METHODS AND COMPOSITIONS FOR TREATING MELANOMA RESISTANT

Abstract

The present invention relates to a method for treating a subject suffering from melanoma resistant by administering to said subject an inhibitor of NAMPT. Using a global metabolic profiling, inventors have showed that in addition to glycolysis, the BRAF inhibitor, PLX4032, promoted a complex metabolic rewiring of melanoma cells, including protein catabolism and fatty acid synthesis. Importantly, they observed that PLX4032 reduced the levels of nicotinamide adenine dinucleotide (NAD+), an important redox co-factor in numerous metabolic processes, including glycolysis, tricarboxylic acid cycle (TCA) cycle, glutamate metabolism and fatty acid betaoxidation. Pharmacological or genetic inhibition of NAMPT impaired melanoma cell growth, whereas the overexpression of NAMPT dampened the antiproliferative effect of PLX4032. In vivo, the inhibition of NAMPT also prevented the xenograft development of PLX4032-sensitive and -resistant melanoma cells, identifying NAMPT as a potential target for BRAFi-resistant melanomas.

Claims

1. A method for predicting the risk of relapse to a treatment in a subject suffering from melanoma comprising the steps of: i) measuring the activity and/or expression level of NAMPT in a biological sample obtained from said subject; ii) comparing the expression level measured at step i) with its predetermined reference value, and iii) concluding that the subject is at risk of relapse to the treatment when the expression level of NAMPT is higher than its predetermined reference value or concluding that the subject is not at risk of relapse when the expression level of NAMPT is lower than its predetermined reference value.

2. The method according to claim 1, wherein, the activity of NAMPT is determined by measuring the production level of NAD.

3. The method according to claim 1 comprising the steps of: i) measuring the activity of NAMPT by determining the production level of NAD+ in a biological sample obtained from said subject; ii) comparing the production level of NAD+ measured at step i) with its predetermined reference value, and iii) concluding that the subject is at risk of relapse to the treatment when the production level of NAD+ is higher than its predetermined reference value or concluding that the subject is not at risk of relapse when the production level of NAD+ is lower than its predetermined reference value.

4. The method according to claim 1, wherein the subject is or is susceptible to becoming resistant to the treatment.

5. The method according to claim 1, wherein the treatment is selected from the group consisting of: PLX4032, immunotherapy and combined treatment.

6. A method for treating a subject who has melanoma and is resistant to one or more melanoma treatments or is at risk of developing resistance to one or more melanoma treatments, comprising administering to said subject a therapeutically effective amount of an inhibitor of nicotinamide phosphoribosyl transferase (NAMPT).

7. The method according to claim 6, comprising, prior to the step of administering, determining whether the subject is resistant to the one or more melanoma treatments or is at risk of developing resistance to the one or more melanoma treatments, by i) measuring the activity and/or expression level of NAMPT in a biological sample obtained from the subject; ii) comparing the activity and/or expression level measured at step i) with a corresponding predetermined reference value, and, when the activity and/or expression level of NAMPT is higher than the corresponding predetermined reference value, then performing the step of administering.

8. The method according to claim 6, wherein the inhibitor of NAMPT is a small organic molecule.

9. The method according to claim 6, wherein the inhibitor of NAMPT is FK866.

10. The method according to claim 6, wherein the inhibitor of NAMPT is CHS 828.

11. The method according to claim 6, wherein the inhibitor of NAMPT is a siRNA.

12. The method according to claim 6, wherein the inhibitor of NAMPT is a MEK inhibitor.

13. The method according to claim 6, wherein the inhibitor of NAMPT is ERK inhibitor.

14. A method of screening a drug suitable for the treatment of resistance to a treatment for melanoma comprising i) providing a test compound and ii) determining the ability of said test compound to inhibit the activity of NAMPT.

Description

FIGURES

[0086] FIG. 1: PLX4032 decreases the levels of NAD+ in BRAFV600E melanoma cells.

[0087] (A) Scatter plot showing the means+/SD of the normalized NAD+ values in control and PLX4032-treated WM9 and UACC62 melanoma cells. (B) Intracellular NAD+ levels in a panel of BRAFV600E and WT BRAF human melanoma cells exposed or not to PLX4032 (5 M, 24 h). The values represent the means+/SD of five independent experiments.

