Abstract 5331: Orthosteric CDK2 kinase inhibitors have a distinctive profile when compared to genetic perturbation of CDK2 in CCNE1-amplified and non-amplified tumor cell lines

Abstract Aim: There has been renewed interest in pursuing cyclin-dependent kinase 2 (CDK2) as a therapeutic target for cancer treatment, given its essential role in driving the survival of CCNE1-amplified tumors and mediating resistance to CDK4/6 inhibitor treatment in estrogen receptor positive breast cancer. This has resulted in the identification of a next generation of orthosteric inhibitors which have increased selectivity for CDK2 kinase versus other CDK-family members. This study aimed to contrast genetic and chemical perturbation of CDK2 in human tumor cell lines with and without CCNE1-amplification. Methods: Cellular responses were assessed using assays for proliferation (CyQUANT), clonogenicity, cell cycle (flow cytometry), and western blotting. NanoBRET tracer displacement assays were used to examine orthosteric kinase inhibition in cells. Genetic perturbation of CDK2 used siRNA and CRISPR-Cas9 editing with sgRNA. Four CDK2 inhibitors currently undergoing clinical trials were profiled. Results: CDK2-targeting siRNA conferred a >75% decrease in the clonogenicity of CCNE1-amplified OVCAR-3, Kuramochi and FUOV1 cells, thus confirming dependence of the cells on CDK2. The non-CCNE1 amplified cells TYK-nu and PEA2 were not sensitive to CDK2-siRNA. The orthosteric CDK2 inhibitors inhibited cellular CDK2 with IC50 values in the range of 1-20 nM. As expected, the compounds inhibited the proliferation of CCNE1-amplified cell lines but they also demonstrated activity against non-CCNE1 amplified TYK-nu cells (one example had GI50 values of 89 ± 8 nM in OVCAR-3 cells, and GI50 values of 122 ± 13 nM in TYK-nu cells, respectively). Consistent with the role of CDK2 in controlling the G1 checkpoint, we observed G1 arrest in the CCNE1-amplified cell line OVCAR-3 following CDK2 CRISPR knock-out. As expected, CDK2 gene deletion did not significantly impact the cell cycle in the TYK-nu non-CCNE1 amplified cell line. In contrast, an orthosteric CDK2 inhibitor induced a G2 arrest in the TYK-nu cell line at doses 2 - 5-fold greater than the compound GI50. Evidence of cyclin E1 accumulation was observed in both CCNE1-amplified and non-amplified cells following orthosteric CDK2 kinase inhibitor treatment (48 - 72 hours). It was also observed following generation of an F80G CDK2 mutant TYK-nu cell line and treatment with the bulky purine analog 3MB-PP1. In contrast, gene-silencing, or gene-editing of CDK2, had no effect on cyclin E1 levels. Conclusion: We demonstrate that the profiled orthosteric CDK2 inhibitors do not reproduce genetic perturbation of CDK2 in CCNE1-amplified and non-amplified cell lines. We propose the need for more selective CDK2 inhibitors and those that inhibit CDK2 in a manner that more closely reflects the genetic perturbation of CDK2. Citation Format: Joanne M. Munck, Susan J. Tudhope, Kleopatra Papa, Alex Howard, Sam Hogan, Suzanne Kyle, Jessica Watt, Luke Gaughan, Maria Ahn, Stephen R. Wedge, John F. Lyons. Orthosteric CDK2 kinase inhibitors have a distinctive profile when compared to genetic perturbation of CDK2 in CCNE1-amplified and non-amplified tumor cell lines [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 5331.