Abstract 2605: BH3 mimetics synergize with the Wee1 inhibitor ZN-c3 by activating caspases which induce DNA damage and degrade key proteins
Authors: Izadi Hooman , Ibrahim Noah , Hoang Tiffany , Ma Jianhui , de Jong Petrus R. , Pinchman Joseph , Bunker Kevin D. , Samatar Ahmed A. , Doñate Fernando 
Published: June 2022

Abstract
Background: Wee1 is a crucial cell cycle checkpoint kinase that regulates the G2/M checkpoint in response to DNA damage, and its inhibition can cause mitotic catastrophe and apoptosis in tumor cells. Inhibitors of BCL-2 have been shown to also induce apoptosis. ZN-d5 and ZN-c3, currently in clinical development for the treatment of cancer, are highly selective and potent inhibitors of Bcl-2 and Wee1, respectively. The combination of ZN-d5 with ZN-c3 induced robust tumor regressions in preclinical models and the mechanism of action of this activity is presented.
Methods: Cell proliferation in tumor cell lines was measured using CellTiter-Glo® (Promega). Cleaved caspase-3, cleaved PARP, phosphorylated CDK1 (p-CDK1), γH2AX, Wee1, RRM2 and other markers were measured by Western blotting, IHC and/or flow cytometry. Anti-tumor efficacy was determined in several xenograft models.
Results: The combination of ZN-c3 and ZN-d5 resulted in additive or synergistic anti-proliferative activity in several tumor cell lines. This enhancement of anti-proliferative activity was also observed at concentrations of ZN-d5 that induced caspase activation, but minimal apoptosis. Treatment of tumor cell lines with ZN-d5 alone at these sublethal concentrations caused induction of γH2AX, a marker of double-stranded DNA breaks, and degradation of caspase-sensitive proteins relevant to the DNA Damage Response (DDR), such as RRM2 (a ribonucleotide reductase subunit) and Wee1. Degradation of Wee1 correlated with decreases in p-CDK1, a marker of Wee1 inhibition. These effects were mainly reversible and were inhibited by pre-treatment with caspase inhibitors. Mechanistically, the combination of ZN-c3 and ZN-d5 resulted in increased levels of γH2AX, enhanced inhibition of p-CDK1, and increased levels of apoptotic markers compared to single agent treatment. Similar results were obtained when ZN-c3 was combined with inhibitors of other BCL-2 family proteins such as inhibitors of MCL-1 and BCL-xL. Finally, the combination of ZN-c3 with ZN-d5 or other BH3 mimetics showed synergistic anti-tumor activity in several xenograft models further supporting the rationale for combining these agents.
Conclusions: ZN-d5 or other BH3 mimetics caused caspase-mediated DNA damage and degraded relevant proteins such as Wee1 and RRM2 resulting in additive or synergistic anti-tumor activity when combined with ZN-c3. These results support a novel use of BH3 mimetics to activate caspase activity, independently of apoptosis induction, to significantly enhance the activity of DDR inhibitors such as ZN-c3
Citation Format: Hooman Izadi, Noah Ibrahim, Tiffany Hoang, Jianhui Ma, Petrus R. de Jong, Joseph Pinchman, Kevin D. Bunker, Ahmed A. Samatar, Fernando Doñate. BH3 mimetics synergize with the Wee1 inhibitor ZN-c3 by activating caspases which induce DNA damage and degrade key proteins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2605.