. When formulated in an ointment, the active compound may optionally be employed with either a paraffinic or a water-miscible ointment base. Alternatively, the active compounds may be formulated in a cream with an oil-in-water cream base. If desired, the aqueous phase of the cream base may include, for example, at least about 30% w/w of a polyhydric alcohol, i.e., an alcohol having two or more hydroxyl groups such as propylene glycol, butane-1, 3-diol, mannitol, sorbitol, glycerol and polyethylene glycol and mixtures thereof. The topical formulations may desirably include a compound which enhances absorption or penetration of the active compound through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethylsulfoxide and related analogues.

When formulated as a topical emulsion, the oily phase may optionally comprise merely an emulsifier (otherwise known as an emulgent) , or it may comprises a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil. Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier which acts as a stabiliser. It is also preferred to include both oil and a fat. Together, the emulsifier (s) with or without stabiliser ( s ) make up the so-called emulsifying wax, and the wax together with the oil and/or fat make up the so-called emulsifying ointment base which forms the oily dispersed phase of the cream formulations. Suitable emulgents and emulsion stabilisers include Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl monostearate and sodium lauryl sulphate. The choice of suitable oils or fats for the formulation is based on achieving the desired cosmetic properties; since the solubility of the active compound in most oils likely to be used in pharmaceutical emulsion formulations may be very low. Thus the cream should preferably be a non-greasy, non-staining and washable product with suitable consistency to avoid leakage from tubes or other containers. Straight or branched chain, mono- or dibasic alkyl esters such as di-isoadipate , isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl

myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2- ethylhexyl palmitate or a blend of branched chain esters known as Crodamol CAP may be used, the last three being preferred esters. These may be used alone or in combination depending on the

properties required.

Alternatively, high melting point lipids such as white soft paraffin and/or liquid paraffin or other mineral oils can be used.

Formulations suitable for rectal administration may be presented as a suppository with a suitable base comprising, for example, cocoa butter or a salicylate. Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray

formulations containing in addition to the active compound, such carriers as are known in the art to be appropriate. Formulations suitable for parenteral administration (e.g. by injection, including cutaneous, subcutaneous, intramuscular, intravenous and intradermal) , include aqueous and non-aqueous isotonic, pyrogen-free, sterile injection solutions which may contain anti-oxidants , buffers, preservatives, stabilisers,

bacteriostats , and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and nonaqueous sterile suspensions which may include suspending agents and thickening agents, and liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more organs. Examples of suitable isotonic vehicles for use in such formulations include Sodium Chloride Injection, Ringer's

Solution, or Lactated Ringer's Injection. Typically, the concentration of the active compound in the solution is from about 1 ng/ml to about 10 g/ml, for example from about 10 ng/ml to about 1 g/ml. The formulations may be presented in unit-dose or multi- dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets. Formulations may be in the form of liposomes or other microparticulate systems which are designed to target the active compound to blood components or one or more organs.

It will be appreciated that appropriate dosages of the active compounds, and compositions comprising the active compounds, can vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects of the treatments of the present invention. The selected dosage level will depend on a variety of factors including, but not limited to, the activity of the particular compound, the route of administration, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds, and/or materials used in combination, and the age, sex, weight, condition, general health, and prior medical history of the patient. The amount of compound and route of administration will ultimately be at the discretion of the physician, although generally the dosage will be to achieve local concentrations at the site of action which achieve the desired effect without causing substantial harmful or deleterious side- effects .

For example, in some embodiments, 0.1 to 10 mg/kg/day, preferably 1 mg/kg/day of MTX, and 0.01 to 1 mg/kg/day, preferably 0.1 mg/kg/day of UCN-01, may be used to reduce melanoma tumours.

Administration in vivo can be effected in one dose, continuously or intermittently (e.g. in divided doses at appropriate intervals) throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician.

In general, a suitable dose of the active compound is in the range of about 100 ]ig to about 250 mg per kilogram body weight of the subject per day. Where the active compound is a salt, an ester, prodrug, or the like, the amount administered is calculated on the basis of the parent compound and so the actual weight to be used is increased proportionately.

Other aspects of the invention relate to methods of screening for compounds which reduce the export of a cytotoxic anti-cancer compound from a cancer cell or reduce drug resistance in a cancer cell .

A method may comprise; contacting a cancer cell with a cytotoxic anti-cancer compound in the presence and absence of a test compound and determining the activation of MyoVa in the cancer cell.

A decrease in activation of MyoVa in the presence relative to the absence of test compound is indicative that the compound is active in reducing the export of an anti-cancer compound from a cancer cell .

Activation of MyoVa may be determined by determining the

phosphorylation of Ser¹⁶⁵⁰ and/or Ser¹⁸¹² of MyoVa. An increase in phosphorylation is indicative of an increase in activation of MyoVa in the cancer cell .

In some preferred embodiments, the cancer cell is a melanoma cell.

Methods may be in vitro methods using isolated cells in culture, Test compound may be contacted with the cells by supplementing the buffer or culture medium with the test compound.

Methods may be in vivo methods using cells which are comprised in a non-human animal, for example a mammal, such as a mouse. Test compound may be contacted with the cells by administering the test compound to the non-human animal .

Compounds which may be screened using the methods described herein may be natural or synthetic chemical compounds used in drug screening programmes. Extracts of plants, microbes or other organisms which contain several characterised or uncharacterised components may also be used.

A method may, for example, comprise identifying the test compound as a compound which inhibits the activation of MyoVa by cytotoxic anticancer compound; and which may be useful in combination with the cytotoxic anti-cancer compound described herein in the treatment of melanoma or other cancers.

A test compound identified using one or more initial screens as having ability to inhibit the activation of MyoVa by cytotoxic anticancer compounds or other activity described above, may be assessed further using one or more secondary screens. A secondary screen may, for example, involve testing for inhibition of the Akt

signalling pathway, preferably the Akt2 signalling pathway, or for a biological function such as an effect on the proliferation of melanoma in combination with a cytotoxic anti-cancer compound described above.

Cytotoxic anti-cancer compounds are described above and include cisplatin and antifolates.

Other aspects of the invention relate to methods of screening for anti-cancer compounds whose cytoxicity may be increased by a melanosome transport inhibitor, for example an Akt pathway

inhibitor, such as UCN-1.

A method may comprise;

contacting a cancer cell with an cytotoxic anti-cancer compound and determining the activation of MyoVa in the cancer cell, wherein an increase in activation of MyoVa in the presence relative to the absence of the compound is indicative that the cytotoxicity of the compound may be increased by melanosome

transport inhibitor, for example an Akt pathway inhibitor, such as UCN-1.

Activation of MyoVa may be determined as described above.

MyoVa undergoes alternative splicing in its medial tail region through alternate usage of three exons named B, D, and F (Lambert et al (1998) Biochem Biophys Res Commun 252:329-333) . Exon F encodes the amino acid sequence that mediates the selective binding of MyoVa to melanosomes (Au JS et al (2002) Cell Motil Cytoskeleton 53(2), 89-102; Van Gele M, et al . (2008) J Invest Dermatol. 128(10), 2474- 2484) . Exon F encodes residues 1414-1438 of the human MyoVa isoform of SEQ ID NO: 2.

MyoVa exon F isoforms are MyoVa splice variants which comprise the amino acid sequence encoded by exon F of the MyoVa gene (SEQ ID NO: 1) and thus bind to melanosomes. Suitable MyoVa exon F isoforms include isoform 1, which comprise the sequences of SEQ ID NO: 2 (NP_000250.3 GI: 215982791) .

Suitable MyoVa isoforms lacking SEQ ID NO:l include isoform 2

(NP_001135967.1 GI : 215982794 )

The presence of MyoVa exon F isoforms or encoding nucleic acids in a cancer cell may be indicative of functional melanosome transport in the cell and resistance to cytotoxic anti-cancer compound. A cancer condition, such as melanoma, suitable for treatment as described above may therefore be a cancer condition characterised by expression of a MyoVa exon F isoform i.e. one or more cancer cells in the individual express MyoVa isoforms that are encoded by nucleic acids comprising exon F.