[0088] FIG. 2: BRAF/MEK/ERK signaling pathway regulates nicotinamide phosphoribosyltransferase (NAMPT) expression. (A) NAMPT mRNA levels in BRAFV600E melanoma cells (A375, WM9, and SKme128) were measured using QPCR, under the same conditions. (B-C) Analysis of publicly available data sets GSE42872 and GSE20051 of melanoma cells exposed to PLX4032. Scatter plots showing the means+/SD of the NAMPT mRNA expression are shown.

[0089] FIG. 3: BRAF regulates NAMPT transcription through STAT5.

[0090] (A-B) Activity of NAMPT promoter reporter in A375 and WM9 cells respectively exposed to increasing doses of PLX4032 (5 M, 48 h). Data are shown as the means+/SD of 4 experiments. For A375, all the PLX4032 doses lead to significant decrease in NAMPT promoter activity (p<0.0034). For WM9, significant decrease in NAMPT promoter activity is observed with PLX4032>0.005, (p<0.014). (C) Activity of NAMPT promoter reporter vector in A375 cells exposed to MEK (GSK1120212 and U0126) or ERK (SCH772984) inhibitors. (D) Activity of NAMPT promoter segments with different lengths in A375 cells exposed to PLX4032. (E) Activity of NAMPT promoter reporter in 501MEL cells transfected with a control vector or a vector encoding BRAFV600E and left untreated or exposed to STAT5 inhibitor (STAT5i, 100 M, 48 h). For A to E, histograms (means+/SD, n=2, excepted for E, n=4) of relative luciferase activity, normalized to -galactosidase are shown. (F) Histograms (means+/SD, n=6) showing the NAD+ level in A375 and WM9 cells exposed to STAT5 inhibitor.

[0091] FIG. 4: NAMPT is required for melanoma proliferation and tumor development.

[0092] (A) Intracellular NAD+ levels in A375 and WM9 melanoma cells are treated with control siRNA or 2 different NAMPT siRNA or exposed to NAMPT inhibitor (FK866). HSP90 was used as loading control. Values represent the means+/SD of three independent experiments. (B) Proliferation of WM9 and A375 melanoma cells treated as in (A). Cells were trypsinized and counted each day. Values represent the means+/SD of three independent experiments. (C) Growth curve of tumor xenografts after subcutaneous injection of WM9 cells. Mice (6 per group) were treated or not with PXL4032 or FK866. Data are shown as the means+/SD of tumor volume. Black arrow indicates beginning of the treatment.

[0093] FIG. 5: NAMPT affects the sensitivity of melanoma cells to BRAF inhibitor.

[0094] (A) Intracellular NAD+ level in A375 and WM9 melanoma cells transduced with a control or NAMPT adenovirus. Values represent the means+/SD of three independent experiments. (B) A375 and WM9 melanoma cells transduced with a control or NAMPT adenovirus were exposed to PLX4032 (5 M). After 72 h, the cells were counted. The histogram represents the means+/SD of 3 independent experiments. (C) A375 melanoma cells resistant to PLX4032 (A375R) were transfected with control or NAMPT siRNA (si #1 and si #2) and subsequently exposed to PLX4032 (5 M). After 48 h, the cells were counted. The histogram represents the means+/SD of 3 independent experiments. (D) WM9 melanoma cells resistant to PLX4032 (WM9R) were transfected with control or NAMPT siRNA (si #1 and si #2) and subsequently exposed to PLX4032 (5 M). After 72 h, the cells were counted. The histogram represents the means+SD of three independent experiments. (E) Growth curve of tumor xenografts of PLX4032-resistant A375 melanoma cells (A375R) after subcutaneous injection). Mice (6 per group) were treated with vehicle, PLX4032 (25 mg/kg) or FK866 (1.5 mg/kg and 15 mg/kg) alone or with the low FK866 dose in combination with PLX4032. Data are presented as the means+/SD. Black arrow indicates beginning of the treatment. (F) Growth curve of tumor xenografts after subcutaneous injection of WM9 cells resistant to PLX4032 (WM9R). Mice (6 per group) were treated with vehicle, PLX4032 or FK866. Data are shown as the means+/SD of tumor volume. Black arrow indicates beginning of the treatment.

