Development of a Novel MEK Mutation Driven Drug Resistant Melanoma Model by CRISPR/Cas9 Technology

Melanoma remains the most lethal form of skin cancer, exhibiting high mortality rates due to a high likelihood of developing metastases and acquiring drug resistance. Approximately 40-50% of melanomas contain oncogenic BRAF mutations, of which 75-90% harbor the BRAF^V600E mutation. This mutation constitutively activates the mitogen-activating protein kinase (MAPK) signaling pathway, leading to uncontrolled cell growth and oncogenesis. Recent combination therapies of BRAF- and MEK- specific inhibitors have shown improved progression-free patient response in phase II clinical trials.  However, in the majority of patients, acquired resistance to MAPK pathway inhibitor therapies develops after approximately 12 months of treatment. Preclinical studies have suggested that MEK1 mutations confer resistance to BRAF and MEK inhibitors. In this study, we used the CRISPR/Cas9 genome-editing technology to generate a drug resistant MEK1^Q56P knock-in mutation within the A375 melanoma cell line, which naturally harbors the BRAF^V600E mutation. We validated this new isogenic cell model using both molecular and biofunctional approaches.

Method and results: Single guide RNAs (sgRNAs) were designed and built into Cas9 plasmids to bind and cut desired regions in the MEK1 target region. The melanoma cell line A375 was co-transfected with single guide-containing Cas9 plasmids and donor plasmids. Single cells were cloned and expanded for subsequent screening of the desired gene mutation events. The introduction of the MEK1^Q56P mutation in the cells was then confirmed at the genetic level via Sanger sequencing and next-generation sequencing (NGS). Validation of the mutation in mRNA transcripts was carried out by RT-PCR followed by sequencing. The cell line maintains permanent and genetically stable resistance characteristics during cell culture expansion without the use of selective pressure. 

Drug responses to BRAF- and MEK1-specific inhibitors and non-specific chemotherapy drugs were compared between the A375 MEK1^Q56P isogenic cell line and the parental cell line in 2-D and 3-D culture environments. Results demonstrated that the isogenic MEK1^Q56P cell line showed significant and specific resistance to BRAF inhibitors in comparison to the parental A375 line.  

Using CRISPR/Cas9 gene-editing technology, we have targeted an endogenous loci within a melanoma cell line, creating a novel in vitro model to aid in basic and translational melanoma research as well as drug screening efforts. This new approach to cell line development provides direct, in vitro, bio-functional evidence of a drug-resistant gene that drives tumor cell survival under targeted anti-cancer treatments. Furthermore, this A375 MEK1^Q56P isogenic cell line represents a new type of drug resistance model that contains a defined genetic resistance mechanism. This model provides an invaluable tool for developing next generation therapeutics that can overcome drug resistance in melanoma.