A method as described above may comprise identifying the presence of one or more MyoVa exon F isoforms (i.e. an MyoVa isoform comprising SEQ ID NO:l) or encoding nucleic acids in one or more cancer cells from a sample obtained from the individual having the cancer condition, such as melanoma.

The presence of a MyoVa exon F isoform or encoding nucleic acid in one or more cancer cells may be indicative that the individual is suitable for treatment using a combination of a cytotoxic anti- cancer compound and a melanosome transport inhibitor, as described herein .

If MyoVa Exon F isoform or encoding nucleic acid is identified in one or more cancer cells from a sample obtained from the individual, the individual may be treated using a combination of a cytotoxic anti-cancer compound and a melanosome transport inhibitor, as described herein. Conversely, the absence of MyoVa Exon F isoforms or encoding nucleic acids in a cancer cell may be indicative of non-functional or inactive melanosome transport in the cell and, thereby, sensitivity to cytotoxic anti-cancer compounds.

A cancer condition, such as melanoma, that is characterised by the absence of expression of MyoVa exon F isoforms i.e. one or more cancer cells in the individual do not express MyoVa isoforms that comprise SEQ ID NO : 1 , may be suitable for treatment as described above with cytotoxic anti-cancer compounds without the need for a melanosome transport inhibitor.

A method as described herein may comprise identifying the absence of MyoVa exon F isoforms (MyoVa isoforms that comprise SEQ ID NO:l) or encoding nucleic acids in one or more cancer cells from a sample obtained from the individual having the cancer condition, such as melanoma .

The absence of MyoVa exon F isoforms or encoding nucleic acids in one or more cancer cells may be indicative that the cancer in the individual is sensitive to a cytotoxic anti-cancer compound.

If MyoVa exon F isoforms or encoding nucleic acids are not expressed in one or more cancer cells from a sample obtained from the

individual, the individual may be treated using a cytotoxic anti- cancer compound.

Other aspects of the invention relate to the treatment of cancer, such as melanoma, in an individual, wherein the cancer is

characterised by the absence of expression of MyoVa exon F isoforms i.e. the cells of the cancer do not express MyoVa exon F isoforms. A cancer cell that does not express MyoVa exon F isoforms may express MyoVa isoforms that lack exon F (e.g. isoform 2) .

A method of treatment of a cancer in an individual may comprise; administering a cytotoxic anti-cancer compound to the

individual ,

wherein the cancer is characterised by the absence of

expression of MyoVa exon F isoforms. In some

as chara

isoforms

In other embodiments, the method may comprise identifying the absence of expression of MyoVa exon F isoforms in one or more cancer cells in a sample obtained from the individual.

Related aspects of the invention provide a cytotoxic anti-cancer compound for use in the above method of treatment of a cancer and the use of a cytotoxic anti-cancer compound in the manufacture of a medicament for use in a method of treatment of a cancer

characterised by the absence of expression of MyoVa exon F isoforms.

Preferably, the cancer is melanoma.

Suitable cytotoxic anti-cancer compounds are well known in the art and are described in detail above.

The presence or absence of MyoVa Exon F isoforms or encoding nucleic acids in a cancer cell may be determined using conventional

immunological or nucleic-acid based methods (Au JS et al (2002); Van Gele M, et al . (2008); Lambert et al (1998)).

Another aspect of the invention provides a method of selecting a treatment for an individual with a cancer condition, preferably melanoma, comprising;

providing a sample of one or more cancer cells obtained from the individual, and

determining the presence of expression of one or more MyoVa exon F isoforms in the cells; or,

determining the absence or expression of MyoVa exon F isoforms in the cells,

wherein the presence of expression is indicative that the individual is suitable for treatment with an cytotoxic anti-cancer compound in combination with a melanosome transport inhibitor and the absence of expression is indicative that the individual is suitable for treatment with an cytotoxic anti-cancer compound.

The method may comprise selecting the treatment from the determined presence or absence of expression; and/or administering the

treatment to the individual. Other aspects and embodiments of the invention provide the aspects and embodiments described above with the term "comprising" replaced by the term "consisting of" and the aspects and embodiments

described above with the term "comprising" replaced by the term "consisting essentially of".

It is to be understood that the application discloses all

combinations of any of the above aspects and embodiments described above with each other, unless the context demands otherwise.

Similarly, the application discloses all combinations of the preferred and/or optional features either singly or together with any of the other aspects, unless the context demands otherwise.

Modifications of the above embodiments, further embodiments and modifications thereof will be apparent to the skilled person on reading this disclosure, and as such, these are within the scope the present invention.

All documents and database entries mentioned in this specification are incorporated herein by reference in their entirety.

The protein symbols and names set out herein are the unique official HUGO Gene Nomenclature Committee (HGNC) symbols and names that have been assigned to that protein (see Gray KA et al Nucleic Acids Res. 2013 Jan 1; 41 (Dl ) : D545-52 ; and the HGNC Database, HUGO Gene

Nomenclature Committee (HGNC) , EMBL Outstation - Hinxton, European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridgeshire, CB10 1SD, UK) .

"and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example "A and/or B" is to be taken as specific

disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described. Thus, the features set out above are disclosed in all combinations and permutations . Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the figures and tables described herein. 1_. Experiments

1.1 Reagents and Antibodies

MTX, UCN-01, cantharidin, and IAKT (Akt inhibitor VIII

trifluoroacetate salt hydrate) were obtained from Sigma-Aldrich

(Madrid, Spain) . Antibodies against the following proteins were used: Aktl, Akt2, phospho-Aktl /2 /3-Thr308 , phospho-Aktl/2/3-Ser473, phospho-H2A.X-Serl39, E2F1, p73, PP2A-C subunit, methyl-PP2A-C subunit (Millipore; Madrid, Spain), Akt3, Aktl/2/3, Melan-A/MARTl , p53, Slac2-a (Santa Cruz Biotechnology, Santa Cruz, CA) , MyoVa (Cell Signaling Tech., Danvers, MA), Apafl (BD Biosciences, Sparks, MD) , Pmell7/HMB45 (Dako Inc., Carpinteria, CA) , Rab27a (Abeam, Cambridge, UK) , and actin (Sigma) .

1.2 Cell Lines, Proliferation and Apoptosis Assays

Melanoma cell lines of human and mouse origin were obtained from ATCC and maintained in the appropriate culture medium supplemented with 10% FBS and antibiotics. Cell viability was evaluated using 3- (4, 5-dimethylthiazol-2-yl ) -2 , 5-diphenyltetrazolium bromide (MTT) . Cells were evaluated for apoptosis using the Cell Death Detection ELISAPLUS kit (Roche Diagnostics, Barcelona, Spain) that detects mono- and oligo-nucleosomes in the cytoplasmic fractions of cell lysates using biotinylated antihistone and peroxidase-coupled anti- DNA antibodies. The amount of nucleosomes is spectrophotometrically quantified at 405 nm by the peroxidase activity retained in the immunocomplexes . Apoptosis was defined as the specific enrichment of mono- and oligonucleosomes in the cytoplasm and was calculated by dividing the absorbance of treated samples by the absorbance of untreated samples after correcting for the number of cells. The induction of apoptosis in each melanoma cell line after a 7 h treatment with 2μΜ staurosporin (100% apoptotic cells) was used to calculate the number of apoptotic cells.

1.3 PCR Analysis

mRNA extraction, cDNA synthesis, and conventional and quantitative real-time PCR (qRT-PCR) were performed as previously described

[ Sanchez-del-Campo , L . ; et al (2008)]. Primers were designed using Primer Express version 2.0 software (Applied Biosystems, Foster City, CA) and synthesized by Life Technologies (Barcelona, Spain) . To quantify the gene expression level of the MyoVa exon F transcripts, primers were designed for exons E and F [accession numbers AF090424 and X57377 for human (h) and mouse (m) transcripts, respectively] using standard techniques. p73 primers were designed to amplify TAp73 (p73 with the transactivating domain; NM 005427.3) using standard techniques. Other primers for Apafl (h) and Actin (h, m) were also designed using standard techniques.

