Abstract 2610: <i>GNAQ/GNA11</i> and <i>BAP1</i> mutant isogenic cell lines engineered by CRISPR/Cas9 gene editing to model ocular melanoma

Abstract 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). These mutations are mostly found in the codon Q209 causing a constitutive activation of different signaling pathways linked to cellular proliferation such as MAPK and PKC. Furthermore, a bi-allelic inactivation of the BRCA1 associated protein-1 (BAP1) is found in 83% of metastatic UM and causes a loss of function of this protein. Our hypothesis is that the genes essential for the survival of mutant UM cells will be ideal therapeutic targets. Using CRISPR/Cas9 technology, 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. 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 with respective RNPs by electroporation into UM cells (Mel285) or CM and favorized the homologous recombination (HR) during DNA repair to knock-in these mutations. After a few days, we genotyped the clones by Sanger sequencing and expanded those of interest into cell lines. 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: Mel285GNAQ-KO, Mel285GNA11-KO and Mel285BAP1-KO. We saw no morphologic changes. A decrease in the proliferation and metabolic activity was observed in Mel285GNAQ-KO and Mel285BAP1-KO cell lines in comparison to the wildtype cells. Furthermore, an increase of phosphorylated ATF2/7 was noted in Mel285GNAQ-KO and Mel285GNA11-KO cell lines by western blotting. 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. Citation Format: Aurélie Fuentes-Rodriguez, Andrew Mitchell, Joël Rousseau, Jacques P. Tremblay, Solange Landreville. GNAQ/GNA11 and BAP1 mutant isogenic cell lines engineered by CRISPR/Cas9 gene editing to model ocular melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2610.

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