EXAMPLE

[0095] Material & Methods

[0096] Cell Cultures and Reagents

[0097] Human melanoma cell lines and short-term cultures derived from different patients with metastatic malignant melanoma cells were grown in DMEM supplemented with 7% FBS at 37 C. in a humidified atmosphere containing 5% CO2. PLX4032-sensitive and PLX4032-resistant melanoma cells were previously described (Bonet et al. 2012; Ohanna et al. 2014). Lipofectamine RNAiMAX and opti-MEM medium were purchased from Invitrogen (San Diego, Calif., USA). FK866 was obtained from Sigma, U0126 and GSK1120212 were purchased from Euromedex, and PD98059 and SCH77294 were obtained from Selleck Chemicals. The STAT5 inhibitor (CAS 285986-31-4) was purchased from Sigma. Intracellular NAD+ was measured using the NAD/NADH Quantitation Kit from Sigma according to the manufacturer's instructions.

[0098] Metabolomic Profiling

[0099] Briefly, samples were prepared using the automated MicroLab STAR system (Hamilton Company). Recovery standards were added prior to the first step in the extraction process for QC purposes. Cell lysates were precipitated using methanol under vigorous shaking for 2 min, followed by centrifugation. The resulting extract was divided into five fractions: two samples for analysis using two separate reversephase (RP)/UPLC-MS/MS methods with positive ion mode electrospray ionization (ESI), one sample for analysis through RP/UPLC-MS/MS with negative ion mode ESI, one sample for analysis through HILIC/UPLC-MS/MS with negative ion mode ESI, and one sample was reserved for backup. The samples were briefly placed on a TurboVap (Zymark) to remove the organic solvent. The sample extracts were stored overnight under nitrogen prior to preparation for analysis. Chromatography analyses were performed using Waters ACQUITY ultra-performance liquid chromatography (UPLC) and a Thermo Scientific Q-Exactive high resolution/accurate mass spectrometer interfaced with a heated electrospray ionization (HESI-II) source and Orbitrap mass analyzer operated at 35,000 mass resolution. The sample extract was dried and subsequently reconstituted in solvents compatible to each method (Miller et al. 2015). The informatics system comprised four major components, the Laboratory Information Management System (LIMS), the data extraction and peak identification software, data processing tools for QC and compound identification, and a collection of information interpretation and visualization tools for subsequent data analysis. The hardware and software foundations for these informatics components were the LAN backbone and a database server running Oracle 10.2.0.1 Enterprise Edition (Evans et al. 2009).

[0100] Western Blot Assays

[0101] Western blotting was performed as previously described (Hilmi et al. 2008; Bertolotto et al. 2011). Briefly, cell lysates (30 g) were separated using SDS-PAGE, transferred onto a PVDF membrane and subsequently exposed to the appropriate antibodies, anti-ERK2 (Santa Cruz Biotechnology, sc-1647 clone D-2), anti-phospho-ERK1/2 (Thr202/Tyr204) (Cell Signaling Technology Inc., #2370), anti-BRAF (Santa Cruz Biotechnology, sc-5284 clone F-7), anti-NAMPT (Sigma, #B5812), antiphospho-STAT5 (Tyr694) (Ozyme #9351), anti-STAT5 (Ozyme #9363), and anti-HSP90 (Santa Cruz biotechnology, #sc-13119). The proteins were visualized using the ECL system (Amersham). The western blots shown are representative of at least 3 independent experiments.

[0102] Transient Transfection of siRNA

[0103] Briefly, a single pulse of 50 nM of siRNA was administered to the cells at 50% confluency through transfection with 5 l of Lipofectamine RNAiMAX in Opti-MEM medium (Invitrogen, San Diego, Calif., USA). NAMPT siRNAs (ON TARGET plus, Dharmacon) were obtained from Thermo Fisher Scientific.

[0104] Cell Proliferation.

[0105] The cells were seeded onto 12-well dishes (10103 cells), and at 48 h post transfection or treatment, the cells were trypsinized from days 1 to 4, counted in triplicate using a hemocytometer. The experiments were performed at least three times.

[0106] Luciferase Reporter Assays

[0107] NAMPT promoter luciferase reporters were provided by Dr. J.G.N. Garcia (University of Arizona). We used 3 constructs containing the following regions of human NAMPT: 2682/+346; 1182/+346; and 582/+346 base pairs (Sun et al. 2014). A375 melanoma cells were transiently transfected as previously described using Lipofectamine reagent (Invitrogen) (Bertolotto et al. 1998). Briefly, the cells were transiently transfected with 0.3 g of NAMPT reporter constructs and 0.05 g of pCMVBGal to control the variability in transfection efficiency. The transfection medium was changed after 6 h, and where indicated, the cells were transfected with an empty vector or a vector encoding BRAFV600E or treated with PLX4032 or STAT5 inhibitor. The cells were assayed for luciferase and -galactosidase activities after 48 h. The experiments were repeated at least three times.