1.4 ChIP Assays .

A chromatin immunoprecipitation (ChIP) assay was performed using the Magna ChlPTM G kit from Millipore according to the manufacturer' s instructions. Briefly, untreated and MTX- and/or UCN-01-treated SK- MEL-28 cells were formaldehyde crosslinked, and the DNA was sheared by sonication to generate an average size of 300 to 3,000 bp. The chromatin was then incubated with anti-E2Fl or mouse IgG antibodies. DNA from lysates prior to immuno-precipitation was used as a positive input control. After washing, elution, and DNA

purification, the DNA solution (2 L) was used as a template for qRT-PCR amplification using specific human primers. The following primer sequences were used for ChlP-PCR: TAp73 promoter region (forward: 5'-TGA GCC ATG AAG ATG TGC GAG-3 ' (SEQ ID NO: 3) and reverse: 5'-GCT GCT TAT GGT CTG ATG CTT ATG-3' (SEQ ID NO: 4)) and GAPDH (forward: 5'-CAA TTC CCC ATC TCA GTC GT-3' (SEQ ID NO: 5) and reverse: 5'-TAG TAG CCG GGC CCT ACT TT-3' (SEQ ID NO: 6)) . Standard curves were generated for all primer set to confirm linearity of signals over the experimentally measured ranges.

1.5 Interference RNA

Specific stealth siRNAs for MyoVa (HSS106896), p53 (HSS129934 and HSS129936) , Aktl (HSS100345 and HSS100346), Akt2 (HSS163235 and

HSS163236), and Akt3 (HSS115177 and HSS115179) were obtained from Life Technologies and transfected into melanoma cells using

Lipofectamine 2000 (Life Technologies) . Treatments began 24 h after siRNA transfection . Stealth RNA negative control duplexes (Life Technologies) were used as control oligonucleotides, and the ability of the stealth RNA oligonucleotides to knock down the expression of the selected genes was analyzed by western blotting 24 h after siRNA transfection . 1.6 Immunoblotting and Immunoprecipitation

Whole cell lysates were collected by adding SDS sample buffer. After extensive sonication, the samples were boiled for 10 min and subjected to SDS-PAGE. Proteins were then transferred to nitrocellulose membranes and analyzed by immunoblotting (ECL Plus, GE Healthcare, Barcelona, Spain) . For Akt2 immuno-precipitation assays, the cells (approximately 5 x 10^s) were lysed in 500 μΐ of lysis buffer (50 mM Tris, pH 8.0, 300 mM NaCl, 0.4% NP40, 10 mM MgC12) supplemented with protease and phosphatase inhibitor

cocktails (Sigma) . Cell extracts were cleared by centrifugation (20,000 x g for 15 min) and then diluted with 500 μΐ of dilution buffer (50 mM Tris, pH 8.0, 0.4% NP40, 2.5 mM CaC12) supplemented with protease and phosphatase inhibitor cocktails and DNase

I (Sigma) . Extracts were pre-cleared by a 30 min incubation with 20 μΐ of PureProteome Protein G Magnetic Beads (Millipore) at 4°C with rotation. An Akt2 antibody was then added to the pre-cleared extracts. After incubation for 1 h at 4°C, 50 μΐ of PureProteome Protein G Magnetic Beads were added, and the extracts were further incubated for 20 min at 4°C with rotation. After extensive washing, the bound proteins were analyzed by western blotting. Unbound extracts were used as positive inputs for protein load

determination .

1.7 Microscopy and FLIM

Electron microscopy was performed as previously described [Watabe H et al 2004] using a Zeiss EM10 electron microscope (Carl Zeiss Microimaging, Inc., Thornwood, NY, USA) . Laser scanning confocal microscopy of fixed cells was performed using a Leica TCS 4D confocal microscope (Wetzlar, Germany) . For indirect

immunofluorescence studies, preparations of cells on glass slides were fixed with cold acetone for 5 min and washed with PBS . The cells were incubated with 3% bovine serum albumin (BSA) for 20 min and then with primary antibodies (diluted 1:200 in PBS containing 1% BSA) for 2 h at room temperature. The cells were washed three times in PBS and incubated for 1 h at room temperature with Alexa Fluor Dyes (Life Technologies) as secondary antibodies. After 3 washes with PBS, the cells were incubated with 0.01% 4 ' -6-diamidino-2- phenylidene (DAPI; Sigma) in water for 5 min. For antibody

specificity, primary antibodies were replaced with specific IgGs (diluted 1:200) during immunofluorescence. Co-localization analysis was performed with the Co-localization Finder plugin of ImageJ-NIH and showed images of the Alexa-Fluor 633 (red) merged with Alexa- Fluor 488 (green) secondary antibodies, and the co-localized pixels in orange. This plugin provides the Pearson^'s overlap coefficient (Rr) ranging from -1 to 1, where 1 represents perfect co- localization, 0 represents random co-localization and -1 represents perfect exclusion.

The confocal imaging of MTX-FITC in live cells (FLIM) was performed using the same confocal microscope. Images were collected following 488 nm laser excitation with an FITC emission filter. Prior to live confocal microscopy, the cells were incubated in serum-free DMEM for 1 h with MTX-FITC and with or without UCN-01. After this time, the cells were extensively washed with DMEM containing MTX (without FITC) and with or without UCN-01 as appropriate. During imaging, the cells were incubated on a heated stage at 37 °C in DMEM containing MTX (without FITC) and with or without UCN-01 as appropriate. Frames were collected at the indicated times. Images were processed with Huygens Essential v. 4.1.0p6 (Scientific Volume Imaging B.V., The Netherlands) and Imaris (Bitplane AG, Switzerland) . Vesicle

movements were traced, and velocities and distances were calculated using the ImageJ 1.6.0 20 (National Institutes of Health, Bethesda, MD, U.S.A.) manual tracking plugin (Fabrice Cordelieres, Institut Curie, France) . The fluorescence intensity levels within selected cell areas were also computed using ImageJ, and the FITC

fluorescence intensity was expressed as integrated density (reactive area multiplied by the mean grey of the same area) .

1.8 Preparation of Melanoma Cell Granular Fractions and Sucrose- density Gradients

Control untreated melanoma cells and those subjected to treatments with MTX and or IAKT were harvested with a mixture of 0.25% trypsin and 0.25 mM EDTA, washed once in 0.25 M sucrose, and centrifuged at 1,000 x g for 10 min at 4°C. Specimens were then homogenized on ice using 20 strokes in a Potter homogenizer and centrifuged at 1,000 ^χ g for 10 min at 4°C. The supernatant was recovered and further centrifuged at 19,000 ^χ g for 30 min at 4°C (14) . The pellet containing melanosome-enriched granular fractions was re-suspended in ice-cold 0.25 M sucrose in 10 mM HEPES (pH 7.0) and examined by electron microscopy. Purified melanoma granular fractions were re- suspended in 2.0 M sucrose and layered at the bottom of a 1.0-2.0 M sucrose step (1.0, 1.2, 1.4, 1.5, 1.6, 1.8 and 2.0 M) gradient. The gradient was centrifuged at 100,000 ^χ g in a Beckman SW 41 swinging- bucket rotor for 1 h at 4°C. 1.9 PP2A Assay

Cells were exposed to the experimental conditions indicated in the results. After two washes with 0.9% NaCl, total cellular proteins were extracted in lysis buffer containing 50 mM Tris-HCl, pH 7.5, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 1% Triton X-100, and 0.5% Nonidet P-40 without phosphatase inhibitors. Specific PP2A activity was measured using the PP2A Immunoprecipitation Phosphatase Assay Kit (Millipore) . All procedures were performed according to the

manufacturer's protocol, and changes in absorbance were measured at 650 nm in a Spectra-MAX 250 (Molecular Devices, Sunnyvale, CA) plate reader .

