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Identification of mechanisms underlying sensitivity and response to targeted therapies, such as the BRAF inhibitor vemurafenib, is critical in order to improve efficacy of these therapies in the clinic and delay onset of resistance. Glycolysis has emerged as a key feature of the BRAF inhibitor response in melanoma cells, and importantly, the metabolic response to vemurafenib in melanoma patients can predict patient outcome. Here, we present a multiparameter genome-wide siRNA screening dataset of genes that when depleted improve the viability and glycolytic response to vemurafenib in BRAFV600 mutated melanoma cells. These datasets are suitable for analysis of genes involved in cell viability and glycolysis in steady state conditions and following treatment with vemurafenib, as well as computational approaches to identify gene regulatory networks that mediate response to BRAF inhibition in melanoma., Measurement(s) cell viability • glycolytic process Technology Type(s) immunofluorescence microscopy assay • absorbance Factor Type(s) drug treatment • gene silencing Sample Characteristic - Organism Homo sapiens Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.12885593

Despite the success of therapies targeting oncogenes in cancer, clinical outcomes are limited by residual disease that ultimately results in relapse. This residual disease is often characterized by non-genetic adaptive resistance, that in melanoma is characterised by altered metabolism. Here, we examine how targeted therapy reprograms metabolism in BRAF-mutant melanoma cells using a genome-wide RNA interference (RNAi) screen and global gene expression profiling. Using this systematic approach we demonstrate post-transcriptional regulation of metabolism following BRAF inhibition, involving selective mRNA transport and translation. As proof of concept we demonstrate the RNA processing kinase U2AF homology motif kinase 1 (UHMK1) associates with mRNAs encoding metabolism proteins and selectively controls their transport and translation during adaptation to BRAF-targeted therapy. UHMK1 inactivation induces cell death by disrupting therapy induced metabolic reprogramming, and importantly, delays resistance to BRAF and MEK combination therapy in multiple in vivo models. We propose selective mRNA processing and translation by UHMK1 constitutes a mechanism of non-genetic resistance to targeted therapy in melanoma by controlling metabolic plasticity induced by therapy.

Despite the success of therapies targeting oncogenes in cancer, clinical outcomes are limited by a residual disease that results in relapse. This residual disease is characterized by drug-induced adaptation, that in melanoma includes altered metabolism. Here, we examined how targeted therapy reprograms metabolism in BRAF-mutant melanoma cells using a genome-wide RNAi screen and global gene expression profiling. This systematic approach revealed post-transcriptional regulation of metabolism following BRAF inhibition, involving selective mRNA transport and translation. As proof of concept we demonstrate the RNA binding kinase UHMK1 interacts with mRNAs that encode metabolic proteins and selectively controls their transport and translation during adaptation to BRAF targeted therapy. Inactivation of UHMK1 improves metabolic response to BRAF targeted therapy and delays resistance to BRAF and MEK combination therapy in vivo. Our data support a model wherein post-transcriptional gene expression pathways regulate metabolic adaptation underpinning targeted therapy response and suggest inactivation of these pathways may delay disease relapse.

Deregulated glucose metabolism fulfills the energetic and biosynthetic requirements for tumor growth driven by oncogenes. Because inhibition of oncogenic BRAF causes profound reductions in glucose uptake and a strong clinical benefit in BRAF-mutant melanoma, we examined the role of energy metabolism in responses to BRAF inhibition. We observed pronounced and consistent decreases in glycolytic activity in BRAF-mutant melanoma cells. Moreover, we identified a network of BRAF-regulated transcription factors that control glycolysis in melanoma cells. Remarkably, this network of transcription factors, including hypoxia-inducible factor-1α, MYC, and MONDOA (MLXIP), drives glycolysis downstream of BRAFV600, is critical for responses to BRAF inhibition, and is modulated by BRAF inhibition in clinical melanoma specimens. Furthermore, we show that concurrent inhibition of BRAF and glycolysis induces cell death in BRAF inhibitor (BRAFi)–resistant melanoma cells. Thus, we provide a proof-of-principle for treatment of melanoma with combinations of BRAFis and glycolysis inhibitors. Significance: BRAFis suppress glycolysis and provide strong clinical benefit in BRAFV600 melanoma. We show that BRAF inhibition suppresses glycolysis via a network of transcription factors that are critical for complete BRAFi responses. Furthermore, we provide evidence for the clinical potential of therapies that combine BRAFis with glycolysis inhibitors. Cancer Discov; 4(4); 423–33. ©2014 AACR. See related commentary by Haq, p. 390 This article is highlighted in the In This Issue feature, p. 377

