Abstract 4194: Inhibiting PELP1 signaling enhances the therapeutic effectiveness of topoisomerase inhibitors in triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive tumor with a poor prognosis and high metastasis, accounting for 15-24% of breast cancer deaths. Proline-, glutamic acid-, and leucine-rich protein 1 (PELP1), an oncogene, is commonly deregulated in TNBC and serves as a biomarker of poor survival rates. The objective of this study is to establish the molecular mechanism of action of PELP1 in the progression of TNBC using IPTG-inducible PELP1 knockdown (PELP1-iKD) models and develop a novel combination therapy targeting PELP1. Methods: TNBC models (MDA-MB-231, BT-549, HCC-1806, SUM-149) with PELP1-iKD were generated through lentiviral transduction. PELP1 signaling was further inhibited with the pharmacological inhibitor SMIP34. SMIP34’s effectiveness was tested with 140 FDA-approved drugs using an MTT assay-based screen, and combination therapy was evaluated using colony formation and Annexin V/PI apoptosis assays. Mechanistic studies were conducted using RT-qPCR, Western blotting, and comet assays. The utility of SMIP34 + Topoisomerase inhibitors (TIs) combination therapy was assessed in cell line-derived xenografts (CDXs), patient-derived organoids (PDOs), and patient-derived xenografts (PDXs). Results: Western blotting analysis confirmed that IPTG induction reduces PELP1 expression in a dose-dependent manner in all four TNBC PELP1-iKD cells. PELP1 KD significantly decreased cell proliferation and colony formation in TNBC cells while enhancing apoptosis. PELP1-iKD cells showed a delayed DNA damage response, characterized by elevated γ-H2AX levels and impaired activation of phospho-ATM, ATR, and DNA-PKcs following DNA damage. Treatment with SMIP34 also resulted in a delayed DNA damage response and accumulation of γ-H2AX, similar to the genetic approach. In an MTT-based screen, SMIP34 enhanced the sensitivity of TNBC cells to TIs. These results were validated in PELP1-iKD cells, where PELP1-iKD further increased sensitivity to TIs, providing genetic evidence of PELP1’s role in the synergy between SMIP34 and TIs. The ability of SMIP34 to enhance the efficacy of TIs was confirmed in additional TNBC cell lines, with synergistic effects observed in cell proliferation, colony formation, and apoptosis assays. Mechanistic investigations demonstrated elevated levels of γ-H2AX in cells treated with the PELP1-iKD+TIs and SMIP34+TIs combinations, compared to monotherapy with TIs. Comet assay results indicated heightened DNA damage with these combination treatments. Furthermore, Combined SMIP34 and Mitoxantrone treatment significantly suppressed PDO proliferation in vitro and reduced CDX and PDX tumor growth compared to monotherapies or controls. Conclusion: Collectively, these findings establish a robust preclinical foundation for the development of PELP1-targeted combination strategies for the treatment of TNBC. Citation Format: Khaled Mohamed Nassar, John R. Sanchez, Durga Meenakshi Panneerdoss, Behnam Ebrahimi, Xue Yang, Uday P. Pratap, Megharani Mahajan, Salvador Cardenas Alejo, Abhi Katabathina, Panneerdoss Subbarayalu, Daohong Zhou, Rajeshwar R. Tekmal, Gangadhara Reddy Sareddy, Manjeet K. Rao, Suryavathi Viswanadhapalli, Ratna K. Vadlamudi. Inhibiting PELP1 signaling enhances the therapeutic effectiveness of topoisomerase inhibitors in triple-negative breast cancer [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 4194.