Abstract 1735: The discovery of PKMYT1 inhibitors to exploit synthetic lethality caused by replication stress

Background: Replication stress is a feature of the hyper-proliferation experienced by many cancers, leading to genome instability and accumulation of potentially lethal DNA damage. During their evolution, many cancer cells inactivate specific DNA-damage response pathways, placing high dependence on the pathways that remain, one of which is regulated by PKMYT1. This protein kinase prevents unscheduled G2 to M transition to allow cells the time needed for DNA repair before division. In cells that rely on PKMYT1, its inhibition forces cells to divide when they are not ready, leading to synthetic lethal killing. PKMYT1 is important when replication stress is driven by Cyclin E amplification and other processes. Thus, PKMYT1 inhibitors offer therapeutic opportunities that can be exploited in combination with DNA damaging agents across a range of cancers. Materials and Methods: We synthesized over 1500 novel PKMYT1 inhibitors and validated their activity in biochemical, biophysical, cell biology and tumor growth analyses. Target engagement and cellular responses were monitored with validated target engagement and cell response biomarkers. Results: We have discovered low nM PKMYT1 inhibitors that are exquisitely selective when assayed against a range of other protein kinases, which includes >1000-fold selective over the closely related protein kinase WEE1. Our inhibitors show extremely slow dissociation kinetics from the target in cells and selectively inhibit proliferation in cells with the genetic features of replication stress. Extensive biomarker analyses confirm target engagement in cells and demonstrate that our inhibitors cooperate with DNA damaging agents to increase accumulation of DNA damage in sensitive cells, but not in insensitive cells. The inhibitors are orally bioavailable, and we confirm target engagement in human tumor xenografts grown in immunocompromised mice. Critically, our inhibitors cooperate with DNA damaging agents to achieve regressions of human tumor xenografts. Conclusions: We have discovered novel orally bioavailable well tolerated PKMYT1 inhibitors. Our inhibitors are exquisitely selective for PKMYT1 over a range of protein kinases, including the closely related protein kinase WEE1. We confirm PKMYT1 engagement in cells and show that our inhibitors display extremely long dissociation kinetics from the target. We show that our PKMYT1 inhibitors cooperate with DNA damaging agents to increase accumulation of DNA damage in cancer cells with high levels of replication stress but not in healthy cells. In human cancer xenografts, our well tolerated inhibitors enhance the anti-tumor efficacy of DNA damaging agents achieving significant tumor growth delay benefits compared to chemotherapy alone. Citation Format: Richard M. Marais, Joanna Grabarek, Myria Ioannou, Leo Leung, Ali Raoof, Mike Brown, Deborah Smithen, Chris Fitzpatrick, Chris Chester, Jacob Leigh, Mohammed Aljarah, Monika Lisauskaite, Oleksandr Grytsai, Ryan Burgin, Dan Niculescu-Duvaz, Graeme Thomson, Caroline Springer. The discovery of PKMYT1 inhibitors to exploit synthetic lethality caused by replication stress [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 1735.