1.10 MTX Conjugation with HRP

MTX (10 mM) was dissolved completely in a pH 7.0 buffer solution (100 mM, phosphate buffer) , and then an equimolar quantity of EDC (10 mM) was added. The mixture was stirred for 40 min at room temperature. The activated MTX (1 mL) was added to 10 μΜ HRPC solutions (1 mL, pH 7.0) and incubated for 18 h at room temperature. The MTX-conj ugated proteins were separated from unreacted MTX using a Sephadex G-25 desalting column equilibrated in PBS (pH 7.4) . The degree of MTX conjugation was spectrophotometrically determined by measuring the difference in absorbance between the conjugated and free proteins at 303 nm. HRP has a molecular mass of 40 kDa and each HRP molecule contains 6 lysine residues. Titration experiments demonstrated that the total protein lysines were conjugated to MTX and this factor was used to determine MTX levels in melanoma cells (Figure 22) . The cellular uptake of MTX-conj ugated HRP was performed by treating SK-MEL-28 melanoma cells (in 96-wells plates) for 5 h with 1 μΜ MTX-HRPC in the absence or presence of 50nM UCN-01. After extensive washing with ice-cold PBS, the cells were disrupted by adding 200 μL of HRP activity medium (150 μΜ ABTS and 75 μΜ H202 in citrate-phosphate buffer, pH 4.5) . The oxidation of ABTS in the presence of HRP was followed by observing the increase in absorbance at 414 nm (Ae414nm = 31.1 mM-lcm-1) in a SpectraMax 340PC384 microplate reader (Molecular Devices, Sunnyvale, CA) .

1.11 dNTP Pool Extraction and Analysis

Asynchronously proliferating SK-MEL-28 cells were seeded in six-well dishes. The extraction and analysis of the dNTP pools in each extract were performed as previously described [Potterf et al

(1996) ] . The reaction mixtures (50 μL) contained 100 mM HEPES buffer, pH 7.5, 10 mM MgC12, 0.1 units of the Escherichia coli DNA polymerase I Klenow fragment (Sigma, Madrid, Spain), 0.25 μΜ oligonucleotide template, and 1 Ci [ 3H] dATP (ARC, St. Louis, MO) or [3H]dTTP (Perkin-Elmer, Waltham, MA) . Incubations were performed for 60 min at 37°C.

1.12 MALDI-TOF Mass Spectroscopy

SK-MEL-28 whole cell lysates were immunoprecipitated as described above. After immunoprecipitation and elution, bound proteins were digested with trypsin according to standard procedures [Angus et al (2002)]. The data were recorded and processed with Agilent

MassHunter Workstation Software to obtain the peptide mass

fingerprint (PMF) . The resulting PMF mass spectra were searched against the MyoVa protein sequence with carbamidomethylation of cysteine as a fixed modification and variable phosphorylation modifications at Serl650 and Serl812 were searched. The peptide mass tolerance was set to 50 ppm, and a maximum of three missed cleavages was considered.

1.13 Mouse Melanoma Models

Animals were bred and maintained according to the Spanish

legislation on the Protection of Animals used for Experimental and other Scientific Purposes' and in accordance with the directives of the European community. For the subcutaneous melanoma model, B16/F10 cells (5.0xl0⁵) were subcutaneously injected into the dorsal flanks of 6-8-week-old female C57BL/6 mice. Animals with tumors greater than 8 mm in diameter on day 8 or with no visible tumor growth by day 12 were excluded. Groups (n =10 mice per group) were subjected to treatments beginning on the 8th day after tumour cell injection. Mice were intradermally treated with MTX (1 mg/kg/day) and/or UCN-01 (0.1 mg/kg/day) 5 times a week for 3 weeks. Hepatic metastases were produced by the intrasplenic injection of 3.0 x 10⁵ B16-F10-Iuc2 mouse melanoma cells (Caliper Life Sciences, Hopkinton, MA) as previously described [Vidal-Vanaclocha et al (1994)] (n = 10-15) . Primary spleen tumors and hepatic metastases at days 12 and 14, respectively, were analyzed using the IVIS Imaging System (Caliper Life Sciences) . Mice were intraperitoneally treated with MTX (1 mg/kg/day) and UCN-01 (0.5 mg/kg/day) from days 1 to 14, and control mice received the same volume of vehicle (DMSO) . Postmortem

histological liver and spleen examination was performed in all animals. Formalin-fixed-paraffin-embedded tissue sections were stained with hematoxylin and eosin (H&E) . A Leica DMRB microscope connected to a Leica DC500 digital camera was used to quantify the number, average diameter, and position coordinates of the metastases [Vidal-Vanaclocha et al (1994)]. For tyrosinase detection, mouse livers (3 per treatment) were cut into approximately 0.2 g slices. Five randomly chosen slices from each liver were used for phenol- chloroform total RNA extraction. The RNA (5 g) was then used to synthesize cDNA, and equal amounts of the five cDNA fractions corresponding with the same liver were pooled and employed for tyrosinase mRNA determinations using qRT-PCR.

2. Results

2.1 MTX stimulates MyoVa-mediated melanosome transport.

We first analysed the changes in the cellular localization of MyoVa after treatment of melanoma with MTX by confocal microscopy (Figure 1) . For this study, we analysed the effect of MTX on the

co-localization of MyoVa with HMB45 (a melanosome marker) , and

Rab27a and melanophilin/Slac2-a (two MyoVa assistant proteins) . In contrast with untreated controls, where MyoVa was mainly located in the perinuclear region and not co-localized with these proteins, in MTX-treated cells, MyoVa was found in the dendritic area of the cells and co-localized with HMB45, Rab27a and melanophilin (Figures 1 and 3) . MTX therefore activated MyoVa-dependent melanosome transport in the amelanotic SK-MEL-28 melanoma cell line (Figure 3) . Activation of MyoVa-dependent melanosome transport by MTX was also confirmed in B16/F10, a highly melanotic murine cell line.

Fluorescence-lifetime imaging microscopy (FLIM) was used to follow MTX-FITC-containing vesicles trafficking in SK-MEL-28 melanoma cells. (Figure 2) . Upon MTX-FITC addition, fluorescent vesicles migrated from the cytosol to the plasma membrane, and before being exported out of the cells, they transiently accumulated in the dendritic tips of melanoma cells. Because MTX-FITC treatment transiently increased fluorescent-containing vesicles in the actin- rich periphery of the dendrites, the effect of MTX on the endogenous MyoVa localization in SK-MEL-28 was further analyzed by

immunocytochemistry . An anti-MyoVa antibody was used in combination with the melanosomal marker HMB45 to analyse the distribution of melanosomes and their possible association with MyoVa in melanoma cells. Melanosomes were found perinuclear but did not co-localize with MyoVa in untreated cells (as determined by Pearson^'s overlap coefficients; Figure 3); in contrast, after the treatment of cells with MTX, MyoVa was localized m the cell periphery and the tips of the dendrites, and associated with melanosomes .

All together, these data indicated that MTX activates the MyoVa mediated melanosome transport.

2.2 MyoVa Silencing Sensitizes Melanoma Cells to MTX-induced

Apoptosis

Additional evidence that MTX activates MyoVa was obtained from experiments designed to specifically silence MyoVa in melanoma cells. We examined whether transient MyoVa silencing could sensitize melanoma cells to MTX by impeding its melanosome-mediated

exportation (Figure 4) . SK-MEL-28 cells were transfected with siRNA- MyoVa or a control siRNA and maintained in culture medium in the presence or absence of MTX. We observed, via sucrose density gradients, that MTX treatment resulted in a significant decrease in the Sk-MEL-28 melanosome content (Figure 6A) which may be related to the cellular exportation of MTX. However the MTX treatment of Sk- MEL-28 in which the expression of MyoVa had been silenced by the use of specific siRNAs did not result in melanosome exportation (Figure 6A) . In fact, MyoVa knockdown significantly increased the

sensitivity of SK-MEL-28 cells to MTX-induced apoptosis. (Figure 5 and 6B) . All together, these results indicated that MyoVa might play a central role in the resistance of melanoma to MTX and that targeting of this protein might result in an increase in sensitivity of melanoma cells to this drug.

