Abstract 4685: The identification of new therapeutic targets in mucinous ovarian carcinoma
Authors: Serena Petrella, Marika Colombo, Mirko Marabese, Chiara Grasselli, Andrea Panfili, Ilaria Craparotta, Maria Chiara Barbera, Giada Andrea Cassanmagnago, Marco Bolis, Giovanna Damia
Journal: Cancer Research (2024)

Abstract Background: Epithelial ovarian cancer (EOC) is the second cause of death among gynaecological cancersworldwide. Mucinous ovarian carcinoma (mEOC) accounts for 3-5% of all EOCs, and when diagnosed at anadvanced stage its prognosis is very poor mainly for its limited chemosensitivity. PLK1 is a member of thewell-conserved serine/threonine kinase family, that plays a key role in the progression of mitosis, in the G2/Mcheckpoint regulation, DNA damage replication, stress response, and cell death pathways. mEOC has beenshown to be sensitive to PLK1 inhibition both with siRNA and small molecule inhibitor (onvansertib). Giventhis background and the need for new therapeutic approaches in mEOC, the purposes of this work were toidentify new therapeutic targets in mEOC using CRISPR/Cas9 lentivirus libraries and to find synthetic lethalpartners with the PLK1 inhibitor onvansertib. Methods: We used three different mucinous cell lines: MCAS,EFO27, TOV2414, overexpressing Cas9 gene. We evaluated the Cas9 expression through Western Blotanalysis, and the Cas9 activity through a Fluorescent Activated Cell Sorter- based-GFP assay. We used twodifferent lentiviral gRNAs Libraries (Bassik Library Human CRISPR Deletion Library - Apoptosis and cancer(Addgene #101926)/Drug targets, kinases, and phosphatases (Addgene #101927)). The EFO27 Cas9 cells wereused for the screening experiment. Cells were infected with the lentiviral libraries at MOI of 0.3 and put inpuromycin selection. Six days after puromycin addition (Time point 0) 30x10^6 cells were collected forsequencing. The remaining 30x10^6 cells were split in two: control cells and cells treated with a subtoxic doseof onvansertib (IC30). After 7 days (Time point 1) 30x10^6 cells for each condition were collected forsequencing. The infection of libraries was performed alone and in combination with sub-toxic onvansertibtreatment, to identify genes important for mucinous carcinoma cell survival, and those genes in syntheticlethality with onvansertib. Results: Bioinformatic analyses comparing control cells (time 1 vs time 0) andPLK1 treated and non -treated cells (time 1) allowed the generation of a gene ranking list, including genesinvolved in survival of EFO27 cells like ZC2HC1C, RPA2, POLE3, KIN, TUBG1, SMC2, CDC26, CDC42, HOXA9,TAF10, DCLRE1C, SENP1, MRPS31, COPS2; and genes in synthetic lethality with PLK1 treatment, like JUND,CARD9, BCL2L2. Validation experiments are ongoing and will be presented and discussed. Conclusion: Using a CRISPR-based approach, we identified genes that either alone or in combination with PLK inhibitioncould have therapeutic value in mEOC. Citation Format: Serena Petrella, Marika Colombo, Mirko Marabese, Chiara Grasselli, Andrea Panfili, Ilaria Craparotta, Maria Chiara Barbera, Giada Andrea Cassanmagnago, Marco Bolis, Giovanna Damia. The identification of new therapeutic targets in mucinous ovarian carcinoma [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(6_Suppl):Abstract nr 4685.