Author Manuscript Published OnlineFirst on August 2, 2019; DOI: 10.1158/1078-0432.CCR-19-0253 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. A fatty acid oxidation-dependent metabolic shift regulates the adaptation of BRAF- mutated melanoma to MAPK inhibitors Andrea Aloia1,*, Daniela Müllhaupt1, Christophe D. Chabbert1, £, Tanja Eberhart1, Stefanie Flückiger- Mangual1, Ana Vukolic1, Ossia Eichhoff2, Anja Irmisch2, Leila T. Alexander3, §, Ernesto Scibona4, Dennie T. Frederick5, Benchun Miao5, Tian Tian6, Chaoran Cheng6, Lawrence N. Kwong7, Zhi Wei6, Ryan J. Sullivan5, Genevieve M. Boland8, Meenhard Herlyn9, Keith T. Flaherty5, Nicola Zamboni3, Reinhard Dummer2, Gao Zhang9, #, Mitchell P. Levesque2, $, Wilhelm Krek1, †, Werner J. Kovacs1, 10, $, * 1ETH Zurich, Institute of Molecular Health Sciences, 8093 Zurich, Switzerland 2University Hospital Zurich, Department of Dermatology, 8091 Zurich, Switzerland 3ETH Zurich, Institute of Molecular System Biology, 8093 Zurich, Switzerland 4ETH Zurich, Institute of Chemical and Bioengineering, 8093 Zurich, Switzerland 5Massachusetts General Hospital Cancer Center, Boston, MA 02114, USA 6Department of Computer Science, New Jersey Institute of Technology, Newark, NJ 07102, USA 7Department of Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX 770303, USA 8Department of Surgery, Massachusetts General Hospital Cancer Center, Boston, MA 02114, USA 9Molecular and Cellular Oncogenesis Program and Melanoma Research Center, The Wistar Institute, Philadelphia, PA 19104, USA 10Lead Contact $Co-senior authors £Current address: Roche Innovation Center Zurich, 8952 Schlieren, Switzerland §Current address: SIB Swiss Institute of Bioinformatics, Personalized Health Informatics, 4056 Basel, Switzerland #Current address: Department of Neurosurgery, The Preston Robert Tisch Brain Tumor Center, and Department of Pathology, Duke University Medical Center, Durham, NC 27710, USA †Deceased 29. August 2018 1 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 2, 2019; DOI: 10.1158/1078-0432.CCR-19-0253 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. *Correspondence to: Werner Kovacs, Institute of Molecular Health Sciences, ETH Zurich, Otto-Stern- Weg 7, HPL H16.1, CH-8093 Zurich, Switzerland. Tel.: +41 44 633 3084. E-mail: [email protected] *Correspondence to: Andrea Aloia, Institute of Molecular Health Sciences, ETH Zurich, Otto-Stern- Weg 7, HPL H23.2, CH-8093 Zurich, Switzerland. Tel.: +41 44 633 3360. E-mail: [email protected] Running title MAPKi induce fatty acid oxidation in BRAFV600E melanomas Keywords BRAF-mutated melanoma, adaptive drug resistance, fatty acid oxidation, CD36, MAPK inhibitors 2 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 2, 2019; DOI: 10.1158/1078-0432.CCR-19-0253 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abstract Purpose: Treatment of BRAFV600E-mutant melanomas with mitogen-activated protein kinase inhibitors (MAPKi) results in significant tumor regression, but acquired resistance is pervasive. To understand non-mutational mechanisms underlying the adaptation to MAPKi and to identify novel vulnerabilities of melanomas treated with MAPKi, we focused on the initial response phase during treatment with MAPKi. Experimental Design: By screening proteins expressed on the cell surface of melanoma cells, we identified the fatty acid transporter CD36 as the most consistently upregulated protein upon short-term treatment with MAPKi. We further investigated the effects of MAPKi on fatty acid metabolism using in vitro and in vivo models and analyzing patients’ pre- and on-treatment tumor specimens. Results: Melanoma cells treated with MAPKi displayed increased levels of CD36 and of peroxisome proliferator-activated receptor (PPAR)-mediated and carnitine palmitoyltransferase 1A (CPT1A)- dependent fatty acid oxidation (FAO). While CD36 is a useful marker of melanoma cells during adaptation and drug-tolerant phases, the upregulation of CD36 is not functionally involved in FAO changes that characterize MAPKi-treated cells. Increased FAO is required for BRAFV600E-mutant melanoma cells to survive under the MAPKi-induced metabolic stress prior to acquiring drug resistance. The upfront and concomitant inhibition of FAO, glycolysis and MAPK synergistically inhibits tumor cell growth in vitro and in vivo. Conclusions: Thus, we identified a clinically relevant therapeutic approach that has the potential to improve initial responses and to delay acquired drug resistance of BRAFV600E-mutant melanoma. 