2.3 MTX activates Akt-dependent phosphorylation of MyoVa in melanoma .

The observation that MyoVa plays an important role in MTX

exportation in melanoma provides indication that its targeting may be therapeutically significant. However, direct targeting of this structural protein is difficult. In order to find possible

strategies to indirectly target MyoVa, we searched for signalling pathways that activate MTX and result in MyoVa activation.

First, we analysed changes in the expression level of MyoVa after treatment of melanoma cells with MTX. Real-time PCR and western blot analysis indicated that MTX did not significantly modified MyoVa mRNA and protein levels respectively in melanoma cells (Figure 7A) . Because Akt2 -mediated phosphorylation of MyoVa at serine 1650 has been shown to enhance the interaction of this protein with the actin cytoskeleton (Yoshizaki, et al . 2007), we next decided to analyse the effect of MTX on this MyoVa posttranslational

modification by using MALDI-TOF mass spectroscopy (Figure 7B; Table 1) . MyoVa has two Akt consensus motifs at Ser¹⁶⁵⁰ (RKRTSS; SEQ ID NO: 7) and Ser¹⁸¹² (RDRKDS; SEQ ID NO: 8), which are highly conserved across mammalian species. By using mass peptide analysis of immunoprecipitated MyoVa, after trypsin digestion, we observed that MTX induced the phosphorylation of this protein at Ser¹⁶⁵⁰.

Since, we observed here that MTX mediated the phosphorylation of MyoVa, the MTX dependent activation of Akt in melanoma cells was further investigated. Western blot and confocal microscopy

experiments showed that MTX induced phosphorylation of Akt at Thr³⁰⁸ and Ser^47j and the nuclear translocation of activated Akt in melanoma (Figures 8 and 9 ) .

MTX activation of the Akt-dependent phosphorylation of MyoVa in melanoma was also demonstrated by co-treatment of melanoma cells with MTX and a specific Akt inhibitor ( IAKT ) . The presence of IAKT during MTX treatments was sufficient to inhibit MyoVa

phosphorylation and melanosome export (Figures 10 and 11), which resulted in a substantial increase in the sensitivity of melanoma cells to MTX-induced apoptosis (Figure 12) .

Although the results indicated that MTX stimulated Akt

phosphorylation, the identification of which Akt isoform(s) were activated by this drug in these experiments is difficult. Therefore, to identify which form of Akt may be involved in MyoVa activation in melanoma, we utilized siRNAs directed against the three Akt

isoforms. For these experiments, SK-MEL-28 cells were transfected with these different siRNAs, and after 48 h, the sensitivity of the transfected cells to MTX-induced apoptosis was determined. Each siRNA led to a marked depletion of the target protein (Figure 13), but only the Akt2 knockdown significantly increased the sensitivity of the melanoma cells to MTX-induced apoptosis. In contrast, Aktl and Akt3 knockdown had only marginal effects on MTX induced

apoptosis .

In addition to nucleotide imbalance, a decrease in cellular

methylation has been overlooked as mechanism for the antiproliferative effect of MTX in cancer cells (Winter-Vann et al 2003) . An attractive hypothesis that could connect the demethylating properties of MTX with Akt activation is that MTX could inactivate protein phosphatase 2A ( PP2A) , an Akt inhibitor that is activated by the methylation o f its catalytic subunit (Guenin et al . , 2008) .

Western blot analysis demonstrated that MTX induced the

demethylation of PP2A catalytic subunit. In agreement with this observation, MTX-dependent PP2A demethylation was also accompanied by a substantial time- and dose-dependent reduction in PP2A activity (Figure 14), which was also accompanied by the lack of cytosolic colocalization of PP2A with Akt as observed in confocal microscopy experiments (Figure 8) .

Cantharidin, a potent and selective inhibitor of PP2A, was used to confirm the consequences of PP2A inhibition on MyoVa activation in melanoma cells (Figure 15) .

2.4 UCN-01 inhibits the MTX-induced activation of MyoVa and prevents MTX exportation in melanoma cells

Although 7 -hydroxystaurosporine (UCN-01) was originally isolated as a PKC-selective inhibitor (Takahashi et al . , 1987), more recent discoveries indicate that this compound may exert its cellular activity by inhibiting the PDKl-Akt survival pathway (Sato et al . , 2002; Kondapaka et al . , 2004) . Since activated Akt seem to play a primary role in MTX induced activation of MyoVa, we next studied the effect of UCN-01 on this activation pathway.

We examined the Akt2 phosphorylation state in total melanoma cell homogenates (Figure 16) . When cells were treated with UCN-01 in the presence of MTX, we observed a significant decrease in Akt2

phosphorylation (with respect to MTX-treated cells) at the Thr308 and Ser473 residues, which is consistent with PDK1 being the UCN-01 molecular target. Thus, UCN-01 showed a great effect on the MTX- induced phosphorylation of MyoVa.

MALDI-TOF mass spectra of MyoVa tryptic peptides demonstrated that UCN-01-mediated Akt phosphorylation inhibition also resulted in a significant reduction in the MTX-induced MyoVa phosphorylation at Serl650 (Figure 17A, Figure 18) . As a consequence of this lack of activation after MTX/UCN-01 treatment, MyoVa was not localized in the periphery of SK-MEL-28 cells and maintained perinuclear

localization as in untreated control cells (Figure 17B) . UCN-01 therefore prevented the MTX-mediated translocation of MyoVa to the melanoma dendritic tips. These results indicated that UCN-01 blocks melanosome transport in melanoma.

To confirm these results, we investigated whether UCN-01 could prevent the sequestration of MTX in melanosomes and its subsequent cellular exportation. To test this, we treated SK-MEL-28 cells with FITC-MTX in the absence and in the presence of UCN-01, and then analyzed the MTX subcellular localization. After 4 h of FITC-MTX treatment, fluorescence was highly reduced in melanoma cells and it was mainly observed in the cell membrane, associated with

fluorescent vesicles, indicating that MTX was being exported out of the cell (Figure 21) .However, fluorescence was highly augmented and localized to the perinuclear regions of the cells after 4 h of a combined treatment of FITC-MTX with UCN-01 (Figure 21) . We also used sub-second frame-rate confocal microscopy to examine the movement of individual MTX-FITC-containing vesicles in basal SK-MEL-28 cells

(Figure 19) . Fluorescent vesicles were observed to make directed movements over short distances at speeds ranging from 0.077 to 0.004 μπι/s (average: 0.035 μιη/s) .However, in the presence of UCN-01, MTX- FITC vesicles remained fairly static over this time course, showing vibrational-type displacements but not moving far from their original location (Figure 19) . Due to this vesicle immobility, fluorescence was mainly associated with cytoplasmic compartments, and it did not migrate to the dendritic tips at longer time periods

(Figures 20 and 21) .

These results were also confirmed by using MTX conjugated to horseradish peroxidase (HRP) , which served as a reporter enzyme (Figure 22) . HRP activity in SK-MEL-28 melanoma cells after 4 h of treatment with MTXHRP was practically unobservable ; however, the inclusion of UCN-01 in this treatment highly elevated intracellular HRP activity in melanoma cells. HRP has a molecular mass of 40 kDa and each HRP molecule contains 6 lysine residues. Titration

experiments demonstrated that the total protein lysines were conjugated to MTX; therefore, calculations based in this observation and HRP activity demonstrated that in the presence of UCN-01 the intracellular concentration of MTX was augmented around 250-times compared with cells treated exclusively with MTX. 2.5 The MTX/UCN-01 combination induces E2Fl-mediated apoptosis in melanoma .

We evaluated whether this higher accumulation of MTX in melanoma cells after MTX/UCN-01 treatment was enough to induce apoptosis in these cells .

