Abstract 5836: CRISPR/Cas9 screening identifies genes affecting the sensitivity to the copanlisib/venetoclax combination in lymphoma cells

Abstract Background: Copanlisib, a PI3K inhibitor with a higher selectivity on PI3Kα and PI3Kδ, is currently being evaluated as a single agent and in combination for lymphoma and solid tumor patients. We and others reported the synergism achieved by adding the BCL2 inhibitor venetoclax, and the combination is in its early clinical assessment (NCT04939272, NCT04572763, NCT03886649). To explore genes affecting the response to the combination, we performed a genome-wide CRISPR Cas9 screen in a marginal zone B cell lymphoma (MZL) cell line under drug exposure. Methods: We generated, via lentiviral infection, a stable Cas9-expressing VL51 line, which was then infected (MOI 0. 3) with the genome-wide Brunello guide RNA library, interrogating 19114 genes. After puromycin selection, 40M cells underwent DNA extraction (T0), 40M were exposed to DMSO (control arm), 40M to copanlisib (0. 5nM) + venetoclax (25nM) (experimental arm) for 2 weeks. DNA at T0 and extracted at 2 weeks underwent Illumina sequencing according to the Broad Institute Genetic Perturbation Platform protocol. MAGeCK-MLE was used to model sgRNA read counts and assess the sgRNAs statistical enrichment (P0. 05 FDR 0. 15), GSEA for genesets enrichments. Results: In the DMSO-treated cells, 160 genes had a positive β value (i. e. , the sgRNAs knocking down the genes were positively enriched after 2 weeks), while 1152 had a negative β value (i. e. , the sgRNAs knocking down the genes were negatively enriched after two weeks), for a total of 1152 differential enriched targets. In the cells exposed to the drug combination, 207 genes had positive and 1324 negative β, for a total of 1531 differentially enriched targets. Genes defined as essential, according to DepMap, represented 768/1152 (67%) and 843/1531 (55%) in control and experimental arms, respectively. The experimental arm did not massively differ from the control arm as shown by 815 genes (615, 74%, defined as essential by DepMap) overlapping between the 2 conditions and by highly significant enrichments at GSEA. A few genes were relevant only in VL51 exposed to copanlisib/venetoclax and not to DMSO. Selected hits were individually validated. Knocking down VKORC1L1, XCL1, RFK, MAP2K6, POC1A, and WTN5A improved the activity of the combination. WTN5A was also validated using recombinant protein (10 ng/mL), which reduced the effect of copanlisib/venetoclax. SPARCL1, MAPK4, ID2, and MSTN silencing reduced the activity of the combination. MSTN, coding for myostatin, was also validated by adding the recombinant protein (10 ng/mL), which increased the drugs anti-tumor activity. Conclusions: A genome-wide genetic screening identified genes modulating the lymphoma cells response to the copanlisib/venetoclax combination, providing novel therapeutic targets or biomarkers. Citation Format: Sara Napoli, Alberto J. Arribas, Eleonora Cannas, Federica Fuzio, Luciano Cascione, Andrea Rinaldi, Emanuele Zucca, Andrea Alimonti, Anastasios Stathis, Francesco Bertoni. CRISPR/Cas9 screening identifies genes affecting the sensitivity to the copanlisib/venetoclax combination in lymphoma cells abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 5836.