3 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 2, 2019; DOI: 10.1158/1078-0432.CCR-19-0253 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Translational Relevance Transiently resistant cells adapting to mitogen-activated protein kinase inhibitors (MAPKi) are responsible for acquired drug resistance. We identified CD36 as a marker of transiently resistant and MAPKi-tolerant BRAFV600E melanoma cells. In addition, we describe an early metabolic reprogramming mechanism induced by MAPKi through increased FAO which is required to survive MAPKi-induced metabolic stress prior to acquiring drug resistance. FAO inhibitors increase the glycolytic flux in untreated and MAPKi-treated melanoma cells as a compensatory mechanism to FAO inhibition. To exploit melanoma cells’ metabolic plasticity for therapeutic intervention, we propose a triple combination of MAPK, FAO and glycolytic inhibitors as a novel treatment option to prevent drug resistance in BRAF-mutated melanomas. DECLARATION OF INTEREST CDC is a full-time employee of Roche AG and a shareholder in AstraZeneca. Other authors declare no competing interests. 4 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 2, 2019; DOI: 10.1158/1078-0432.CCR-19-0253 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Introduction Malignant melanoma is an aggressive skin tumor with poor prognosis due to metastasis and therapeutic resistance (1). Approximately 50% of melanoma patients carry gain-of-function mutations in the BRAF gene (2). The most frequent mutation is the substitution of valine at position 600 by glutamic acid (BRAFV600E) resulting in constitutive activation of the mitogen-activated protein kinase (MAPK) signal transduction pathway (3). BRAF plays a pivotal role in the MAPK pathway by promoting cell division, proliferation, and survival (4). Since BRAF is a critical mediator of melanomagenesis, BRAFV600E inhibitors (BRAFi) and MEK/ERK kinase inhibitors (MEKi) were developed to target key components of the MAPK signaling pathway and induce cell death (5,6). BRAFi alone or in combination with MEKi have shown clinical efficacy in BRAF-mutated metastatic melanoma patients (7,8). However, drug resistance inevitably develops and most patients relapse within a few months with limited benefits. Drug resistance represents a common complication of targeted therapies hampering long-term treatment success (9). To date, numerous mechanisms of acquired drug resistance have already been identified in metastatic melanoma patients (10). Mechanisms of therapy resistance are quite heterogeneous because they can be different among patients (inter-patient heterogeneity), co-exist in tumors of the same patient (intra-patient heterogeneity), and be identified within a single tumor (intra-tumor heterogeneity) (11). Resistance can be classified as pre-existing in cells that are insensitive to treatments (primary or intrinsic resistance), as acquired when progressive disease occurs after clinical benefits (secondary or acquired resistance) or as initial attenuation of therapeutic interventions preceding acquired resistance (adaptive resistance). When MAPKi-treated melanoma cells develop adaptive resistance, they become transiently resistant by modifying their molecular phenotype through non-genetic mechanisms such as phenotype switching or metabolic reprogramming (12). Continuous drug treatment drives transiently resistant cells into a stable drug-tolerant state (13). Phenotype switching is the transition from a proliferative (epithelial-like) state to an invasive (mesenchymal-like) one which has been described as a characteristic feature of drug-tolerant melanoma cells and linked to increased resistance to MAPKi (14). Metabolic reprogramming is an essential aspect in the emergence of the drug-tolerant state, in particular as MAPK inhibition reduces glycolysis (15), induces oxidative phosphorylation (OXPHOS) (16,17), and triggers the expression of a mitochondrial biogenesis gene signature (18). Mitochondria are the location of essential energy producing 5 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 2, 2019; DOI: 10.1158/1078-0432.CCR-19-0253 Author manuscripts have