Although UCN-01 exhibits potent antitumor activity in several in vivo and in vitro tumor models (Akinaga et al . , 1991; Seynaeve et al . , 1993), it is inactive against melanoma (Fecher et al., 2007) . We confirmed that UCN-01 induces Gl phase arrest but not apoptosis in all the studied melanoma cell lines, which included lines harbouring wild-type p53 (A375 , G361, and B16/F10) and mutant p53 (SK-MEL-28) . Melanomas are also intrinsically resistant to MTX, which acts as a cytostatic agent in melanoma cells ( Sanchez-dei- Campo et al, 2009a) .

However, combination of MTX and UCN-01 strongly induced apoptosis in SK-MEL-28 melanoma cells (Figure 23) . Next, we analysed the possible participation of p53 in MTX/UCN-01 -induced apoptosis. Several experiments indicated that the induction of apoptosis by this drug combination was p53-independent . First, the p53 status of melanoma cells did not influence the apoptotic effect of the drug combination (Figure 23) . Additionally, the silencing of p53 in G361 cells did not affect MTX/UCN-01-mediated apoptosis (Figure 24) . Importantly, the MTX/UCN-01 combination was able to induce apoptosis not only independently of the mutational status of p53 but also of the mutational state of genes such as BRAF or PTEN (Figure 24) .

We analyzed whether the accumulation of MTX in the presence of UCN- 01 could modify dTTP and E2F1 cellular levels and induce E2F1- mediated apoptosis in melanoma. The treatment of melanoma cells with MTX/UCN-01 generated a nucleotide imbalance that favored dTTP depletion and the subsequent increase of E2F1 protein (Figures 25) . Contrary to previously reported data showing the induction of SSBs upon MTX treatment, the treatment of melanoma cells with MTX/UCN-01 thus induced double strand breaks (DSBs) .

Thymidine depletion induces DNA double strand break (DSB) formation characterized by phosphorylation of histone H2AX at Serl39 (yH2AX) by ATM/ATR kinases and the subsequent rapid formation of yH2AX foci at the DSB sites (Sedelnikova et at., 2003) . Western blot and localization analyses of yH2AX (Figures 26 & 27) in SK-MEL-28 cells after MTX/UCN-01 treatment indicated that this combination induced massive DNA damage in melanoma cells.

In addition, western blot and real-time-PCR analysis (Figures 27 and 28E) indicated that when combined with UCN-01, MTX induced the expression of both the transactivating form of p73 (TAp73) and the apoptosis protease-activating factor 1 (Apafl), two pro-apoptotic targets of E2F1. ChIP assays also indicated that, compared with untreated cells or those subjected to individual treatments; the combination MTX/UCN-01 significantly increased the occupancy of E2F1 at the TAp73 promoter of SK-MEL-28 (Figure 28) .

DBS induction was also accompanied by early phosphorylation of Chkl and Chk2 (Figure 27) . In response to DNA damage, the E2F1 protein is stabilized through distinct mechanisms, including direct

phosphorylation by Chk2 at Ser364 (Urist et al . , 2004) . MTX/UCN-01 treatment induced a substantial increase in E2F1 protein levels (Figure 27), which was accompanied by an increase in the protein expression of its apoptotic effectors p73 and Apafl (Figure 27) . All together, the results indicated that accumulation of MTX in cells co-treated with UCN-01 activates the E2F1 machinery required for E2Fl-mediated but p53-independent cell death signalling in melanoma . 2.6 UCN-01 Enhances In Vivo Melanoma Sensitivity to MTX.

B16/F10 cells (5.0 x 105) were subcutaneously injected into the dorsal flanks of 6-8 week-old female C57 /B16 mice, a syngeneic melanoma model in which host mice retain intact immune systems.

Animals with tumors greater than 8 mm in diameter on day 8 or with no visible tumor growth by day 12 were excluded. Groups (10 mice per group) were subjected to treatments starting at day 8 after tumor cell injection. Mice were treated intraperitoneally with MTX (1 mg/kg/day) and/or UCN-01 (0.1 mg/kg/day) 5 times a week for 3 weeks. Animals were bred and maintained according to the Spanish

legislation on the 'Protection of Animals used for Experimental and other Scientific Purposes' (RD 1201/2005) and in accordance with the directives of the European community.

Visual examination revealed that the MTX/UCN-01 combination acted synergistically to inhibit tumor growth (Figure 29 and Table 2) . Using B16 cells that express a luciferase reporter (Figure 29), quantification of the in vivo luciferase signal confirmed that the MTX/UCN-01 combination was highly effective at reducing the primary tumor burden. A histological study of primary splenic tumors suggested that MTX/UCN-01 treatment induced necrosis in B16/F10 tumors. As vehicle controls, B16/F10 tumors treated with DMSO showed their usual histological appearance of poor differentiation and limited necrosis (Figure 29) . In contrast, 14 days treatment with MTX/UCN-01 induced obvious haemorrhagic necrosis, with necrotic areas of approximately 85%. Necrosis in splenic tumors was less evident in mice treated with MTX or UCN-01 (4% ± 2%; and 2% ± 0.5%, respectively) .

As an alternative approach, we tested whether MTX/UCN-01

administration after the injection of melanoma cells could prevent melanoma dissemination from the spleen to the liver, one of the preferential metastatic locations for melanomas. Luciferase-tagged B16/F10 cells were injected into the spleens of C57BL/6 mice, and after 14 days of treatment, the number of macroscopic liver

metastases was assessed using a dissecting microscope (Figure 30) . Luciferase imaging demonstrated that mice treated with MTX/UCN-01 had an appreciably lower burden of macroscopic liver metastases compared with the untreated control (Figure 30) . To confirm the bioluminescent results, the livers were analyzed by histology at necropsy. MTX/UCN-01 treatment reduced the hepatic metastasis volume by 50% compared with control mice treated with vehicle. Interesting, MTX/UCN-01 eliminated sinusoidal-type metastasis, and only a limited number of portal-type metastases were observed after treatment.

These data were also confirmed by a real-time PCR analysis designed to detect melanoma specific tyrosinase in mouse livers (Figure 31) .

The data above demonstrate that MyoVa plays a key role in the resistance of melanoma to MTX, and show targeting this protein in the presence of MTX prevents drug exportation. Signalling pathways shown above to converge during the MTX-induced activation of MyoVa allow the design of drug-based therapies that indirectly target MyoVa in melanoma.

Akt2-mediated MyoVa activation, discovered here as a consequence of MTX action, may represent a general drug resistance mechanism of melanoma cells in response to Akt-activating chemotherapeutic agents [Chen et al (2006); Xie et al (2009); Jiang et al (2009; Huang et al (2012)] and/or in melanoma with pre-existing high, endogenously up regulated, Akt2 activity. It is well known that endoplasmic reticulum (ER) stress leads to Akt activation [Hu et al (2004)], and recently, the adaptation of melanoma cells to ER stress has been proposed as a resistance mechanism of these cells to

chemotherapeutic drugs [Jiang et al (2009)]. Interestingly, melanoma cells are highly resistant to ER stress-inducing drugs such as cisplatin and adriamycin [Huang et al (20012), Jiang et al (2009)], but also to drugs that did not cause ER stress such as docetaxel and vincristine, two microtubule-targeting drugs [Jiang et al (2009)]. In the latter case, it was also reported that human melanoma under ER stress were more resistant to apoptosis induced by these drugs due, at least in part, to the activation of the PI3K/Akt pathway. Therefore our results, showing the molecular link between the Akt signaling pathway and the cellular export of chemotherapeutic drugs might offer a global explanation for multidrug resistance in melanoma and might be useful to open new avenues for exploring therapeutical combinations of Akt2 inhibitors with MTX or other cytotoxic drugs.

Because of the potential for entering the clinical arena, MTX/UCN01 combination, irrespective of the precise action mechanism, must be taken into consideration in the future design of melanoma therapies. MTX is in widespread clinical use for a variety of steroid- recalcitrant inflammatory diseases, and UCN-01 has been included in multiple clinical trials regimes [Dees et al (2005), Schenk et al (2012)]; thus, MTX/UCN-01 therapy has the potential for rapid application in the human setting.

