Rb1 deficiency induces synthetic lethality with ATR and PKMYT1 co-inhibition in breast cancer

Targeting cell cycle checkpoints has emerged as a promising strategy in cancer therapy, yet single-agent inhibitors often fail due to compensatory mechanisms. Here, we demonstrate that co-inhibition 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, resulting in replication stress, premature mitotic entry, and DNA damage accumulation. In vitro, Rb1-deficient breast cancer cell lines exhibited marked apoptosis and loss of clonogenic potential, whereas Rb1-proficient models remained largely resistant to combination treatment. Genetic manipulation of Rb1 confirmed this dependency: Rb1 knockdown sensitized resistant models, and overexpression conferred protection. In vivo, patient-derived xenograft (PDX) models recapitulated these findings. Rb1-deficient tumors underwent complete regression, while Rb1-proficient tumors showed limited response with RP-3500/RP-6306 treatment. Combination therapy was well tolerated in vivo, without significant toxicity or weight loss. 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 non-homologous 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 following ATR/PKMYT1 inhibition in Rb1-deficient cells. Clinically, analysis of stage IV breast cancer patient datasets revealed that Rb1-low tumors display reduced DNA repair pathway activity and are enriched in triple-negative and CDK4/6 inhibitor-resistant luminal breast cancers. These findings establish Rb1 loss as a functional biomarker for ATR/PKMYT1-targeted therapy, offering a precision treatment strategy for advanced breast cancers.