[0108] Colony Formation Assay

[0109] Human melanoma cells were seeded onto 6-well plates. The cells were subsequently placed in a 37 C., 5% CO2 incubator. Colonies of cells were grown before being stained with 0.04% crystal violet/2% ethanol in PBS for 30 min. Photographs of the stained colonies were captured. The colony formation assay was performed in duplicate.

[0110] Cell Death Analysis by Flow Cytometry

[0111] Cells were seeded at a density of 50 000 cells/well, in 24-well plate and treated with FK866 for indicated time. Cells were harvested using Accutase enzyme, washed twice with ice-cold phosphate-buffered saline, resuspended in medium with DAPI (1 g/ml) and incubated for 15 minutes at room temperature (25 C.) in the dark. Samples were immediately analyzed by a flow cytometer (MACS QUANT) using a laser at 405 nm excitation with a bandpass filter at 425 nm and 475 nm for DAPI detection.

[0112] mRNA Preparation and Real-Time/Quantitative PCR

[0113] The mRNA was isolated using TRIzol (Invitrogen) according to a standard procedure. QRT-PCR was performed using SYBR Green I (Eurogentec, Seraing, Belgium) and Multiscribe Reverse Transcriptase (Applied Biosystems) and subsequently monitored using the ABI Prism 7900 Sequence Detection System (Applied Biosystems, Foster City, Calif.). The detection of the SB34 gene was used to normalize the results. Primer sequences for each cDNA were designed using either Primer Express Software (Applied Biosystems) or qPrimer depot (http://primerdepot.nci.nih.gov), and these sequences are available upon request.

[0114] Animal Experimentation

[0115] Animal experiments were performed in accordance with French law and approved by a local institutional ethical committee. The animals were maintained in a temperature controlled facility (22 C.) on a 12-h light/dark cycle and provided free access to food (standard laboratory chow diet from UAR, Epinay-S/Orge, France). Human WM9 melanoma cells, responsive or resistant to PLX4032 (2106 cells), were subcutaneously inoculated into 8-week-old female, immune-deficient, athymic, nude FOXN1nu mice (Harlan Laboratory). When the tumors became palpable (0.1-0.2 cm3), the mice received an intraperitoneal injection of PLX4032 (25 mg/kg), FK866 (15 mg/kg) or both drugs dissolved in a mixture of Labrafil M1944 Cs, dimethylacetamide, and Tween 80 (90:9:1, v/v/v) three times per week. Control mice were injected with Labrafil alone. The growth tumor curves were determined after measuring the tumor volume using the equation V=(LW2)/2. At the end of the experiment, the mice were euthanized by cervical dislocation, and the tumors were harvested for immunofluorescence.

[0116] Immunofluorescence Studies

[0117] Frozen sections of melanoma xenografts were fixed with 4% paraformaldehyde (PFA, Sigma-Aldrich) for 15 min and subsequently blocked with 10% normal goat serum (Vector) with or without 0.1% Triton X-100 (Bio-Rad) in PBS for 30 min at room temperature. The samples were incubated with primary antibodies overnight at +4 C. followed by the appropriate secondary fluorescent-labeled antibodies (Invitrogen Molecular Probes) for 1 h at room temperature and mounted using Gel/Mount (Biomeda Corp., Foster City, Calif.). The nuclei were counterstained with DAPI. Apoptosis in melanoma xenografts was detected through a TUNEL assay using an in-situ cell apoptosis kit (R&D Systems). Immunofluorescence was examined and photographed using a Zeiss Axiophot microscope equipped with epifluorescence illumination.

[0118] Statistical Analysis

[0119] The data are presented as the meansSD and analyzed using two-sided Student's test with Prism or Microsoft Excel software. The difference between both conditions was statistically significant at p<0.05. For the metabolomics analysis, the p values were adjusted using the Benjamini-Hochberg procedure (Anastats). Supplementary information is available at Cell research's website.

[0120] Results

[0121] Alteration of Metabolism by PLX4032 in BRAFV600E Melanoma Cells.

