Abstract Background: Rlip (aka RLIP76 protein encoded by RALBP1) is a Ral-regulated, membrane-associated ATPase enzyme that facilitates Phase 2 detoxification, plays a role in clathrin-dependent endocytosis (CDE), and is vital for cancer cell formation and survival. DNA repair is essential for both normal and cancer cell survival, as it protects cancer cells from DNA damage caused by chemotherapy or radiation. Poly (ADP-ribose) polymerases (PARPs) play a crucial role in the repair of DNA damage. By targeting these repair mechanisms in cancer cells, it is possible to improve the effectiveness of cancer treatments. A notable example is dysfunctional homology-directed repair (HDR), which results from inactivating mutations in the BRCA1 and BRCA2 genes, contributing to radiation resistance in human cancer cells. Therefore, identifying new targets within double-strand break (DSB) repair pathways is crucial. A core concept in using PARP inhibitors (PARPi) for treating patients with BRCA1/2 mutations is the concept of synthetic lethality. Our research found that Rlip depletion or inhibition, combined with PARPi, works synergistically in both BRCA1/2-mutated and BRCA1/2-wild-type breast cancer cells. Methods: Two PARP inhibitors, AZD2281 (Olaparib) and AZD2461, were tested on the proliferation and survival of different cancer cell lines, including MDA-MB-231 (triple negative), MCF-7 (ER+), SKBR-3, AU565 (HER-positive), and MDA-MB-436 (BRCA1 mutated triple negative), with and without Rlip depletion using an antisense locked nucleic acid (LNA). For all experiments, 2.5 µM AZD2461 and 10 µM AZD2281 were used for 24 hours in complete growth medium. Cell cycle analysis was performed by flow cytometry. Expression levels of γH2AX and Rad51 proteins were assessed via Western blotting and immunofluorescence staining. The comet assay was used to measure DNA damage and fragmentation. Results: Rlip depletion significantly increased the reduction in cell survival caused by PARPi treatments across all tested cell lines. Combination index (CI) values were calculated using the Hill equation and Chou-Talalay’s method with CompuSyn software. The effects were highly synergistic (CI 1) with both inhibitors in SK-BR-3 and MCF-7 cells. Olaparib showed additive effects in MDA-MB-231 cells, whereas AZD2461 exhibited an antagonistic effect. The combination of PARPi and Rlip depletion was antagonistic in AU565 and MDA-MB-436 (CI 1), but the doses of both inhibitors were significantly reduced (p 0.001) after Rlip depletion. Cell cycle analysis with Rlip depletion showed either an increase in the sub-G1 population (cell death) or G2/M phase arrest, suggesting cells attempt to undergo mitosis with unrepaired DNA damage, leading to elevated DNA double-strand break signaling, cell cycle arrest, and apoptosis. Alkaline comet assay confirmed more DNA damage with Rlip depletion compared to treatments with the inhibitor alone. Western blot analysis for Rad51 and γ-H2AX showed defects in protein levels following Rlip depletion. The number of γ-H2AX foci, characteristic of DNA damage in nuclei, increased significantly (p 0.01) after Rlip depletion compared to inhibitor treatment alone. Conclusion: Our findings suggest that drugs that inhibit or deplete Rlip could help sensitize breast cancer cells, regardless of their BRCA status or whether they are wild-type ER+ or triple-negative breast cancers, to PARP inhibitors. The immunogenicity of HRD-associated malignancies and the immunomodulatory effects of PARP inhibition could be combined with Rlip inhibitors to treat HRD cancers. These findings clarify the underlying mechanisms and are expected to lead to the development of first-in-class therapies. Citation Format: C. Bose, S. Awasthi, T. Hutson, F. Sardela de Miranda, M. F. Mahecha, M. W. Melkus, R. Layeequr Rahman, S. P. Singh. Ralbp1 depletion downregulates DNA repair and potentiates PARP inhibitor activity in BRCA wild type and mutated breast cancer cell lines 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 PS4-04-20.
Bose et al. (Tue,) studied this question.