Date of Award
Open Access Thesis
Medical Doctor (MD)
ARID1A DEFICIENCY AS A BIOMARKER FOR SENSITIVITY TO IONIZING RADIATION AND ATR INHIBITION IN GYNECOLOGIC MALIGNANCIESJessie Y. Li, Shari Damast, MD (Department of Therapeutic Radiology, Yale University, Yale School of Medicine, New Haven, CT), and Gloria Huang, MD (Section of Gynecologic Oncology, Department of Obstetrics, Gynecology, and Reproductive Sciences, Yale University, Yale School of Medicine, New Haven, CT) Objective: ARID1A, which encodes a protein that participates in the SWI/SNF chromatin remodeling complex, is frequently mutated in ovarian and endometrial malignancies. The emergence of the role of ARID1A in gynecologic cancer progression reveals potential implications for therapeutic targets. Our primary purpose was to investigate ARID1A deficiency as a potential biomarker conferring sensitivity to ionizing radiation and ATR inhibitors. Methods: Paired isogenic cell lines ES2 and HCT116 were used containing wild-type ARID1A or homozygous loss of function ARID1A mutations. Ionizing radiation doses were delivered using Precision X-ray 320-kV orthovoltage unit at a dose rate of 2 Gy/45 seconds. Cytotoxicity assays were performed to evaluate cell survival following various doses of ATR inhibitor AZD6738. Clonogenic survival assays were performed with or without 50 nM of AZD6738 1 hour prior to radiation at doses of 0, 1, 2, 4, or 6 Gy. Survival curves were generated and compared using the linear quadratic equation on GraphPad Prism. gH2AX foci formation following radiation at 5 Gy was observed using immunofluorescence (IF) and Western blot. IF images were analyzed using ImageJ. Cell cycle distribution following radiation at 5 Gy was performed using flow cytometry and analysis was performed using FlowJo software. Results: ARID1A-/- cells exhibited greater sensitivity to radiation alone compared to ARID1A+/+ cells as measured by clonogenic survival and demonstrated a diminished DNA damage response following radiation compared to ARID1A+/+ cells, as measured by gH2AX foci formation by IF and Western blot. ARID1A-/- cells exhibited an impaired G2/M cell cycle checkpoint following radiation, as exhibited by delayed cell cycle arrest at G2/M compared to wild-type cells. ARID1A-/- cells demonstrated sensitization to AZD6738 alone compared to wild-type cells by cytotoxicity assay. Preliminary results suggest that ARID1A-/- cells greater sensitization to AZD6738 treatment prior to radiation compared to radiation alone. Further evaluation of combinatorial strategies is in progress. Conclusions: Our in vitro findings confirm that ARID1A deficiency confers sensitivity to radiation and ATR inhibition, suggesting a biologically informed treatment strategy for ARID1A-deficient malignancies. Further work regarding dosing and timing of combination treatment will be explored in the future.
Li, Jessie, "Arid1a Deficiency As A Biomarker For Sensitivity To Ionizing Radiation And Atr Inhibition In Gynecologic Malignancies" (2022). Yale Medicine Thesis Digital Library. 4095.
This Article is Open Access