It is well known that the amino acid sequence encoded by an

alternatively spliced exon, exon F, is necessary for the selective binding of MyoVa to melanosomes [Van Gele M (2008); Au et al

(2002)]. The MyoVa isoforms lacking this amino acid sequence are not targeted to the melanosomes, but localized to the perinuclear region instead [Van Gele M (2008), Au et al (2002)] . Although all the melanoma cell lines used in this study presented observable levels of MyoVa exon F expression, determination of the levels of this MyoVa spliced variant in melanoma biopsy samples may be of interest from a clinical point of view. Based on the data above, MyoVa may be considered as an oncogenic protein that promotes melanoma resistance to anticancer drugs; therefore, determination of MyoVa exon F in clinical samples of melanoma patients could help in the design of personalized therapies Although combined therapies to target the Akt2/MyoVa pathway would be functional in melanoma cells having an operative melanosome trafficking system, melanomas showing low or no expression of MyoVa exon F, or related trafficking protein would be sensitive to classical monotherapy treatments. Therefore, oncologists could decide the most appropriate treatment of melanoma patients in function of MyoVa exon F expression levels in biopsy samples.

In addition to the promising clinical and therapeutic perspectives of these findings, our results may also have implications for understanding the mechanism of action of antifolates in melanoma and identifying the functionality of the pro-apoptotic pathways in these cancer cells. Although E2F1 has been identified as a component of pathways that link the DNA damage response to cancer cell death

[Engelmann et al (2010)], this signalling pathway has been barely explored for melanoma therapy. Here, we report that the E2F1 apoptotic pathway is functional in melanoma and that its induction activates p73 and Apafl following a p53-autonomous pro-apoptotic signalling event. In addition, melanoma is often driven by mutations that activate MAPK signalling, such as BRAF [Fecher et al (2007) , Tap et al (2010)]; therefore, the observation that targeting

Akt2/MyoVa in the presence of MTX, a compound that blocks essential cell metabolic pathways, induces apoptosis in BRAF-mutated cells may be of potential importance when designing new treatment strategies to improve chemosensitivity, one of the most important obstacles for the management of patients with malignant melanoma.

In conclusion, this application describes a multidrug resistance mechanism that may serve as a link between other resistance

mechanisms described in melanoma [Chen et al (2006) ; Sanchex-del- Campo et al (2009); Huang et al (2012); Xie et al (2009); Jiang et al (2009)] . First, MyoVa was identified as a novel and specific target of Akt2 in melanoma; second, siRNA or pharmacological blockade of the Akt2/MyoVa pathway in melanoma was observed to suppress MTX and other drug resistance in these cancer cells; third, novel druggable targets were identified in this molecular pathway, which could be of interest from a clinical point of view; and fourth, the functionality of pro-apoptotic pathways in melanomas were analysed in response to antifolate treatment, by avoiding the cellular export of MTX. SEQUENCES

YFEELYADDPKKYQSYRISLYKRMI SEQ ID NO: 1 - amino acid sequence encoded by exon F of MyoVa

MAASELYTKFARVWIPDPEEVWKSAELLKDYKPGDKVLLLHLEEGKDLEYHLDPKTKELPHLRNPDILVGENDLT ALSYLHEPAVLHNLRVRFIDSKLIYTYCGIVLVAINPYEQLPIYGEDI INAYSGQNMGDMDPHIFAVAEEAYKQM ARDERNQSI IVSGESGAGKTVSAKYAMRYFATVSGSASEANVEEKVLASNPIMESIGNAKTTRNDNSSRFGKYIE IGFDKRYRI IGANMRTYLLEKSRWFQAEEERNYHIFYQLCASAKLPEFKMLRLGNADNFNYTKQGGSPVIEGVD DAKEMAHTRQACTLLGISESHQMGIFRILAGILHLGNVGFTSRDADSCTIPPKHEPLCIFCDLMGVDYEEMCHWL CHRKLATATETYIKPISKLQATNARDALAKHIYAKLFNWIVDNVNQALHSAVKQHSFIGVLDIYGFETFEINSFE QFCINYANEKLQQQFNMHVFKLEQEEYMKEQIPWTLIDFYDNQPCINLIESKLGILDLLDEECKMPKGTDDTWAQ KLYNTHLNKCALFEKPRLSNKAFI IQHFADKVEYQCEGFLEKNKDTVFEEQIKVLKSSKFKMLPELFQDDEKAIS PTSATSSGRTPLTRTPAKPTKGRPGQMAKEHKKTVGHQFRNSLHLLMETLNATTPHYVRCIKPNDFKFPFTFDEK RAVQQLRACGVLETIRISAAGFPSRWTYQEFFSRYRVLMKQKDVLSDRKQTCKNVLEKLILDKDKYQFGKTKIFF RAGQVAYLEKLRADKLRAACIRIQK IRGWLLRKKYLRMRKAAI MQRYVRGYQARCYAKFLRRTKAA I IQKYW RMYWRRRYKIRRAA IVLQSYLRGFLARNRYRKILREHKAVI IQKRVRGWLARTHYKRSMHAI IYLQCCFRRMM AKRELKKLKIEARSVERYKKLHIGMENKIMQLQRKVDEQNKDYKCLVEKLTNLEGIYNSETEKLRSDLERLQLSE EEAKVATGRVLSLQEEIAKLRKDLEQTRSEKKCIEEHADRYKQETEQLVSNLKEENTLLKQEKEALNHRIVQQAK EMTETMEKKLVEETKQLELDLNDERLRYQNLLNEFSRLEERYDDLKEEMTLMVHVPKPGHKRTDSTHSSNESEYI FSSEIAEMEDIPSRTEEPSEKKVPLDMSLFLKLQKRVTELEQEKQVMQDELDRKEEQVLRSKAKEEERPQIRGAE LEYESLKRQELESENKKLKNELNELRKALSEKSAPEVTAPGAPAYRVLMEQLTSVSEELDVRKEEVLILRSQLVS QKEAIQPKDDKNTMTDSTILLEDVQKMKDKGEIAQAYIGLKETNRSSALDYHELNEDGELWLVYEGLKQANRLLE SQLQSQKRSHENEAEALRGEIQSLKEENNRQQQLLAQNLQLPPEARIEASLQHEITRLTNENLYFEELYADDPKK YQSYRISLYKRMIDLMEQLEKQDKTVRKLKKQLKVFAKKIGELEVGQMENISPGQI IDEPIRPVNIPRKEKDFQG MLEYKKEDEQKLVKNLILELKPRGVAVNLIPGLPAYILFMCVRHADYLNDDQKVRSLLTSTINSIKKVLKKRGDD FETVSFWLSNTCRFLHCLKQYSGEEGFMKHNTSRQNEHCLTNFDLAEYRQVLSDLAIQIYQQLVRVLENILQPMI VSGMLEHE IQGVSGVKPTGLRKRTSSIADEGTYTLDSILRQLNSFHSVMCQHGMDPELIKQWKQMFYI IGAI LNNLLLRKDMCSWSKGMQIRYNVSQLEEWLRDKNLMNSGAKETLEPLIQAAQLLQVKKKTDDDAEAICSMCNALT TAQIVKVLNLYTPVNEFEERVSVSFIRTIQMRLRDRKDSPQLLMDAKHIFPVTFPFNPSSLALETIQIPASLGLG FISRV

SEQ ID NO: 2 Human MyoVa isoform 1 - exon F underlined,

Modification MyoVa Status Peptide Sequence^* Measured Theoretical Control" MTX"

Im/z} {mm (Intensity)" (Intensity)'

Phosphorylation Non-phosphorylatrd (RVrSSIADEGTYTLDSILR(Q) 1841.91 1S41.99 101324 9512

(Si 650} Ptio¾phorylated (R)TS.pSIADEGTYTLDSILR(Q) 1 21.88 1921.99 7835 98451

Phosphorylation Noti-phosphorylatco. ( ) DSPQLLMDA (H) 1245.65 12.45.46 112843 10514

(S1812) PhosphoTvlated (R)KDSpPOLLMDAK(H> 132.5.25 1325.46 2305 2525 aTlie characteristics peptides involving phosphorylation of MyoVa (phosphorylation = P), as well as their measured and theoretical m/z are shown; ^Peptides were analyzed in untreated SK-MEL-28 cells (control) and those treated with 1 μΜ MTX for 10 h.; 'Relative intensities of specific tryptie peptides were normalized with respect to an internal matrix control.