[0122] To fully elucidate the effect of PLX4032 on the metabolism of melanoma cells, we performed global metabolic profiling using Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectroscopy of two distinct human melanoma cell lines (UACC62 and WM9) that harbor the BRAFV600E mutation. Analysis of more than 500 biochemicals identified 93 metabolites altered by treatment with PLX4032. A total of 45 metabolites were downregulated, whereas 48 metabolites were upregulated. The heat map shows the 40 most significantly regulated metabolites. Further analysis identified (data not shown) the downregulation of 4 biochemicals in glycolysis and 3 compounds in the TCA cycle indicating the inhibition of the carbohydrate metabolism and energy production. Twelve dipeptides, 6 monoacylglycerols, 6 long chain fatty acids (PUFA) and 4 sphingolipids were upregulated upon PLX4032 treatment, suggesting a global increase in protein catabolism and lipid synthesis. In addition, 6 metabolites in the methionine, cysteine and taurine pathways and 7 in the purine metabolic pathway were either up or down regulated by PLX4032. A schematic view of the metabolic consequences of BRAF inhibition is provided (data not shown).

[0123] Among the other metabolites, we observed that nicotinamide adenine dinucleotide (NAD+) levels were reduced in both melanoma cell lines exposed to PLX4032 (FIG. 1A). We extended these analyses to a larger panel of melanoma cell lines and short-term melanoma cell cultures. As expected, PLX4032 inhibited cell proliferation in BRAFV600E but not in BRAFWT human melanoma cells (data not shown). Importantly, PLX4032 reduced intracellular NAD+ levels in all BRAFV600E-mutated melanoma cell lines and short-term melanoma cell cultures but not in BRAFWT human melanoma cells (FIG. 1B). Further, additional BRAF inhibitors as well as MEK and ERK inhibitors also decreased NAD+ levels, indicating that ERK pathway inhibition impacted NAD+ metabolism in A375 melanoma cells (data not shown).

[0124] BRAFV600E Regulates NAD+ Levels and Controls NAMPT Expression.

[0125] The essential of NAD+ as co-factor for multiple metabolic pathways, prompted us to investigate the mechanism by which PLX4032 regulate the level of NAD+ in melanoma cells. The NAD+ level is primarily maintained in human cells via the salvage pathway in which NAMPT is the rate-limiting enzyme (Canto et al. 2015). Our results indicated that PLX4032 reduced NAMPT expression at both the protein (data not shown) and mRNA levels (FIG. 2A) in 4 different BRAFV600E melanoma cells. No inhibition of NAMPT expression by PLX4032 was observed in BRAFWT human melanoma cells (data not shown). Analysis of publicly available microarray data sets (GSE20051 and GSE42872) from other sources confirmed the inhibition of NAMPT mRNA expression in BRAFV600E melanoma cell lines exposed to BRAF inhibitor (FIGS. 2B-2C). The inhibition of NAMPT expression in A375 melanoma cells was also observed with the MEK inhibitors U0126, PD98059 and GSK1120212 (Trametinib) and the ERK inhibitor, SCH77294, which efficiencies were shown on the inhibition of ERK phosphorylation levels (data not shown). We also observed the inhibition of NAMPT expression in publicly available microarray data (GSE51115) of melanoma cell lines exposed to the MEK inhibitor, PD0325901 (data not shown). Additionally, forced expression of BRAFV600E in normal human melanocytes stimulated the ERK signaling pathway and increased levels of NAMPT (data not shown), which were prevented using MEK and ERK inhibitors. Altogether, our data demonstrate that the BRAF/MEK/ERK signaling cascade plays a key role in the control of NAMPT expression and the regulation of NAD+ metabolism in melanoma cells.

[0126] Regulation of NAMPT Transcription Through the BRAF-STAT5 Signaling Cascade.

[0127] Changes in NAMPT mRNA levels suggested that the BRAF/ERK pathway controlled NAMPT at the transcriptional level. Using a human NAMPT promoter luciferase reporter construct, we showed that PLX4032 induced a dose-dependent decrease in NAMPT promoter activity in both A375 and WM9 cells (FIGS. 3A-B). MEK and ERK inhibitors also strongly reduced NAMPT promoter activity (FIG. 3C). To identify the regulatory elements, we assessed the effect of PLX4032 on human NAMPT promoter constructs of different lengths. The results revealed that the BRAF/ERK responsive element was localized between 1182 and 2682 base pairs upstream of the transcriptional start site (FIG. 3D). Within this region, Sun et al. identified a STAT5 site that mediated the mechanical stress signal to upregulate NAMPT gene transcription (Sun et al. 2014). Therefore, we hypothesized that in melanoma cells, the ERK pathway might control NAMPT expression through the activation of STAT5. We showed that STAT5 inhibitor decreased both basal and BRAFV600E-stimulated NAMPT promoter activity, thereby demonstrating the involvement of STAT5 in the BRAFV600E-induced stimulation of NAMPT transcription (FIG. 3E). In both A375 and WM9 cells, we observed that PLX4032 inhibited both ERK and STAT5 phosphorylation, while STAT5 inhibitor efficiently reduced STAT5 phosphorylation but did not affect ERK phosphorylation (FIG. 3F). Both BRAF and STAT5 inhibitors decreased NAMPT expression (data not shown) and NAD+ levels (FIG. 1D and FIG. 3F). Taken together, these data demonstrate that the BRAF/ERK pathway regulates NAMPT expression, and consequently NAD+ levels, at the transcriptional level through STAT5 activation.

