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

SIMPLE SUMMARY: BRAF inhibitor drug resistance has been a long-time challenge in the treatment of melanoma with BRAF V600E mutation. This study employed the CRISPR/Cas9 technology to generate three isogenic A375 melanoma cell lines with point mutations of NRAS Q61K, KRAS G13D and MEK1 Q56P, respectively. They recapitulated the resistance to BRAF inhibitors in vitro as such mutations have been found in patients with acquired resistance to BRAF inhibitors during treatment. Hence, these novel isogenic cell lines become extremely useful tools for upcoming research in this field. Additionally, we determined 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. The KRAS G13D isogenic line displays elevated PD-L1 expression suggesting the KRAS G13D mutation could be a potential indication for immunotherapy. ABSTRACT: BRAF V600E mutation drives uncontrolled cell growth in most melanomas. While BRAF V600E tumors are initially responsive to BRAF inhibitors, prolonged treatment results in inhibitor resistance and tumor regrowth. Clinical data have linked the NRAS Q61K, KRAS G13D and MEK1 Q56P 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 NRAS Q61K, KRAS G13D and MEK1 Q56P mutations into the A375 melanoma cell line with endogenously high expression of BRAF V600E. The resulting isogenic cell lines are resistant to BRAF inhibitors. The A375 MEK1 Q56P 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 KRAS G13D isogenic line displays elevated PD-L1 expression suggesting the KRAS G13D mutation could be a potential indication for immunotherapy. Overall, these three novel isogenic cell models with endogenous level RAS and MEK1 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.