Molecular basis of cell-cycle plasticity and robustness

Project Summary The mammalian cell cycle is commonly conceived as a well-understood, hardwired, invariant pathway. Emerging work, however, indicates that the cell cycle is much more plastic than generally believed, with multiple adaptive routes through the cell cycle under different conditions. This plasticity makes the cell cycle robust to environmental perturbations, but also

drives adaptive drug resistance to targeted cell-cycle inhibitors. A fresh look at the dynamics and pliability of cell-cycle progression will reveal new principles that predict dependence on a particular cell-cycle node and new strategies to suppress adaptive cell-cycle rewiring. Cyclin-Dependent Kinases (CDKs) are key enzymes that drive cell proliferation, and

consequently, multiple CDK inhibitors are in development to suppress unwanted cell proliferation. However, cells eventually find a way around these drugs to resume proliferation. A plausible hypothesis is that cells leverage cell-cycle plasticity to pursue alternative paths through the cell cycle. In one striking example, inhibition of CDK2 leads to rapid loss of substrate phosphorylation

as expected, but then CDK2 substrate phosphorylation rebounds within several hours. This rebound depends on CDK4 and CDK6, which insulate the cell from fluctuations in CDK2 activity by maintaining Rb hyper-phosphorylation and E2F transcription. This enables CDK2 re-activation and eventual cell-cycle completion, even in the presence of potent CDK2 inhibitors.

My lab has pioneered the development of a set of powerful time-lapse microscopy tools to visualize rapid drug responses in single, living cells. Here, we will apply our technology to determine the mechanisms driving this unusual rebound in CDK2 activity observed upon CDK2 inhibition. First, we will test the role of the p16 CDK4/6 inhibitor protein on the CDK2 activity

rebound. Second, we will test whether CDK2-dependent degradation of Cyclin D modulates the CDK2 activity rebound. Third, we will identify additional mechanisms underlying the robustness of Rb phosphorylation to inhibition of CDK2 and show how long-term drug pressure unleashes the plasticity of CDKs. Since our findings are likely to be broadly applicable beyond CDK2

inhibitors, our proposed work will fill long-standing gaps in our understanding of how the cell cycle is wired for success in the face of perturbations.