[0128] NAMPT Controls Melanoma Cell Proliferation.

[0129] To determine the impact of NAD+ metabolism on melanoma cell proliferation, we used FK866, a highly specific non-competitive inhibitor of NAMPT, and NAMPT siRNA. As expected, 2 different NAMPT siRNAs efficiently inhibited NAMPT expression in both A375 and WM9 cells (data not shown). FK866 did not affect NAMPT expression. Both FK866 and NAMPT siRNAs dramatically decreased the intracellular levels of NAD+, in both A375 and WM9 melanoma cell lines (FIG. 4A) and impaired cell proliferation (FIG. 4B). Interestingly, the addition of exogenous NAD+ to the culture medium reversed the effects of FK866, thereby demonstrating that the effect of FK866 was causally associated with NAD+ depletion and not to non-specific effects (data not shown). Further, flow cytometry analysis of DAPI permeable cells showed that NAMPT inhibition by FK866 or siRNA increased cell death (data not shown). We next monitored the effect of FK866 in vivo. WM9 cells were subcutaneously engrafted into 6-week-old female athymic nude mice. When the tumors became palpable (0.1-0.2 cm3), the mice were treated with FK866 at a dose of 15 mg/kg or with its vehicle (labrafil) every two days. Compared with vehicle treatment, FK866 treatment impaired WM9 melanoma cell xenograft growth (FIG. 4C). FK866 caused no weight loss in the treated mice (not shown). Thus, the inhibition of NAMPT has clear anti-melanoma effects in vitro and in vivo.

[0130] NAMPT Affects the Sensitivity of Melanoma Cells to BRAF Inhibitor.

[0131] We next examined whether NAMPT might affect the response to PLX4032. Forced expression of NAMPT (data not shown) enhanced the intracellular NAD+ level (FIG. 5A) but did not significantly affect proliferation (data not shown). As expected, PLX4032 inhibited the viability of A375 and WM9 melanoma cells. Forced expression of NAMPT markedly decreased the efficiency of PLX4032 to inhibit the proliferation of melanoma cells (FIG. 5B). These observations prompted us to hypothesize that NAMPT inhibition might impact the sensitivity of BRAFi-resistant melanoma cells to PLX4032. In agreement with this hypothesis, we observed that NAMPT silencing strongly inhibited the proliferation of BRAFi-resistant A375 melanoma cells (A375R) and BRAFi-resistant WM9 melanoma cells (WM9R) (FIG. 5C and FIG. 5D). Interestingly, although NAMPT inhibition had a marked effect on BRAFi-resistant cells, we observed the statistically significant restoration of PLX4032 sensitivity in BRAFi-resistant cells, upon depletion of NAMPT (FIG. 5C and FIG. 5D). In vivo, xenografts derived from BRAFi-resistant WM9 melanoma cells were not affected by PLX4032 but were highly sensitive to NAMPT inhibition by FK866: FK866 inhibited tumor growth (FIG. 5F). We also performed in vivo experiments on A375R cells. In contrast to PLX4032, increasing doses of FK866 dose-dependently reduced melanoma progression (FIG. 5E). As expected, a reduction in NAD.sup.+ was observed in the FK866-treated tumors (data not shown). The combination of both PLX4032 and FK866 resulted in a synergistic inhibition of melanoma growth. Thus, both in vitro and in vivo, FK866 treatment caused melanoma cell death and restored PLX4032 sensitivity in BRAFi-resistant melanoma cells.

[0132] Nevertheless, the striking effect of NAMPT inhibition on BRAFi-resistant melanoma xenografts suggests that targeting NAMPT might be a valid therapeutic option to overcome BRAFi resistance.

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