Abstract 3935: CRISPR/Cas9 screens to identify proliferation and resistance mechanisms in uveal melanoma
Authors: Bennett Richard L. , Monagle Darby , Smith Katelyn R. Raburn , Sobh Amin , Smalley Keiran S. , Harbour J William , Licht Jonathan D. 
Published: April 2023

Purpose: Uveal (ocular) melanoma (UM) is a highly aggressive and frequently fatal cancer of the eye. While only 2-4% of patients have metastases at diagnosis, up to 50% of develop metastatic disease, overwhelmingly to the liver. Almost all UM harbor an initiating mutation in the Gαq signaling pathway, most commonly in GNAQ. Most UM also contain secondary mutations that drive tumor progression, the most common being BAP1 loss. Targeted therapies such as MEK inhibitors have been utilized in UM, but tumors rapidly become refractory. The aims of this study were to identify novel mechanisms specifically activated in UM cells that drive proliferation and escape from MEK inhibitor therapy.

Procedures: We performed CRISPR-Cas9 mediated gene disruption screening of UM cell lines 92.1, Mel202 and Mel270 using the Brunello sgRNA library targeting the whole genome. Cells were transduced at 0.3 MOI and 500X coverage then split into two groups to compare treatment with the MEK inhibitor (MEKi) trametinib (6nM) vs. untreated control cells. Cell pellets were collected at day 2 (baseline) and after every 3 cell divisions for 21 cell divisions (treated and control). In addition, we generated 92.1 and Mel270 cell lines stably expressing Cas9 and transduced cells at 1000x coverage with a sgRNA library targeting either 192 epigenetic regulators or 482 kinases. At least two replicates for each screen were performed. sgRNA representation between samples was compared using MAGeCK. Genes specifically essential in UM cell lines were distinguished from commonly essential genes using the Broad institute dependency map (DepMap). These hits were validated by individual sgRNAs and pharmacological inhibition.

Results: Whole genome screens revealed a set of 84 genetic dependencies unique for UM cells that were required for proliferation. Among these genes were those required for the lipoic acid biosynthesis pathway: MCAT, MECR, LIPT2, LIAS as well as genes encoding the MITF-SOX10-TFAP2 transcription factors, the H3K9 methyltransferase SETDB1, and members of the MLL3/4 complex. Screening of UM cell lines with a focused guide RNA library targeting epigenetic regulators revealed that BRD2, TRIM28, KMT2D, SETD1B and HDAC1 were specifically required for UM survival. Screens with a sgRNA library targeting the kinome revealed CDK2, CDK4 and TRRAP were required for UM survival. After 1 week of treatment with MEKi trametinib, we identified 112 genes whose disruption increased the anti-proliferative effects of trametinib in Mel202 and Mel270 cells and 10 of these genes were essential only in UM. These genes included metabolic enzymes critical for fatty and lipoic acid synthesis such as MECR, MCAT and TAZ. By contrast disruption of TP53, NF1, or PTEN led to MEKi resistance.

Conclusions: These results identify novel mechanisms of proliferation and therapy resistance specifically required in GNAQ mutant UM cells that may represent new therapeutic targets.

Citation Format: Richard L. Bennett, Darby Monagle, Katelyn R. Raburn Smith, Amin Sobh, Keiran S. Smalley, J William Harbour, Jonathan D. Licht. CRISPR/Cas9 screens to identify proliferation and resistance mechanisms in uveal 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 3935.