Abstract Cyclin-dependent kinase 2 (CDK2) is a critical effector of cell-cycle progression, including during late G1, S, and G2 phases. While CDK4/6 inhibitors have become a backbone in HR+/HER2- breast cancer therapy, resistance frequently emerges through activation of alternative CDK/Cyclin complexes. Notably, the CDK2/Cyclin E axis is frequently associated with resistance to CDK4/6 inhibitors and has driven the development of catalytic CDK2 inhibitors for breast cancer therapy. Here, we sought to: 1, delineate the mechanistic determinants of response to pharmacologic CDK2 inhibition in breast cancer models; 2, define biomarkers predictive of response; and 3, interrogate rational combination strategies to maximize therapeutic efficacy. Using a panel of breast cancer models, it was found that CDK2 catalytic inhibitors induced a bimodal response with two fundamentally distinct phenotypic responses. A minority of cell lines underwent G1 arrest while most accumulated 4N DNA content consistent with a G2/M block. Co-expression of p16INK4A and high Cyclin E1 strongly correlated with the G1 arrest phenotype and marked sensitivity to CDK2 inhibition that was also observed in vivo. Models lacking this co-expression adopted the 4N arrest response and displayed upregulation of phospho-CDK1 (Y15) and Cyclin B1. Critically, this latter phenotype was observed in all HR+/HER2- models tested, including those with engineered resistance to CDK4/6 inhibitors. Using whole-genome CRISPR screens, loss of CDK2 was identified as a top hit conferring resistance to catalytic inhibitors, underscoring the concept that inhibitor binding to CDK2 (rather than simple loss) is essential for the cytostatic effect. Further, deletion of CDK2 reversed the G2/M arrest induced by the inhibitor and restored proliferation. Ahost of potential targets were identified that enhanced sensitivity to CDK2 inhibitors through CRISPR and drug screening approaches. These included CDK4/6 inhibitors that were broadly synergistic with CDK2 inhibitors irrespective of breast cancer subtype. The basis of this cooperation involved arrest in multiple phases of the cell cycle. Several additional combinatorial strategies have recently been defined that further credential CDK2 as a key therapeutic target moving forward. Together this work addresses the complexity of targeting CDK2 for the treatment of breast cancer and multiple opportunity areas for combinations. Citation Format: E. Knudsen, V. Kumarasamy, A. Dommer, J. Wang, A. Witkiewicz. Discrete Vulnerabilities to CDK2 Inhibition in Breast Cancer: Genetic Determinants and Therapeutic Opportunities 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 PS2-13-01.
Knudsen et al. (2026) studied this question.