BRAF and MEK inhibitor combinations induce potent molecular and immunological effects in NRAS-mutant melanoma cells – insights into mode of action and resistance mechanisms
About 25% of melanoma harbor activating NRAS mutations, which are associated with aggressive disease therefore requiring a rapid anti-tumor intervention. However, no efficient targeted therapy options are currently available for patients with NRAS-mutant melanoma. MEK inhibitors (MEKi) appear to display a moderate anti-tumor activity and also immunological effects in NRAS-mutant melanoma, providing an ideal backbone for combination treatments. In this study, the MEKi binimetinib, cobimetinib, and trametinib combined with the BRAF inhibitors (BRAFi) encorafenib, vemurafenib, and dabrafenib were investigated for their ability to inhibit proliferation, induce apoptosis and alter the expression of immune modulatory molecules in sensitive NRAS-mutant melanoma cells using 2D and 3D cell culture models and transcriptome analyses. Furthermore, NRAS-mutant melanoma cells resistant to the three BRAFi/MEKi combinations were established to characterize the mechanisms contributing to their resistance. All BRAFi induced a stress response in the sensitive NRAS-mutant melanoma cells thereby significantly enhancing the anti-proliferative and pro-apoptotic activity of the MEKi analyzed. Furthermore, BRAFi/MEKi combinations upregulated immune relevant molecules, such as ICOS-L, components of antigen-presenting machinery and the “don't eat me signal” molecule CD47 in the melanoma cells. The BRAFi/MEKi-resistant, NRAS-mutant melanoma cells counteracted the molecular and immunological effects of BRAFi/MEKi by upregulating downstream mitogen-activated protein kinase pathway molecules, inhibiting apoptosis and promoting immune escape mechanisms. Together, this study reveals potent molecular and immunological effects of BRAFi/MEKi in sensitive NRAS-mutant melanoma cells that may be exploited in new combinational treatment strategies for patients with NRAS-mutant melanoma. Overall design: Bulk RNA sequencing was carried out  from the sensitive WM1366 melanoma cell line and 3 BRAF and MEK inhibitor-resistant daugther cell lines. The resistant cell lines were cultured in presence of the BRAF and MEK inhibitors, while the sensitive cell line was cultured in DMSO. Samples from 3 independent experiments were sequenced.