ARID1A deficiency as a biomarker for sensitivity to radiation and ATR inhibition (232)

Objectives: 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 one 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-deficient cells exhibited greater sensitivity to radiation alone compared to wild-type cells as measured by clonogenic survival and demonstrated a diminished DNA damage response following radiation compared to wild-type cells, as measured by gH2AX foci formation by IF and Western blot. ARID1A-deficient cells exhibited an impaired G2/M cell cycle checkpoint following radiation, as exhibited by diminished arrest at G2/M compared to wild-type cells. ARID1A-deficient cells demonstrated sensitization to AZD6738 alone compared to wild-type cells by cytotoxicity assay. Preliminary results suggest that ARID1A-deficient cells have 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. Objectives: 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 one 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-deficient cells exhibited greater sensitivity to radiation alone compared to wild-type cells as measured by clonogenic survival and demonstrated a diminished DNA damage response following radiation compared to wild-type cells, as measured by gH2AX foci formation by IF and Western blot. ARID1A-deficient cells exhibited an impaired G2/M cell cycle checkpoint following radiation, as exhibited by diminished arrest at G2/M compared to wild-type cells. ARID1A-deficient cells demonstrated sensitization to AZD6738 alone compared to wild-type cells by cytotoxicity assay. Preliminary results suggest that ARID1A-deficient cells have 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.