GNAQ/11 and BAP1 mutant isogenic cell lines engineered by CRISPR/Cas9 to model ocular melanoma

Abstract

Purpose : Uveal melanoma (UM) is the most frequent intraocular cancer in adults and 50% of patients develop liver metastases for which no treatment is effective. According to The Cancer Genome Atlas, 92% of cases are characterized by early mutations in two genes coding for alpha subunits of G protein coupled receptors (GPCR): G protein subunit alpha q (GNAQ) or 11 (GNA11). Furthermore, a bi-allelic inactivation of the BRCA1 associated protein-1 (BAP1) is found in 83% of metastatic UM. Our hypothesis is that the genes essential for the survival of mutant UM cells will be ideal therapeutic targets. Using CRISPR/Cas9, we engineered GNAQ/11 and BAP1 mutant isogenic cell lines derived from wildtype UM cells or choroidal melanocytes (CM) to better understand the molecular mechanisms involved in UM development.

Methods : We have designed three ribonucleoprotein complexes (RNPs) composed of single-guide RNA (sgRNA) and Streptococcus pyogenes Cas9 (spCas9) specific to the GNAQ/11 and BAP1 genes. This allowed to target the DNA sequence on the exon 5 of these proteins to induce a double-strand break. To introduce the desired mutations Q209L in GNAQ/11 or C91G in BAP1, we have also designed DNA templates called single-strand oligo DNA nucleotides (ssODNs) that were co-transfected by electroporation into UM cells (Mel285) or CM. After a few days, we genotyped the clones by Sanger sequencing and expanded those of interest into cell lines.

Results : We obtained an electroporation efficiency of 50% for CM and 70% for Mel285 using an eGFP plasmid. The first CRISPR transfections showed a mean edition of 20-60% of targeted regions with 4% HR. To increase our percentage of HR, we have also used the Prime Editing method; nine plasmids per mutation have been designed and constructed. The first transfection tests in HEK293T cells are currently underway. We obtained three KO cell lines: Mel285^GNAQ-KO, Mel285^GNA11-KO and Mel285^BAP1-KO. We saw no morphologic changes. A decrease in the proliferation and metabolic activity was observed in Mel285^GNAQ-KO and Mel285^BAP1-KO cell lines in comparison to the wildtype cells. Furthermore, an increase of phosphorylated ATF2/7 was noted in Mel285^GNAQ-KO and Mel285^GNA11-KO cell lines by western blotting.

Conclusions : A better understanding of the altered pathways in our GNAQ/11 or BAP1 mutant isogenic cell lines will help to identify new drugs targeting specifically metastatic UM cells.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.