DEFINING MECHANISTIC TARGETS AND CIRCULATING BIOMARKERS OF THERAPEUTIC RESPONSE AND RESISTANCE TO CLINICAL ATR AND CHK1 INHIBITORS IN HIGH-GRADE OVARIAN CANCER

Background: Small molecule inhibitors targeting DNA damage response (DDR) factors, such as ataxia telangiectasia and Rad3-related protein (ATRi) and checkpoint kinase 1 (Chk1i) represent new strategies to combat disease recurrence in ovarian cancer patients. There are currently 19 active clinical trials assessing ATRi and Chk1i for the treatment of diverse malignancies, including ovarian cancers (clinicaltrials.gov). Understanding mechanisms of drug resistance to these therapies remains largely unexplored. We have generated isogenic models of ATRi (Chk1i) resistance by metronomic treatment of ovarian cancer cell lines (OVCAR3, OV90 and TOV-21G) with the ATR inhibitor AZD6738 (Astra Zeneca). All ATRi (Chk1i)-resistant cells exhibit a protective G1/S-phase cell cycle arrest following ATRi treatment, suggesting conserved alterations in G1/S-phase checkpoint signaling. To this end, we have identified that loss of cyclin-dependent kinase 6 (CDK6) expression, a key G1/S regulatory protein, confers resistance to ATRi and Chk1i (LY2606368). This is noteworthy as CDK6 is amplified in ~3% of ovarian cancers and exhibits a copy number gain in ~20% of ovarian cancers (The Cancer Genome Atlas, n=300 patients). Further, a recent study of over 1,000 ovarian cancer patients has shown that elevated CDK6 correlates with disease recurrence, chemoresistance, and poor progression-free survival (Dall’Acqua A et al., 2017). Additionally, we find that proteins secreted by ATRi (Chk1i)-resistant versus ATRi-treated wildtype cells are largely unique, suggesting that these models could be used to establish circulating protein biomarkers that can predict resistance and response to ATRi (Chk1i) as well as to potent ATRi combinations, namely with poly (ADP-ribose) polymerase inhibitors (PARPi).Hypothesis: Our preliminary findings motivate this application, which is focused on understanding mechanisms underlying loss of CDK6 as this will provide needed insights into a novel ATRi (Chk1i) drug resistance mechanism. The conserved response of ATRi (Chk1i) models to ATRi treatment, i.e., G1/S-phase arrest, also motivates leveraging these models to establish circulating biomarkers of resistance and response to ATRi (Chk1i) and ATRi and PARPi combinations.Specific Aim 1: Investigate mechanisms underlying CDK6 stability, and the associated role of UBC9 in models of ATRi-resistant ovarian cancer cells.Specific Aim 2: Establish circulating biomarkers of therapeutic response and resistance to clinical ATRi (Chk1i) and ATRi/PARPi combinations.Study Design: Specific Aim 1 studies will focus on mechanistic evaluation of CDK6 protein stability as loss of CDK6 is not due to altered mRNA levels in ATRi-resistant cells. We will investigate the role of ubiquitin-conjugating enzyme9 (UBC9)-mediated SUMOylation, an event recently described to stabilize CDK6, in ATRi-resistant models, i.e., wildtype/control and ATRi-resistant cells. We will silence expression of UBC9 by siRNA, analyze CDK6 expression, and perform phenotypic analyses to discern impact on ATRi (Chk1i) sensitivity. We will further apply transient metabolic labelling and mass spectrometry-based proteomic techniques to investigate alterations in CDK6 translational rate as well as to characterize binding partners that impact CDK6 stability. Specific Aim 2 will focus on defining proteins actively secreted by ATRi (Chk1i)-resistant and wildtype cells in response to ATRi, Chk1i, and ATRi/PARPi combinations by similar metabolic labeling and proteomic analyses as described above. We will verify these candidates as circulating biomarkers in subsequent proteomic analyses of blood and tumor tissues harvested from xenograft models established from ATRi (Chk1i)-resistant ovarian cancer cells treated with ATRi, Chk1i, and ATRi/PARPi.Innovation: We aim to characterize a novel mechanism of treatment resistance to ATRi (Chk1i), i.e., loss of CDK6 expression, that will be of future benefit to ovarian cancer patients with high CDK6 expression as these patients may be more responsive to ATRi (Chkli) treatment. Since we have uncovered conserved phenotypic responses to ATRi-treatment in ATRi (Chk1i) resistant cells, i.e., protective G1/S-phase arrest, we will use these models to establish novel circulating biomarkers of resistance and response to ATRi (Chk1i) and potent ATRi/PARPi combinations using cutting-edge quantitative proteomic methods.Impact: This application is responsive to the CDMRP OCRP FY17 Areas of Encouragement focused on Treatment resistance as mechanisms of resistance to ATRi and Chk1i remain largely unexplored. A future benefit of these studies will be improved treatment of CDK6-high disease and the management of treatment response and resistance to ATRi (Chk1i) therapeutics that will support the ongoing use of these agents for the treatment of ovarian cancer.