Abstract 2906: Functional and therapeutic significance of DNA ligase I (LIG1) depletion in triple-negative breast cancer (TNBC)

Abstract Background: We recently reported a proteogenomic study of TNBCs that demonstrated a correlation between chemoresistance and heterozygous LIG1 loss, a critical ligase serving DNA replication and repair (PMID: 36001024). Thus, this translational study aims to functionally characterize and investigate therapeutic opportunities driven by LIG1 depletion. Methods: Heterozygous LIG1 knockouts (LIG1het-KO) were engineered into the non-tumorigenic breast epithelial cell line, MCF-12A. Since TP53 was also mutated in almost all TNBC cases with LIG1 het deletion, LIG1het-KO was attempted in both TP53wt and TP53null MCF-12A. In contrast to LIG1het-KO in TP53null model, LIG1het-KO in TP53wt cells were not viable. Roles of LIG1 in cellular transformation, cell cycle dynamics, and the effect of carboplatin and PARP inhibition (PARPi) on these cells were investigated. RNA-seq was also employed to determine the impact of LIG1het-KO loss at a molecular level. Results : LIG1het-KOin MCF-12A TP53null cell lines enhanced cell migration, decreased epithelial and increased mesenchymal gene expression, suggesting LIG1 depletion induced epithelial-to-mesenchymal transition (EMT). Recapitulating patient data, LIG1het-KOcells were less responsive to carboplatin than LIG1 intact cells. RNA-seq and western blotting demonstrated upregulated homologous recombination (HR) genes in MCF-12A TP53null, LIG1het-KOversus MCF-12A TP53null LIG1wtcells. Consistent with enhanced DNA repair, lower DNA repair foci (yH2AX) were observed in TP53nullLIG1het-KO and the proportion of S-phase cells remained unchanged. This suggests that progression through cell cycle was not delayed despite LIG1het-KO, due to compensatory upregulation of HR. To further assess therapeutic vulnerabilities in LIG1het-KO cells, a published CRISPR KO screen of PARPi was reanalyzed to identify LIG1 loss as a robust determinant of PARPi sensitivity (PMID: 29973717). This was confirmed in genome-wide CRISPR screenings with different PARPi(s) in the MCF-10A TP53nullcells. Experimentally, MCF-12A TP53nullLIG1het-KOcells displayed a significant increase in PARPi sensitivity, which was reversed upon over-expressing LIG1. Knocking down LIG1 in the LIG1 intact, HR-proficient TNBC line (MDA-MB-468) induced PARPi sensitivity, while overexpressing LIG1 in the LIG1-low, HR-deficient TNBC cell line (MDA-MB-436) reduced PARPi sensitivity. PARPi and carboplatin in combination was more effective than either drug alone in MCF-12A TP53nullLIG1het-KO cells. Conclusion: LIG1 depletion drives chemotherapy resistance through EMT and HR pathways activation. LIG1 depleted TNBC tumors could benefit from combination of PARPi and carboplatin, potentially driving future studies to investigate biomarker-guided TNBC treatment strategies. Citation Format: Anh M. Tran Huynh, Sumimasa Arimura, Lacey E. Dobrolecki, Mariah J. Berner, Xuxu Gou, Junkai Wang, Michael T. Lewis, Jason Mills, Jun Xu, Gloria V. Echeverria, Susan M. Rosenberg, Christopher J. Lord, Simon Powell, Jonathan T. Lei, Matthew J. Ellis, Meenakshi Anurag. Functional and therapeutic significance of DNA ligase I (LIG1) depletion in triple-negative breast cancer (TNBC). [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2906.