Epigenetic CRISPR screen identifies chromatin regulators essential for ovarian cancer spheroid formation

Background: Cancer stem-like cells (CSCs) contribute to recurrence and chemoresistance in high-grade serous ovarian cancer (HGSOC). Spheroid formation in ultra-low attachment (ULA) cultures models CSC self-renewal, yet the epigenetic regulators and molecular dependencies sustaining this 3D phenotype remain largely undefined. Methods: A focused CRISPR knockout screen targeting 800 epigenetic and chromatin regulators was performed in OVCAR-3 spheroids. sgRNA enrichment after 3D selection identified genes essential for spheroid growth. Top candidates were functionally validated in BRCA1-wildtype, BRCA1-LOH, and BRCA1-mutant cell lines by assessing spheroid morphology, compaction, viability, and proliferative behavior. Results: The screen identified a high-confidence set of regulators required for spheroid formation, including DNA damage response genes (BRCA1, PARG), Polycomb complex members (SUZ12, BMI1), RNA-binding proteins (NCL, SYNCRIP), and cell cycle regulators (CDK2, PBRM1). These hits converge on chromatin remodeling, histone methylation, and transcriptional control pathways, linking epigenetic regulation with DDR signaling.Functional assays revealed a striking BRCA1 dependency: while BRCA1-WT and LOH cell lines formed compact, viable spheroids, BRCA1-mutant cells produced disorganized, loosely aggregated, hyperproliferative structures with poor architectural integrity. This indicates that BRCA1 is essential not for proliferation per se, but for establishing and maintaining 3D stem-like organization. Integration of the CRISPR hits with phenotypic outcomes suggests a cooperative network between DDR factors, RNA-binding regulators, and chromatin modifiers in sustaining CSC-like behavior. Conclusions: Our findings demonstrate that BRCA1 function underpins the epigenetic and chromatin-based control of spheroid architecture, revealing a mechanistic link between DNA repair fidelity, chromatin integrity, and cancer stemness. The identified vulnerabilities highlight potential therapeutic targets for BRCA1-deficient ovarian and breast cancers.