Table 1 showing SEQ ID NOS: 9 and 10

Table 2

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Claims

Claims

1. A method of treatment of melanoma comprising;

administering a cytotoxic anti-cancer compound and a

melanosome transport inhibitor to an individual in need thereof.

2. A melanosome transport inhibitor for use in the treatment of melanoma in combination with a cytotoxic anti-cancer compound.

3. A cytotoxic anti-cancer compound for use in the treatment of melanoma in combination with an melanosome transport inhibitor

4. A combination of an melanosome transport inhibitor and a cytotoxic anti-cancer compound for use in the treatment of melanoma

5. Use of a melanosome transport inhibitor in the manufacture of a medicament for use in the treatment of melanoma in combination with a cytotoxic anti-cancer compound.

6. Use of a cytotoxic anti-cancer compound in the manufacture of a medicament for use in the treatment of melanoma in combination with an melanosome transport inhibitor.

7. Use of a combination of a melanosome transport inhibitor and a cytotoxic anti-cancer compound in the manufacture of a medicament for use in the treatment of melanoma.

8. A pharmaceutical formulation comprising an melanosome transport inhibitor and a cytotoxic anti-cancer compound, optionally for use in the treatment of melanoma.

9. A pharmaceutical formulation according to claim 8 comprising a pharmaceutically acceptable carrier and optionally one or more additional active compounds.

10. A method, use, inhibitor, compound or formulation according to any one of claims 1 to 9 wherein the melanosome transport inhibitor is an inhibitor of MyoVa activation.

11. A method, use, inhibitor, compound or formulation according to any one of claims 1 to 10 herein the melanosome transport inhibitor is an Akt2 signalling pathway inhibitor.

12. A method, use, inhibitor, compound or formulation according to claim 11 wherein the Akt signalling pathway inhibitor is 7- hydroxystraurosporine (UCN-01), triciribine, honokiol or perifosine (1 , l-dimethylpiperidinium-4-yl octadecyl phosphate) or IAKT.

13. A method, use, inhibitor, compound or formulation according to claim 12 wherein the Akt signalling pathway inhibitor is 7- hydroxystraurosporine .

14. A method, use, inhibitor, compound or formulation according to any one of claims 1 to 12 wherein the cytotoxic anticancer compound activates melanosome transport in a melanocyte.

15. A method, use, inhibitor, compound or formulation according to any one of claims 1 to 13 wherein the cytotoxic anticancer compound activates myoVa-mediated melanosome transport in a melanocyte.

16. A method, use, inhibitor, compound or formulation

according to any one of claims 1 to 15 wherein the melanoma is characterised by expression of one or more MyoVa exon F isoforms .

17. A method, use, inhibitor, compound or formulation according to any one of claims 1 to 16 wherein the cytotoxic anticancer compound is a DNA crosslinking agent or an antifolate.

18. A method, use, inhibitor, compound or formulation according to claim 17 wherein DNA crosslinking agent is an anthracycline .

19. A method, use, inhibitor, compound or formulation according to claim 17 wherein DNA crosslinking agent is a platinum containing compound .

20. A method, use, inhibitor, compound or formulation according to claim 17 wherein the antifolate compound is methotrexate (MTX) , aminopterine (AMT) , trimetrexate (TMQ) fluorouracil , lometrexol (LMTX) , pemetrexed, raltitrexed or prelatrexate .

21. A method, use, inhibitor, compound or formulation according to claim 20 wherein the antifolate compound is methotrexate (MTX) .

22. A method, use, inhibitor, compound or formulation according to any one of the preceding claims wherein the cytotoxic anti-cancer compound is methotrexate (MTX) and the melanosome transport

inhibitor is 7-hydroxystraurosporine (UCN-01) .

23. A method, use, inhibitor, compound or formulation according to any one of claims 1 to 16 wherein the cytotoxic anticancer compound is a DNA alkylating agent.

24. A method, use, inhibitor, compound or formulation according to claim 23 wherein the DNA alkylating agent is dacarbazine.

25. A method, use, inhibitor, compound or formulation according to any one of claims 1 to 16 wherein the cytotoxic anticancer compound is a BRAF inhibitor.

26. A method, use, inhibitor, compound or formulation according to claim 25 wherein the BRAF inhibitor is vemurafenib.

27. A method, use, inhibitor, compound or formulation according to claim 25 wherein the BRAF inhibitor is dabrafenib

28. A method of screening for a compound with activity in increasing the sensitivity of a melanoma cell to a cytotoxic anti-cancer compound comprising;

contacting a cancer cell with a cytotoxic anti-cancer compound and determining the activation of MyoVa in the melanoma cell,

wherein a decrease in activation of MyoVa in the presence relative to the absence of test compound is indicative that the compound is active in increasing the sensitivity of a melanoma cell to the cytotoxic anti-cancer compound.

29. A method according to claim 28 wherein the cytotoxic

anticancer compound is a DNA crosslinking agent.

30. A method according to claim 29 wherein DNA crosslinking agent is an anthracycline .

31. A method according to claim 29 wherein DNA crosslinking agent is a Pt containing compound.

32. A method according to claim 28 wherein the cytotoxic anticancer compound is an antifolate.

33. A method according to claim 32 wherein the antifolate compound is methotrexate (MTX) , aminopterine (AMT) , trimetrexate (TMQ) fluorouracil , lometrexol (LMTX) , pemetrexed, raltitrexed or prelatrexate .

34. A method according to claim 32 wherein the antifolate compound is methotrexate (MTX) .

35. A method of treatment of a melanoma comprising;

administering a cytotoxic anti-cancer compound to an

individual in need thereof,

wherein the melanoma is characterised by the absence of expression of MyoVa isoforms comprising SEQ ID NO:l.

36. A cytotoxic anti-cancer compound for use in the treatment of melanoma in an individual, wherein the melanoma is characterised by the absence of expression of MyoVa isoforms comprising SEQ ID NO: 1

37. Use of a cytotoxic anti-cancer compound in the manufacture of a medicament for use in the treatment of melanoma characterised by th absence of expression of MyoVa isoforms comprising SEQ ID NO: 1.

38. A method compound, use or formulation according to any one of claims 35 to 37 wherein the treatment comprises identifying one or more melanoma cells from the individual as devoid of expression of MyoVa isoforms comprising SEQ ID NO: 1.

39. A method compound, use or formulation according to any one of claims 35 to 38 wherein the melanoma in the individual has been previously identified as a melanoma characterised by the absence of expression of MyoVa isoforms comprising SEQ ID NO: 1.

40. A method compound, use or formulation according to any one of claims 35 to 39 wherein the cytotoxic anti-cancer compound is methotrexate (MTX) and the melanosome transport inhibitor is 7- hydroxystraurosporine (UCN-01) .

41. A method of selecting a treatment for an individual with melanoma, comprising; providing a sample of one or more melanoma cells obtained from the individual, and

determining the presence of expression of one or more MyoVa isoforms comprising SEQ ID NO:l in the cells; or,

determining the absence of expression of MyoVa isoforms comprising SEQ ID NO : 1 in the cells, and;

selecting treatment with an cytotoxic anti-cancer compound in combination with a melanosome transport inhibitor if expression is present, or

selecting treatment with an cytotoxic anti-cancer compound if expression is absent.

42. A method according to claim 41 comprising administering the selected treatment.