LAUSR

Introduction Malignant melanoma is an aggressive tumour characterised by poor prognosis and therapeutic resistance. It is partly driven by the MAPK signalling pathway, providing a rationale for the development of inhibitors (MAPKi) targeting key components of the activated MAPK pathway. MAPKi induce tumour regression and improve progression-free and overall survival of metastatic melanoma patients. However, due to the development of drug resistance, even in patients displaying complete response, eventually the tumour progress. Both genetic and non-genetic resistance mechanisms have been described during MAPK inhibition. A better understanding of these resistance mechanisms could uncover novel cancer cell vulnerabilities to target in combination strategies for enforcing the efficacy and long-term response to MAPKi. Material and methods To identify mechanisms and biomarkers of adaptive MAPKi-resistance in BRAF-mutated melanoma cells, we performed a cell surface marker screening in A375P cells. A panel of 361 antibodies was used to analyse membrane antigens by flow cytometry at various time points following treatment with MAPKi. Candidates were validated by FACS and qPCR in established cell lines, in a panel of primary patient-derived melanoma cells and in xenograft tumours. Results and discussions Our screening approach identified 42 membrane epitopes changing expression in A375P in response to MAPKi treatment at 36 and 60 hour. In vitro and in vivo validations highlighted CD36, a fatty acid transporter involved in fatty acid oxidation (FAO), as the most consistently up-regulated protein during treatments. Analysis of RNAseq and qPCR data revealed increased expression of FAO genes and unveiled MAPKi-induced metabolic reprogramming involving PPARα and CPT1α. Functional assays confirmed metabolic reprogramming in multiple human melanoma cell lines as well as association between increased FAO rates and enhanced resistance to MAPKi. However, while pharmacological inhibition of FAO failed to boost cell death during MAPKi treatments due to compensatory up-regulation of glycolysis, inhibition of both, FAO and glycolysis, was sufficient. Conclusion Our study identified CD36 as the most consistent cell surface protein marking BRAF-mutated melanoma cells in the early stage of the development of resistance. Results implicate adaptive metabolic reprogramming as a potential resistance mechanism and indicate that combined inhibition of glycolysis and FAO are critical to induce cell death and overcome MAPKi resistance in melanoma cells.