Abstract ND06: First disclosure of AZD8421, a highly selective CDK2 inhibitor to address resistance to CDK4/6 inhibitors in breast and CCNE1-high cancers
Authors: Denz Christopher R. , Grondine Michael , Fan Jun , Hsu Jessie Hao-Ru , Jackson Anne , Robinson James , Guo Grace , Li Wen , Wang Yanjun , Martin Maryann San , Prickett Laura , Johannes Jeffrey , Ghosh Avipsa , Song Kun , Sudhan Dhivya , Vasalou Christina , Richards Ryan , Beaumont Kevin , Peters Matthew , Drew Lisa , Fawell Stephen , Goldberg Frederick W. 
Published: April 2024

Abstract
Cyclin-dependent kinases (CDKs) are an attractive class of cancer targets due to their role in cell-cycle regulation. CDK2 is a Ser/Thr kinase activated via interaction with its cyclin partners CCNE and CCNA, driving G1/S progression of the cell cycle. CDK2 inhibition has the potential to address multiple resistance mechanisms to CDK4/6 inhibitors in breast cancer. In addition, CCNE1 is frequently amplified and over-expressed in cancers including ovarian, with pre-clinical data linking CDK2 inhibition sensitivity to high CCNE1. First-generation inhibitors targeting CDK2 suffered from poor tolerability in the clinic likely due to off-target activities across both the CDK family and across the protein kinome. As well as achieving CDK family selectivity in cells versus key off-targets (CDK1, CDK4/6, CDK9), it’s noteworthy that AZD8421 had no significant kinase inhibition outside the CDK family. X-ray co-crystal structures of AZD8421 with CDK2 provided the basis for the observed selectivity among the CDK-family. For example, hydrogen bonding interaction of AZD8421 with Lys89 (a CDK2-specific residue) near the solvent region of the ATP-binding pocket was the key requisite for achieving &gt;300-fold selectivity over CDK9. In nanoBRET assays measuring cellular target engagement, AZD8421 had an IC50 against CDK2 of 9nM with selectivity over CDK1, CDK4 and CDK6. Further kinetic characterization of AZD8421 binding revealed a long residence time at CDK2 (2 hours) but not CDK1 (7 minutes). These kinetic studies showed that AZD8421 has a low Kd,app for CDK2 and thus even higher selectivity vs CDK1 than was measured by single timepoint assays. In a CCNE1 amplified cell line, AZD8421 potently inhibited cell proliferation (69nM, OVCAR3), correlated with inhibition of pRB, arrest in G1/S phase of the cell cycle and induction of senescence. In vitro combination assays in CDK4/6 inhibitor resistant breast cancer cell lines showed combination benefit with AZD8421 plus approved CDK4/6 inhibitors. In vivo, AZD8421 potently suppressed phosphorylation of Rb, and demonstrated robust monotherapy and CDK4/6i combination activity in breast and ovarian in vivo models. In vivo PD marker suppression and efficacy was demonstrated in CDK4/6 inhibitor resistant breast PDXs in combination with palbociclib (CDK4/6i). AZD8421 showed robust monotherapy activity in a CCNE1 amplified ovarian model OVCAR3 with regressions seen with monotherapy and in combination with palbociclib. These data, along with suitable physical and pharmacokinetic properties, supported progression of AZD8421 into clinical studies [NCT06188520].
Citation Format: Christopher R. Denz, Michael Grondine, Jun Fan, Jessie Hao-Ru Hsu, Anne Jackson, James Robinson, Grace Guo, Wen Li, Yanjun Wang, Maryann San Martin, Laura Prickett, Jeffrey Johannes, Avipsa Ghosh, Kun Song, Dhivya Sudhan, Christina Vasalou, Ryan Richards, Kevin Beaumont, Matthew Peters, Lisa Drew, Stephen Fawell, Frederick W. Goldberg. First disclosure of AZD8421, a highly selective CDK2 inhibitor to address resistance to CDK4/6 inhibitors in breast and CCNE1-high cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr ND06.