Abstract 5418: Targeting PKMYT1 to preferentially kill tumor cells that have CCNE1 amplifications
Authors: Samantha S. Hodge, Natalie Hill, Kayla Walzer, Ritu Kushwaha, Yihong Guan, Jeremy Fournier, Ester Fernandez-Salas
Journal: Cancer Research (2025)

Abstract BACKGROUND: Cyclin E1 (CCNE1) amplification occurs in endometrial (40%), ovarian (20%), stomach (11%) and breast (3%) cancers and is associated with poor clinical outcomes. CCNE1 amplification deregulates the G1/S checkpoint and causes premature entry into S phase. Consequently, CCNE1 amplified cells depend on the G2/M checkpoint to ensure genomic stability before mitosis. PKMYT1, a member of the WEE1 family of enzymes, phosphorylates and inhibits CDK1 to regulate the G2/M checkpoint and prevent entry into mitosis. Here, we show that PKMYT1 inhibition in CCNE1-high cancer cells results in premature entry into mitosis, catastrophic DNA damage, and cell death. PKMYT1 inhibition also synergizes with chemotherapy to increase CCNE1-high cancer cell killing. Importantly, non-tumorigenic cells are not dependent upon PKMYT1 and do not exhibit DNA damage in response to PKMYT1 inhibition. METHODS: An isogenic pair of CCNE1-low and CCNE1-high expressing cell lines was generated using lentiviral transduction. PKMYT1 was inhibited using Compound A, a tool small molecule PKMYT1 inhibitor. Inhibition of PKMYT1 was measured by CDK1 pT14 via western blot and HTRF. Cell cycle was monitored by flow cytometry using EdU and DNA content stains. DNA damage was measured by western blot and flow cytometry for yH2AX. Viability was measured with AlamarBlue and Caspase3/7. Bliss Synergy scores were calculated and used to assess synergy after combination treatments. RESULTS: CCNE1 overexpression increases entry into S phase and dependence on the G2/M checkpoint (CDK1 pT14 and pY15 levels) without changing basal levels of DNA damage. While PKMYT1 inhibition decreases CDK1 pT14 in both CCNE1-low and CCNE1-high cells, only CCNE1-high cells exhibit decreased S phase and increased SubG1 (apoptotic) and G2/M phases. CCNE1-amplified breast and ovarian cancer cells display similar inhibition of CDK1 pT14, reduction of S phase, and increased DNA damage and cell death as a consequence of PKMYT1 inhibition. Combination treatment of PKMYT1 inhibition and chemotherapy synergistically reduced cell viability in CCNE1-amplified cancer cells. Non-tumorigenic cells are not dependent upon PKMYT1 and do not exhibit changes in the cell cycle or DNA damage after PKMYT1 inhibition. In contrast, WEE1 inhibition is detrimental to non-tumorigenic cells and induces cell cycle dysregulation and DNA damage. CONCLUSIONS: The data presented here provide a rationale for targeting PKMYT1 in cancer cells that are dependent on the G2/M checkpoint to maintain genomic stability, such as CCNE1-amplified tumors. PKMYT1 inhibition selectively kills CCNE1-amplified cancer cells while sparing non-tumorigenic cells that have intact G1/S checkpoints. Our results highlight the importance of developing selective PKMYT1 inhibitors to avoid toxicities related to WEE1 inhibition. Citation Format: Samantha S. Hodge, Natalie Hill, Kayla Walzer, Ritu Kushwaha, Yihong Guan, Jeremy Fournier, Ester Fernandez-Salas. Targeting PKMYT1 to preferentially kill tumor cells that have CCNE1 amplifications [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 5418.