Rb1 deficiency induces synthetic lethality with ATR and PKMYT1 coinhibition in breast cancer cell lines and patient-derived xenografts.

Targeting cell cycle checkpoints has emerged as a promising strategy in cancer therapy, yet single-agent inhibitors often fail because of compensatory mechanisms. Here, we demonstrate that coinhibition of ATR (RP-3500) and PKMYT1 (RP-6306) induces synthetic lethality in Rb1-deficient breast cancers by disrupting both S-G2 and G2-M checkpoints. This leads to replication stress, premature mitotic entry, and accumulation of DNA damage. In vitro, Rb1-deficient breast cancer cells exhibited marked apoptosis and loss of clonogenic survival, whereas Rb1-proficient models remained resistant to combination treatment. Genetic manipulation confirmed this dependency: Rb1 knockdown sensitized resistant models, whereas reexpression conferred protection. In vivo, patient-derived xenograft (PDX) models recapitulated these findings. Rb1-deficient tumors regressed after ATR/PKMYT1 coinhibition, whereas Rb1-proficient tumors showed only modest responses. Combination therapy was well tolerated without weight loss or measurable toxicity. Biomarker analysis revealed increased γH2AX and reduced Ki67 staining exclusively in Rb1-deficient PDX models, underscoring the specificity of this response. Mechanistically, Rb1 loss impaired double-strand DNA repair by attenuating homologous recombination and nonhomologous end joining, leading to replication fork collapse, chromosomal instability, and mitotic catastrophe. Proteogenomic analysis identified JNK/p38 stress response pathway activation as a key driver of apoptosis after ATR/PKMYT1 inhibition in Rb1-deficient cells. Clinically, a retrospective analysis of stage IV breast cancer datasets revealed that Rb1-low tumors display reduced DNA repair pathway activity in triple-negative and CDK4/6 inhibitor-resistant luminal breast cancers. These results identify Rb1 loss as a predictive biomarker for ATR/PKMYT1-targeted therapy, offering a potential precision treatment strategy for advanced breast cancers.