Dependence On Glycolysis Sensitizes BRAF-mutated Melanomas For Increased Response To Targeted BRAF Inhibition

www.nature.com/scientificreports OPEN received: 24 October 2016 accepted: 11 January 2017 Published: 16 February 2017 Dependence On Glycolysis Sensitizes BRAF-mutated Melanomas For Increased Response To Targeted BRAF Inhibition Keisha N. Hardeman1,2, Chengwei Peng1,2, Bishal B. Paudel2,3, Christian T. Meyer2, Thong Luong4, Darren R. Tyson1,2, Jamey D. Young5, Vito Quaranta1,2 & Joshua P. Fessel1,4 Dysregulated metabolism can broadly affect therapy resistance by influencing compensatory signaling and expanding proliferation. Given many BRAF-mutated melanoma patients experience disease progression with targeted BRAF inhibitors, we hypothesized therapeutic response is related to tumor metabolic phenotype, and that altering tumor metabolism could change therapeutic outcome. We demonstrated the proliferative kinetics of BRAF-mutated melanoma cells treated with the BRAF inhibitor PLX4720 fall along a spectrum of sensitivity, providing a model system to study the interplay of metabolism and drug sensitivity. We discovered an inverse relationship between glucose availability and sensitivity to BRAF inhibition through characterization of metabolic phenotypes using nearly a dozen metabolic parameters in Principle Component Analysis. Subsequently, we generated rho0 variants that lacked functional mitochondrial respiration and increased glycolytic metabolism. The rho0 cell lines exhibited increased sensitivity to PLX4720 compared to the respiration-competent parental lines. Finally, we utilized the FDA-approved antiretroviral drug zalcitabine to suppress mitochondrial respiration and to force glycolysis in our cell line panel, resulting in increased PLX4720 sensitivity via shifts in EC50 and Hill slope metrics. Our data suggest that forcing tumor glycolysis in melanoma using zalcitabine or other similar approaches may be an adjunct to increase the efficacy of targeted BRAF therapy. Melanoma is the most malignant form of skin cancer, and roughly 50% of clinical isolates have a mutation in the BRAF kinase of the mitogen-activated protein kinase (MAPK) pathway1,2. Ninety percent of those BRAF mutations are missense mutations that change the valine at position 600 to glutamic acid (V600E) or aspartic acid (V600D)3. The mutation confers constitutive activation of the BRAF kinase and drives oncogenic signaling through MEK phosphorylation. Targeted therapies against the mutant BRAF have prolonged progression-free survival and overall survival in Phase III clinical trials4. Unfortunately, most patients will exhibit some degree of disease progression while treated with a BRAF inhibitor, with nearly 50% of patients progressing after only 6 to 7 months of initial treatment5. There have been a variety of mechanisms that underlie initial and acquired drug resistance described in the literature. Generally, mechanisms of resistance to anti-BRAF therapies are put into MEK-dependent and MEK-independent categories. MEK-dependent mechanisms include mutations in NRAS, MEK1 and MEK26, loss of RAS regulation by NF17,8, COT overexpression driving MEK signaling9, and genetic alterations in BRAF itself, such as truncation or amplification10. MEK-independent mechanisms of resistance include receptor tyrosine kinase protein and ligand overexpression, such as cMET, IGF1R, and PDGFRβ6, and signaling through PI3K11. Unfortunately, more than 40% of the resistance found in patients who progressed on targeted therapy cannot be 1 Department of Cancer Biology, Vanderbilt University School of Medicine, 2220 Pierce Avenue, Nashville, TN 37232, USA. 2Center for Cancer Systems Biology at Vanderbilt, Vanderbilt University School of Medicine, 2220 Pierce Avenue, Nashville, TN 37232, USA. 3Chemical and Physical Biology Graduate Program, Vanderbilt University, Nashville, TN, 37232, USA. 4Departments of Medicine & Pharmacology, Division of Allergy, Pulmonary, and Critical Care Medicine, Vanderbilt University School of Medicine, 21st Avenue South, Nashville, TN 37232, USA. 5 Departments of Chemical Biomolecular Engineering, and Molecular Physiology & Biophysics, Vanderbilt University, Nashville, TN 37232, USA. Correspondence and requests for materials should be addressed to J.P.F. (email: ) Scientific Reports | 7:42604 | DOI: 10.1038/srep42604 1 www.nature.com/scientificreports/ attributed to any of these mechanisms12. One of the features common to all of the known pathways that contribute to resistance is that they exert direct or indirect control of multiple cellular metabolic pathways—contributing to the single “hallmark” of metabolic reprogramming. In the last several years, there has been an increasingly intense focus on tumor metabolism as an exploitable therapeutic avenue13–16, with the success of asparaginase in the treatment of acute lymphoblastic leukemia (ALL) being just one example that has achieved widespread clinical use17,18, and with many other metabolism-based therapies under active development19,20. Dysregulated metabolism in cancer has been shown to affect treatment outcome via multiple pathways, including the activation of compensatory receptor tyrosine kinase signaling to bypass molecular targeted therapies, the repression of pro-apoptotic signaling, and limitation of drugs’ access to molecular targets through active and passive mechanisms20. Komurov et al. showed chronic lapatinib treatment of HER2+breast cancer cell lines produced cells with an advanced nutrient starvation phenotype21. Furthermore, the cells were sensitive to the antihelminthic pyrvinium pamoate, which targets mitochondrial function under various conditions22, particularly glucose deprivation23. Recently, it has been shown in BRAF-mutated melanoma that chronic treatment with BRAF inhibitor induces glutamine dependence that correlates with drug resistance24,25. We were interested in the prospect that the molecular metabolic landscape of any individual tumor might have a direct relationship to its sensitivity to targeted therapies. The same metabolic pathways that have been targets for investigation in other malignancies have also been explored in BRAF-mutated melanoma, but a consensus of the major metabolic program exhibited by BRAF-mutated melanomas, or even whether a single dominant metabolic program exists, is lacking. BRAF-mutated melanomas have conversely been characterized as exhibiting primarily aerobic glycolysis26 or oxidative phosphorylation27,28. Moreover, the relationship between metabolic program and therapeutic response in BRAF-mutated melanoma is poorly understood, so we set out to probe the phenotypic relationship of metabolism and responses to the BRAF inhibitor vemurafenib. In the present study, we used a panel of human BRAF-mutated melanoma cell lines to demonstrate in vitro variability in response to PLX4720, a BRAF inhibitor and analogue of vemurafenib. Utilizing our previously described method for measuring proliferative rate under various treatment conditions29, we calculated a metric describing the depend (...truncated)