The role of p16 expression in sensitivity and resistance to CDK2 inhibitors - PROJECT SUMMARY Emerging work indicates that the cell cycle is much more plastic than previously thought, with multiple routes to cell-cycle progression seen in different cancers. Cyclin-Dependent Kinase 2 (CDK2) is a key driver of the cell cycle and is also hyper-activated, often by Cyclin E1 overexpression, in numerous cancers, making it an attractive therapeutic target. Work from our lab discovered that CDK2 inhibitors cause rapid loss of substrate phosphorylation, but unexpectedly, CDK2 substrate phosphorylation rebounds within several hours. We showed that this rebound depends on CDK4/6, which backstops the proliferative program, enabling rapid reactivation of CDK2. While many cancer cells exhibit this rapid adaptation to CDK2 inhibition, this is not a universal response. Recent studies have shown that Cyclin E1-amplified ovarian cancers have a high dependence on CDK2 and thus are particularly sensitive to CDK2 inhibition. The reason for this sensitivity is thought to be that inhibition of the ‘driver’ of these cancers, Cyclin E1, by CDK2 inhibitors blocks proliferation. Instead, my work shows that p16 expression sensitizes cells to CDK2 inhibitors by inhibiting CDK4/6 activity, thus preventing compensation for CDK2 inhibition. Aim 1 will explore how p16-expressing cells develop resistance to CDK2 inhibitors over time. We hypothesize that chronic exposure to CDK2 inhibitors may lead to a loss of p16 expression, allowing CDK4/6 to compensate and drive cell cycle progression. To test this, we will chronically treat p16-expressing cells with CDK2 inhibitors over several months, analyzing changes in p16 expression, cell cycle markers, and CDK activity. Aim 2 will investigate the role of p16 in Cyclin E-amplified ovarian cancer cells, where p16 is often paradoxically expressed despite its tumor-suppressive function. We hypothesize that p16 may act to restrain excessive cell cycle progression in these cells. We will modulate p16 expression in ovarian cancer cells and examine its effects on CDK2/4/6 activity, Rb phosphorylation, and overall cell cycle regulation. Aim 3 will determine whether cell- cycle regulatory markers can predict in vivo sensitivity to CDK2 inhibition. While in vitro data suggest that p16 sensitizes cells to CDK2 inhibition, it is unclear whether this relationship holds in patient tumors. To address this, we will profile expression of key cell-cycle regulators in a cohort of ovarian cancer patient samples. We will use patient-derived xenografts (PDX) and assess tumor response to CDK2 inhibitors to identify predictive biomarkers associated with CDK2 dependence. Using approaches such as long-term drug adaptation, live-cell CDK activity sensors, in vivo drug testing, and multiplexed immunofluorescence imaging of patient tumors, this research will uncover how p16 expression influences response to CDK2 inhibition, define mechanisms of resistance, and identify new biomarkers for therapeutic response. This research significantly advances our understanding of cell cycle regulation, particularly how cells adapt to perturbation, revealing new insights into cell cycle plasticity. Ultimately, these findings aim to improve the clinical efficacy of CDK2 inhibitors, contributing to the development of more effective therapeutic strategies and better patient outcomes.
