105P Discovery of potent and selective PKMYT1 inhibitors

Methods:We employed a tiered experimental approach encompassing cell line panels, isogenic Rb1 manipulated cell line systems, and patient derived xenograft models, stratified by Rb1 status. Results: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.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 re-expression conferred protection.In vivo, patient-derived xenograft (PDX) models recapitulated these findings.Rb1-deficient tumors regressed following ATR/PKMYT1 co-inhibition, whereas Rb1-proficient tumors showed only modest responses.Combination therapy was well tolerated without weight loss or measurable toxicity.Biomarker analysis revealed increased H2AX 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, 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.Conclusions: These results identify Rb1 loss as a predictive biomarker for ATR/ PKMYT1-targeted therapy, offering a potential precision treatment strategy for advanced breast cancers.