Abstract 41: Tumor resistance to CDK4/6 inhibitors and degraders determined by the expression state of CDK6
Authors: Wu Xuewei , Yang Xiaobao , Xiong Yan , Li Ruitong , Ito Takahiro , Ahmed Tamer , Karoulia Zoi , Adamopoulos Christos , Wang Hong , Wang Li , Xie Ling , Ueberheide Beatrix , Aaronson Stuart , Chen Xian , Sellers William , Buchanan Sean , Jin Jian , Poulikakos Poulikos I. 
Published: July 2021

Abstract
CDK4 and CDK6 kinases are master regulators of cell cycle progression, and they have thus been attractive targets for cancer therapy. CDK4/6 inhibitors (CDK4/6i) showed significant clinical effectiveness in ER+ breast cancers, and three CDK4/6i are now FDA-approved for this indication. However, in several tumor types, CDK4/6i have only been modestly effective, but the underlying mechanism(s) accounting for the discrepancy have remained elusive. To identify the basis of resistance to CDK4/6i, we employed a variety of biochemical, genetic and proteomic approaches, we developed a potent and selective CDK4/6-directed degrader (PROTAC) to investigate in-cell binding of compounds and validated our findings in large tumor and clinical-based data. Our studies revealed that tumor response to CDK4/6i is critically determined by the expression of CDK6. Tumors with low CDK6 expression (CDK6-low) depend on CDK4, and are uniformly sensitive to CDK4/6i. CDK6-low tumors include both entire cancer types (e.g. Ewing Sarcomas, MCL), as well as subgroups of large tumor types, such as portion of non-small cell lung adenocarcinomas (NSCLC). We further validated this finding in NSCLC patients previously treated with CDK4/6i in a clinical trial. In contrast, tumors that express both CDK4 and CDK6 universally depend on CDK6, which is expressed as either CDK4/6i-sensitive CDK6 (CDK6-S) or CDK4/6-resistant CDK6 (CDK6-R) in different cells. Using a CDK4/6-directed PROTAC, we further found that CDK4/6i binds strongly CDK6-S but weakly CDK6-R, indicating different conformations adopted by CDK6 in the two states. An unbiased proteomic analysis identified binding of components of the HSP90/CDC37 complex associated with the state of CDK6: in tumors in which CDK6 is expressed as a thermo-unstable, strong HSP90/CDC37-client, CDK4/6i (and consequently CDK4/6 PROTACs) bind and potently inhibit CDK6 and downstream Rb/E2F signaling. In contrast, tumors resistant to CDK4/6i express CDK6 as a thermostable, weak HSP90/CDC37-client that binds weakly to CDK4/6i. Our data uncover the expression state of CDK6 and its dependence on the HSP90/CDC37 complex as a critical determinant of tumor response to CDK4/6i. We further identify low CDK6 expression as a molecular predictor of tumor sensitivity to current CDK4/6i, indicating a potential biomarker for selection of patients that are more likely to benefit from these drugs. Finally, our findings underline the need for novel inhibitors targeting the thermostable CDK6-R to be used for the treatment of the large number of patients with of Rb-proficient solid tumors that are resistant to current clinical CDK4/6i. Thus, this unexpected dualism in the dependence of CDK6 kinase on HSP90 explains resistance of a large portion of tumors to current CDK4/6i, and provides a roadmap for developing more effective CDK4/6-directed pharmacologic strategies for cancer therapy.
Citation Format: Xuewei Wu, Xiaobao Yang, Yan Xiong, Ruitong Li, Takahiro Ito, Tamer Ahmed, Zoi Karoulia, Christos Adamopoulos, Hong Wang, Li Wang, Ling Xie, Beatrix Ueberheide, Stuart Aaronson, Xian Chen, William Sellers, Sean Buchanan, Jian Jin, Poulikos I. Poulikakos. Tumor resistance to CDK4/6 inhibitors and degraders determined by the expression state of CDK6 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 41.