Abstract: A mutant BRAF gene can lead to uncontrolled cell growth through overactivation of the RAS-RAF-MAPK signaling pathway. The BRAFV600E mutation occurs in approximately 40% to 50% of melanomas. Although current BRAF inhibitors have been used to successfully treat melanomas containing the BRAFV600E mutation, patients often become resistant to BRAF inhibitors within a few months. A number of clinical studies have indicated that secondary mutations in RAS or NF1 are associated with BRAF resistance. However, due to the genetic heterogeneity commonly observed in tumors, it is unclear if those secondary mutations already existed within low percentage subclones, or if they were acquired through drug treatment. Further, it has yet to be determined whether such genetic variants are only associated with resistance, or whether they actually cause the BRAF inhibitor resistance. In this study, we used genome editing CRISPR technology to generate two drug-resistant melanoma cell lines (ATCC® CRL-1619IG-2™ and CRL-1619IG-1™) that contain NRASQ61K or KRASG13D mutations. These isogenic lines were derived from the parental A375 (ATCC® CRL-1619™) melanoma cell line, which naturally contains BRAFV600E. When compared to the parental line, the isogenic cell models demonstrated that genetically modified NRAS or KRAS genes at the endogenous level directly leads to significant resistance to BRAF inhibitors.
Single guide RNAs (sgRNAs) were designed and built to guide Cas9 to bind and cut desired regions in the NRAS or KRAS gene targets. The parental cell line A375 was co-transfected with the single guide and CRISPR all-in-one plasmid alongside a donor plasmid. Transfected cells were sorted into single cells and expanded for subsequent screening of desired gene mutation events. The introduction of the NRASQ61K or KRASG13D mutation in the cells was then confirmed via Sanger sequencing and NGS at the genetic and transcriptional levels. Drug responses to BRAF-specific inhibitors and non-specific chemotherapy drugs were compared between RAS isogenic A375 cell lines and parental A375 cell line in 2D and 3D culture environments. Testing results demonstrated that the isogenic cell lines created by CRISPR showed significant resistance to BRAF inhibitors in comparison to the parental control in both 2D and 3D culture environments. These two novel in vitro cell models with endogenous level RAS mutants provide direct biofunctional evidence that acquiring a drug-resistant gene drives tumor cell survival under targeted therapeutic treatment.