Douglas A. Whittington

LIG1 Is a Synthetic Lethal Target in BRCA1 Mutant Cancers

Abstract Synthetic lethality approaches in BRCA1/2-mutated cancers have focused on PARP inhibitors, which are subject to high rates of innate or acquired resistance in patients. In this study, we used CRISPR/Cas9-based screening to identify DNA ligase I (LIG1) as a novel target for synthetic lethality in BRCA1-mutated cancers. Publicly available data supported LIG1 hyperdependence of BRCA1 mutant cells across a variety of breast and ovarian cancer cell lines. We used CRISPRn, CRISPRi, RNAi, and protein degradation to confirm the lethal effect of LIG1 inactivation at the DNA, RNA, and protein level in BRCA1 mutant cells in vitro. LIG1 inactivation resulted in viability loss across multiple BRCA1-mutated cell lines, whereas no effect was observed in BRCA1/2 wild-type cell lines, demonstrating target selectivity for the BRCA1 mutant context. On-target nature of the phenotype was demonstrated through rescue of viability with exogenous wild-type LIG1 cDNA. Next, we demonstrated a concentration-dependent relationship of LIG1 protein expression and BRCA1 mutant cell viability using a titratable, degradable LIG1 fusion protein. BRCA1 mutant viability required LIG1 catalytic activity, as catalytically dead mutant LIG1K568A failed to rescue viability loss caused by endogenous LIG1 depletion. LIG1 perturbation produced proportional increases in PAR staining in BRCA1 mutant cells, indicating a mechanism consistent with the function of LIG1 in sealing ssDNA nicks. Finally, we confirmed LIG1 hyperdependence in vivo using a xenograft model in which LIG1 loss resulted in tumor stasis in all mice. Our cumulative findings demonstrate that LIG1 is a promising synthetic lethal target for development in patients with BRCA1-mutant cancers.