Abstract 359: Rb loss-related DNA damage repair deficiency mediates the synthetically lethal effect of ATR/PKMYT1 co-inhibition in TNBC and CDK4/6i-resistant ER+ breast cancer

Abstract Purpose: This study explores the therapeutic potential of dual ATR and PKMYT1 inhibition for treatment-resistant breast cancers, particularly triple-negative breast cancer (TNBC) and ER-positive breast cancer resistant to cyclin-dependent kinase 4/6 inhibitors (CDK4/6i). By investigating the synthetic lethality of ATR/PKMYT1 co-inhibition, we aimed to identify biomarkers predictive of treatment response, specifically examining Rb status and its effect on DNA damage repair mechanisms. Methods: The study tested the combination of ATR inhibitor (RP3500) and PKMYT1 inhibitor (RP6306) in TNBC and ER-positive parental and palbociclib-resistant breast cancer cell lines, as well as in patient-derived xenograft (PDX) models. RNA sequencing (RNAseq), reverse phase protein array (RPPA) analyses, and western blot were employed to evaluate treatment efficacy, synergy, and biomarkers associated with response, particularly focusing on Rb1 expression. Results: In vitro, both TNBC and palbociclib-resistant breast cancer cells—characterized by higher replicative stress—showed heightened dependency on ATR and PKMYT1 pathways. ATR inhibition disrupted DNA replication, while PKMYT1 inhibition induced premature mitosis, collectively leading to increased DNA damage and apoptosis in these models. In vivo, the combination treatment produced significant anti-tumor effects in TNBC and palbociclib-resistant PDX models. The loss of Rb strongly predicted treatment response; Rb-deficient cell lines (MCF-7 palbociclib-resistant, BT-549, and MB-436) and PDX models (PR4-T141 and TNBC2-BCX070) demonstrated pronounced apoptosis and tumor regression. Conversely, Rb-proficient MCF-7 parental cells, MB-231 and MB453 were less responsive. Rb1 re-expression in sensitive BT-549 cells mitigated apoptosis under the combination treatment, while Rb1 knockdown in MB-231 and MCF-7 cells increased sensitivity to the therapy. Comparative RNAseq and RPPA analyses showed diminished DNA damage repair activity in Rb-deficient cell lines and PDX models compared to Rb-proficient models, suggesting an Rb-dependent regulation of various DNA damage repair pathways. This was further supported by neutral comet assays in the MB-231 parental and Rb1 knockdown cell lines, which demonstrated longer DNA double stranded-break repair times with Rb1 knockdown. Conclusions: ATR and PKMYT1 co-inhibition is a promising therapeutic strategy for TNBC and CDK4/6i-resistant breast cancer. The synthetic lethality of this combination is enhanced in Rb-deficient models, where Rb1 loss impairs DNA damage repair capabilities, amplifying the anti-tumor effects. Citation Format: Xiao-Ting Jiang, Mei-Kuang Chen, Jiwang Lee, Xiayu Rao, Jing Wang, Kelly Hunt, Khandan Keyomarsi}. Rb loss-related DNA damage repair deficiency mediates the synthetically lethal effect of ATR/PKMYT1 co-inhibition in TNBC and CDK4/6i-resistant ER+ 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 359.