Abstract 3836: New type of drug resistant isogenic cancer cell model created by CRISPR genome editing

Mutant BRAF gene can lead to uncontrolled cell growth through overactivation of RAS-RAF-MAPK signaling pathway. BRAF V600E mutation occurs in approximately 40% to 50% of melanomas. Although BRAF inhibitors have been used to successfully treat melanomas containing the BRAF V600E mutation, patients often become resistant to BRAF inhibitors within a few months. A number of studies have indicated that secondary mutations such as NRAS or NF1 are significantly associated with BRAF resistance by sequencing patient samples. 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. It9s yet to be determined whether such genetic variants are only associated with resistance, or they actually cause the resistance. In this study, we used genome editing CRISPR technology to generate a drug resistant mutation NRAS Q61K within A375 melanoma cell line which naturally contains BRAFV600E. When compared to the parental line, this isogenic cell model demonstrated that genetically modified NRAS gene at the endogenous level directly leads to a significant resistance to BRAF inhibitors. Method and results: Single guide RNAs (sgRNAs) were designed and built to guide Cas9 to bind and cut desired regions in the NRAS gene target. Melanoma cell line A375 was co-transfected with the single guide and CRIPSR all-in-one plasmid alongside donor plasmid. Transfected cells were sorted into single cells and expanded for subsequent screening of desired gene mutation events. The introduction of NRAS Q61K mutation in the cells was then confirmed via Sanger sequencing and NGS at the genetic level and transcriptional level. Drug responses to BRAF specific inhibitors and non-specific chemotherapy drugs were compared between A375 NRAS Q61K isogenic cell line and parental cell line in 2D and 3D culture environment. Testing results demonstrated that the isogenic cell line created by CRISPR showed significant resistance to BRAF inhibitor in comparison to the parental control in both 2D and 3D culture environment. In summary, we utilized the CRISPR/Cas9 genome editing platform to target endogenous loci in human cells and create the intended genetic mutation event. This new approach provides direct bio-functional evidence of acquiring a drug resistant gene drives tumor cells survival under targeted therapeutic treatment. Furthermore, unlike conventional drug resistant cell models that have been developed through drug selection, the A375 NRAS Q61K isogenic cell line represents a new type of drug resistant model that contains a defined genetic resistance mechanism. It reserves the permanent and genetically stable resistance characteristics without being maintained in drug selection culture environment. Therefore, it provides a valuable tool for developing next generation therapeutics that can overcome BRAF drug resistant in melanoma. Citation Format: Lysa-Anne Volpe, Metewo Selase Enuameh, Luping Chen, Michael Jackson, Catherine Nguyenngo, John Foulke, Fang Tian. New type of drug resistant isogenic cancer cell model created by CRISPR genome editing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3836. doi:10.1158/1538-7445.AM2017-3836