Deciphering the mechanism of non-canonical cell cycle entry

Title: Deciphering the mechanism of non-canonical cell cycle entry PROJECT SUMMARY The decision to enter the cell cycle is a fundamental cellular process that is critical to development and tissue maintenance, with dysregulation of this process being responsible for the development of cancer. Initiation of the cell cycle is regulated by cyclin-dependent kinase 4 and 6 (CDK4/6), which is activated by mitogenic signaling. Activating mutations are commonly found in mitogenic signaling in cancer cells, leading to overactivation of CDK4/6 and abnormal cell proliferation. Thus, specific CDK4/6 inhibitors show immense promise as treatments for various types of cancer. In addition to the canonical CDK4/6-dependent pathway, emerging evidence suggests that mammalian cells can enter the cell cycle in the absence of CDK4/6 activity. Although the mechanism by which CDK4/6 regulates cell cycle entry is well established, the molecular mechanisms underlying non-canonical cell cycle entry remain elusive. The objective of the proposed research program is to determine the mechanisms of the non-canonical pathway for cell cycle entry. Given cell-to-cell heterogeneity and the highly interconnected regulatory processes of cell cycle entry, elucidating how and when mammalian cells bypass CDK4/6 and enter the cell cycle is challenging. To overcome these challenges, we will apply live-cell sensors for CDK activity in combination with our innovative single-cell methodologies. We will also use a variety of pharmacological and genetic approaches to manipulate potential regulators of non-canonical cell cycle entry. Our preliminary data point to a theoretical mechanism for non-canonical cell cycle entry that may at first appear paradoxical: a tumor suppressor gene that is normally inactivated by CDK4/6 is routed for degradation in response to sustained CDK4/6 inhibition, thereby providing a means for non-canonical cell cycle entry. Moreover, our preliminary data implicate multiple factors in the regulation of this process, suggesting a dynamic interplay of factors that further integrates extrinsic and intrinsic signals to control non-canonical cell cycle entry. Completion of the proposed research will provide new insight into the dynamic regulation of cell cycle entry and a greater understanding of adaptations to CDK4/6 inhibition in mammalian cells. In addition, as non-canonical cell cycle entry severely limits the success of CDK4/6 inhibitor therapy in cancer, outcomes from the proposed research have the potential to lead to novel therapeutic interventions targeting the cell cycle in cancer.