Abstract IA20: Exploiting CDK2-driven replication stress to repurpose cancer chemotherapy

Abstract

Abstract Cyclin-dependent kinase 2 (CDK2) coordinates diverse process, including cell cycle entry and progression, DNA replication, and DNA damage and replication stress responses. Cancers frequently contain mutations in genes that regulate CDK2, which is also activated by oncogenic signaling. Because cancers have abnormally high CDK2 activity, most therapeutic strategies seek to inhibit CDK2. Our studies of CDK2 function during replication stress suggest an alternate approach: to harness high CDK2 activity as a therapeutic modality to cause irreparable DNA damage. In order to study the functions of CDK2 inhibitory phosphorylation without perturbing other cell cycle proteins, we used gene targeting in human cells to mutate the endogenous CDK2 T14 and Y15 Wee1 phosphorylation sites. CDK2AF cells exhibited abnormal G1 progression, abnormal DNA replication dynamics, and genome instability, indicating essential regulatory roles for CDK2 inhibitory phosphorylation in these processes. Most strikingly, cells that cannot inhibit CDK2 during stalled S-phase rapidly accumulate massive and irreparable DNA damage and double stranded DNA breaks (termed CDK2-dependent replication stress failure). Importantly, even transient S-phase arrest produces irreversible DNA damage and permanent cell cycle exit, and genetic and pharmacologic studies revealed that that persistent CDK2 activity is both necessary and sufficient to drive replication stress failure. CDK2 inhibitory phosphorylation is thus essential for S-phase checkpoint function. Because cancers do not contain CDK2AF mutations, we used MK1775, a pharmacologic Wee1 inhibitor, to prevent CDK2 inhibitory phosphorylation during replication stress. Pharmacologic Wee1 inhibition of cells treated with S-phase toxins (e.g. hydroxyurea-HU, aphidicolin) recapitulated the CDK2AF replication stress failure phenotype, and brief (5hr) treatment of HU-phase arrested cells caused catastrophic DNA damage and permanent cell cycle exit, and this was seen in multiple cell types. Wee1 inhibition thus converts HU from an agent that reversibly inhibits S-phase to one that acts irreversibly through DNA damage. Moreover, HU/MK-1775 combination therapy exhibited synergy at HU and MK-1775 concentrations that were ineffective as single agents. Because CDK2-driven replication stress failure requires CDK2 activity to be maintained in S-phase arrested cells, rapidly dividing cells are extremely sensitive to this approach compared with slowly cycling cells, such as stem cells and many normal tissues. Importantly, we have identified common oncogenic mutations that greatly sensitize cancer cells to CDK2-driven replication stress failure, thus providing an even greater therapeutic index that favors killing cancer cells while sparing normal tissues, and identifying tumor genotypes appropriate for clinical trials of induced replication stress failure. These concepts are now being employed in mouse leukemia and solid tumor models. Citation Format: Hannah Richards, Hui Zhao, Bridget T. Hughes, Bruce E. Clurman. Exploiting CDK2-driven replication stress to repurpose cancer chemotherapy. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr IA20.