Abstract 591: Discovery of ZM-2322, a highly selective, potent inhibitor of membrane-associated tyrosine- and threonine-specific cdc2-inhibitory kinase (PKMYT1)

Abstract

Abstract The genome DNA is consistently threatened by a variety of intrinsic and exogenous DNA damage factors. Cell cycle checkpoint is one important mechanism in response to DNA damage and maintaining genome stability. In tumor cells, gene alterations, such as CCNE1 amplification (encoding for Cyclin E1), result in G1/S checkpoint loss and replication stress, leading to high dependency on the G2/M checkpoint. PKMYT1 inhibits crucial checkpoint protein CDK1 activity by phosphorylating CDK1 at the residues of Thr-14 and Tyr-15 in the G2/M phase. In CCNE1 amplification cells, the inhibition of PKMYT1 leads to the reduction of pCDK1 and loss of G2/M checkpoint, consequently results in premature mitosis and the accumulation of DNA damage, then finally induces cell death. Here we identify a novel small molecular ZM-2322, which displays robust intracellular binding to PKMYT1 and inhibits the specific downstream phosphorylation of CDK1 Thr-14 with IC50 values of 14 nM and 29 nM, respectively. ZM-2322 strongly inhibits the proliferation of the HCC1569 cell line with CCNE1 high-level amplification and shows a >400 × selectivity folds over a CCNE1 wildtype cell line SKOV3. ZM-2322 also shows significant inhibition of tumor growth in a dose-dependent manner in the HCC1569 xenograft model, and the efficacy correlates well with PK and target inhibition. There is evidence showing that loss-of-function mutations of FBXW7, a cyclin E E3 ligase, lead to cyclin E accumulation and are synthetic-lethal with PKMYT1 inhibition. Consistently, a potent anti-proliferation of ZM-2322 is observed in DLD-1 FBXW7 -/- cells, but not DLD-1 parental cells. These data suggest a high potency of ZM-2322 on PKMYT1 inhibition and a potential good kinase selectivity which is further confirmed by using a kinase panel biochemical assay. As expected, when compared head-to-head with another clinical PKMYT1 inhibitor RP-6306 which shows inhibitory effect on 27 of 217 human kinases with inhibition rates >90% at 1 μM, ZM-2322 is found to be a much more PKMYT1-selective inhibitor and hits only several individual kinases under the same assay condition, suggesting a potentiality in better safety profile and higher dose tolerance. Furthermore, our data illustrates that in combination with gemcitabine, ZM-2322 elicits strong synergetic anti-proliferation activities on HCC1569 cells in vitro and results in tumor regression with a minor effect on body weight in vivo. Besides, a strong synergy effect is also observed in the combo of ZM-2322 and ATR inhibitor in HCC1569 in vitro. These data indicate that ZM-2322 has the potential of application expansion and good tolerance under continuous administration. Taken together, our data suggest that ZM-2322 is a potent and highly selective PKMYT1 inhibitor. Citation Format: Feng Zhou, Lu Liu, Lei Jiang, Baoying Cheng, Zhentao Li, Dongxing Zhu, Jianbin Xue, Liting Xue, Renhong Tang. Discovery of ZM-2322, a highly selective, potent inhibitor of membrane-associated tyrosine- and threonine-specific cdc2-inhibitory kinase (PKMYT1) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 591.