Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype associated with poor prognosis and high rates of metastasis, contributing to 15-24% of breast cancer-related deaths. The oncogene proline-, glutamic acid-, and leucine-rich protein 1 (PELP1) is frequently overexpressed in TNBC and is associated with poor survival. This study aimed to elucidate the critical role of PELP1 in DNA repair mechanisms of TNBC and to evaluate a novel therapeutic strategy using a PELP1 small-molecule inhibitor, SMIP34, in combination with topoisomerase inhibitors (TIs). Methods: PELP1-inducible knockdown (iKD) TNBC models (MDA-MB-231, BT-549, HCC-1806, SUM-149) were generated via lentiviral transduction using IPTG-inducible shRNA constructs. PELP1 signaling was inhibited pharmacologically using SMIP34. A drug screen of 140 FDA-approved compounds was performed using MTT assays to identify synergistic agents with SMIP34. Functional assays, including colony formation and Annexin V/PI apoptosis assays, were used to assess therapeutic efficacy. Mechanistic studies utilized RT-qPCR, Western blotting, comet assays, and DNA Damage Response Phosphorylation Arrays. Combination treatments were validated in cell line-derived xenografts , patient-derived organoids , and patient-derived xenografts . Results: Western blot analyses confirmed that IPTG induction resulted in a dose-dependent suppression of PELP1 expression in all four PELP1-iKD TNBC cell lines. PELP1 knockdown significantly impaired cell proliferation and colony formation while inducing apoptosis. In parallel, pharmacological inhibition using SMIP34 produced similar phenotypic outcomes, validating the functional relevance of PELP1 in TNBC cell survival. Importantly, PELP1 suppression led to a delayed DNA damage response (DDR), marked by elevated γ-H2AX and activation of key DDR kinases including p-ATM, p-ATR, and DNA-PKcs. SMIP34 treatment replicated these effects, confirming that both genetic and pharmacologic inhibition of PELP1 compromises DNA repair efficiency. A high-throughput MTT-based screen of 140 FDA-approved agents identified topoisomerase inhibitors (TIs) as potent synergistic agent with SMIP34. Co-treatment with SMIP34 and TIs resulted in significantly enhanced growth inhibition and apoptosis compared to monotherapy. This synergy was confirmed in PELP1-iKD cells, where knockdown further sensitized cells to TIs, providing genetic validation of PELP1’s role in TI resistance. Mechanistic studies showed that combination treatments (PELP1-iKD+TIs or SMIP34+TIs) markedly increased DNA damage and apoptotic signaling. Western blotting and phospho-antibody arrays revealed upregulation of DDR and checkpoint proteins, including γ-H2AX, p-CHK2, p-ATR, p-CHK1, p-BRCA1, and p21, indicating enhanced replication stress and cell cycle arrest. Comet assays demonstrated significantly greater DNA fragmentation with combination therapy compared to single agents. In preclinical models, SMIP34+TI treatment significantly suppressed organoid growth in patient-derived organoid cultures. In vivo, this combination led to marked tumor growth inhibition in both cell line-derived xenografts and patient-derived xenografts, outperforming either agent alone. No significant toxicity was observed, supporting the therapeutic potential of this approach. Conclusion: These findings highlight PELP1 as a critical modulator of DNA damage response in TNBC and support the development of combination strategies using SMIP34 and topoisomerase inhibitors. This approach offers a promising therapeutic avenue for improving outcomes in patients with TNBC. Citation Format: K. Nassar, J. Sanchez, D. Panneerdoss, E. Behnam, X. Yang, U. Pratap, M. Mahajan, S. Alejo, P. Subbarayalu, D. Zhou, G. Sareddy, M. Rao, S. Viswanadhapalli, R. K. Vadlamudi. Pelp1 inhibition disrupts dna repair and enhances topoisomerase inhibitor efficacy in triple negative breast cancer abstract. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS4-04-23.
Nassar et al. (Tue,) studied this question.