Abstract 5705: Discovery of a selective slow-off CDK2 inhibitor NKT3447 with distinct features of suppressing pCDK2, downregulating cyclin E, and achieving prolonged pathway inhibition
Authors: Geng Jianlin , Liu Ke , Yu Zhiyong , Sun Wenfeng , Wei Hairong , Li Wenjun , Lu Jing , Deng Juan , Geng Liqing , Liu Zhihong , Gao Zhenhai , Lou Yan 
Published: March 2024

Abstract
CDK2 is a cell cycle kinase that plays a critical role in controlling the G1 to S-phase transition and its abnormal activation through cyclin E1(CCNE1) amplification has been indicated as the primary oncogenic driver in advanced cancers including ovarian, endometrial, and gastric cancers. In addition, cyclin E1 overexpression has emerged as a key resistance mechanism to CDK4/6 inhibitors in hormone receptor-positive breast cancer patients. Therefore, CDK2 represents a promising novel oncology target. However, development of a potent and selective CDK2 inhibitor has been challenging due to the high homology of CDK2 with other CDKs, particularly CDK1, which is essential in normal cell cycle progression. NKT3447 is a novel, orally bioavailable small molecule CDK2 inhibitor with slow-off kinetics and is highly selective against CDK1 and other CDKs. In addition, NKT3447 exhibits a distinct mechanism of action, including suppression of the Thr160 activating phosphorylation on CDK2 and downregulation of cyclin E1. In biochemical assays, NKT3447 drives a concentration dependent disruption of the CDK2/cyclin E1 and CDK2/cyclin A2 complexes, but not the CDK1/cyclin B1 complex. Consequently, NKT3447 potently inhibits the phosphorylation of Rb (pRb) and induces G1 cell cycle arrest in CCNE1 amplified OVCAR3 cells, while showing minimal or no effect on G2/M phase distribution in all the cell lines tested, including OVCAR3 and KYSE520 (CDK1-dependent) cells. Importantly, NKT3447 has a slow-off kinetics, as demonstrated by NanoBRET target engagement assay, which leads to sustained pRb inhibition after treatment withdrawal. In addition, NKT3447 treatment reduces cyclin E1 protein levels and suppresses phospho-CDK2 (Thr160), which further represses CDK2 activity and prolongs the inhibition of CDK2 signaling. NKT3447 also demonstrates a selective anti-proliferation effect in a panel of human cancer cell lines with elevated cyclin E1 expression and/or dependency on CDK2 or cyclin E1 (DepMap). In line with cellular studies, pharmacodynamic study of NKT3447 in mouse tumor models recapitulates a significant repression of pRb, pCDK2 and cyclin E1. Furthermore, NKT3447 treatment results in a dose-dependent tumor growth inhibition or tumor regression in CCNE1 amplified OVCAR3 and MKN1 tumor models. In addition, the combination of NKT3447 with carboplatin exhibits enhanced anticancer effects in OVCAR3 and MFE-280 models. These results demonstrate that NKT3447 is a potent, selective CDK2 inhibitor with distinct properties and mechanism of action, which holds great potential to effectively treat patients with aberrant CDK2/Cyclin E activation in a variety of cancer types. NKT3447 is currently being investigated in a Phase 1 clinical trial in patients with advanced solid tumors.
Citation Format: Jianlin Geng, Ke Liu, Zhiyong Yu, Wenfeng Sun, Hairong Wei, Wenjun Li, Jing Lu, Juan Deng, Liqing Geng, Zhihong Liu, Zhenhai Gao, Yan Lou. Discovery of a selective slow-off CDK2 inhibitor NKT3447 with distinct features of suppressing pCDK2, downregulating cyclin E, and achieving prolonged pathway inhibition [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 5705.