CRISPR/Cas9 Edited RAS & MEK Mutant Cells Acquire BRAF and MEK Inhibitor Resistance with MEK1 Q56P Restoring Sensitivity to MEK/BRAF Inhibitor Combo and KRAS G13D Gaining Sensitivity to Immunotherapy.

mutation drives uncontrolled cell growth in most melanomas. While tumors are initially responsive to BRAF inhibitors, prolonged treatment results in inhibitor resistance and tumor regrowth. Clinical data have linked the , and mutations to the BRAF inhibitor resistance. However, development of novel therapeutics is hindered by the lack of relevant isogeneic cell models. We employed CRISPR/Cas9 genome engineering to introduce , and mutations into the A375 melanoma cell line with endogenously high expression of . The resulting isogenic cell lines are resistant to BRAF inhibitors. The A375 isogenic cells are additionally resistant to MEK inhibitors as single agent, but interestingly, these cells become sensitive to MEK/BRAF inhibitor combo. Our results suggest that resistance in the NRAS and MEK isogenic lines is driven by constitutive MEK/ERK signaling, while the resistance in the KRAS isogenic line is driven by EGFR overexpression. Interestingly, the isogenic line displays elevated PD-L1 expression suggesting the mutation could be a potential indication for immunotherapy. Overall, these three novel isogenic cell models with endogenous level and point mutations provide direct bio-functional evidence demonstrating that acquiring a drug-resistant gene drives tumor cell survival and may simultaneously introduce new indications for combo therapy or immunotherapy in the clinic.