Background/Aims: After BRAF, NRAS is the most commonly mutated oncogene in melanoma. However therapies targeting NRAS remain elusive. Recent investigations have demonstrated that mutated RAS can increase glycolytic metabolism in cancer cells, and our previous work has elucidated a role for glycolysis in the sensitivity of BRAF-mutant melanoma cells to inactivation of BRAF(1). This study aims to identify the molecular pathways controlling glycolysis in NRAS-mutant melanoma in the context of therapeutic targeting of critical oncogenic pathways. Methods: A panel of NRAS and BRAF-mutant melanoma cell lines were screened for changes in viability and glycolysis following treatment with a panel of small molecule inhibitors targeting the MAPK and PI3K pathways. Glycolytic responses of melanoma cells, following MEK, ERK, selective-PI3K, AKT, mTOR or combined PI3K/mTOR inhibition (alone or in combination), were investigated by assessing cell proliferation, glucose uptake, lactate production, and known transcriptional regulators of glycolysis. Results: In NRAS-mutant melanoma, MEK and ERK inhibition resulted in a reduction in lactate production, and similar responses were observed following treatment with mTORC1/2 and PI3K/mTOR inhibitors but not AKT or selective PI3K inhibitors. Treatment with MEK or ERK inhibitors resulted in a consistent reduction in glucose uptake, with a more modest effect being observed following combined PI3K/mTOR inhibition. Treatment with a combination of MEK or ERK inhibitor with either an mTORC1/2 or PI3K/mTOR inhibitor, resulted in synergy with respect to a reduction in cell proliferation. However, synergy was not demonstrated when lactate production and glucose uptake were assessed indicating that inhibition of MEK or ERK was sufficient to inhibit glycolysis in NRAS-mutant cells. Notably, treatment with these inhibitors also resulted in altered expression of transcriptional regulators of glycolysis (reduced MYC and HIF1α, increased TXNIP protein expression via MondoA), similar to that seen in BRAF-mutant melanoma cells. The role of each individual transcriptional regulator of glycolysis in the response of NRAS-mutant cells to MEK/ERK inhibition is under investigation. Conclusion: NRAS-mutant melanoma cells demonstrate a similar glycolytic phenotype to BRAF-mutant melanoma cells, with MAPK pathway or mTOR inhibition resulting in decreased lactate production, glucose uptake and modulation of the MYC/HIF1α/MondoA network of transcriptional regulators of glycolysis. These findings identify the mechanism of oncogene-driven glycolysis in NRAS-mutant melanoma, and open new possibilities for targeting glycolysis as a therapeutic target in the treatment of patients with NRAS-mutant melanoma. References: 1. Parmenter TJ, Kleinschmidt M, Kinross KM et al. Response of BRAF mutant melanoma to BRAF inhibition is mediated by a network of transcriptional regulators of glycolysis. Cancer Discovery, 2014;4:423-433. Citation Format: Aparna D. Rao, Lorey K. Smith, Tiffany J. Parmenter, Grant A. McArthur. Mutant NRAS regulates glycolysis in melanoma through ERK, mTOR and the MYC/HIF1α/MondoA network of transcriptional regulators. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr B23.

The BRAF mutant, BRAF(V600E), is expressed in nearly half of melanomas, and oral BRAF inhibitors induce substantial tumor regression in patients with BRAF(V600E) metastatic melanoma. The inhibitors are believed to work primarily by inhibiting BRAF(V600E)-induced oncogenic MAPK signaling; however, some patients treated with BRAF inhibitors exhibit increased tumor immune infiltration, suggesting that a combination of BRAF inhibitors and immunotherapy may be beneficial. We used two relatively resistant variants of Braf(V600E)-driven mouse melanoma (SM1 and SM1WT1) and melanoma-prone mice to determine the role of host immunity in type I BRAF inhibitor PLX4720 antitumor activity. We found that PLX4720 treatment downregulated tumor Ccl2 gene expression and decreased tumor CCL2 expression in both Braf(V600E) mouse melanoma transplants and in de novo melanomas in a manner that was coincident with reduced tumor growth. While PLX4720 did not directly increase tumor immunogenicity, analysis of SM1 tumor-infiltrating leukocytes in PLX4720-treated mice demonstrated a robust increase in CD8(+) T/FoxP3(+)CD4(+) T cell ratio and NK cells. Combination therapy with PLX4720 and anti-CCL2 or agonistic anti-CD137 antibodies demonstrated significant antitumor activity in mouse transplant and de novo tumorigenesis models. These data elucidate a role for host CCR2 in the mechanism of action of type I BRAF inhibitors and support the therapeutic potential of combining BRAF inhibitors with immunotherapy.