Abstract
Despite aggressive multimodal treatment with chemoradiation or surgery and adjuvant radiation therapy (RT) +/- chemotherapy, recurrence rates approach 50% for locally advanced human papillomavirus (HPV)-negative HNSCC. Thus, more effective therapeutics are needed in both the upfront and recurrent settings to improve outcomes for HNSCC patients. Ataxia telangiectasia and Rad 3-related (ATR) is a potential therapeutic target that may enhance RT response. ATR is a DNA damage response protein activated by replication stress and single-strand DNA breaks, such as those caused by RT. We hypothesize that ATR inhibition radiosensitizes tumors by preventing DNA repair and forcing HNSCC cells with unrepaired DNA damage into mitosis, leading to cell death. We evaluated the effects of ATR inhibitor BAY 1895344 (BAY) in preclinical models of HPV-negative HNSCC using murine and human cell lines, including MOC1, MOC2, JHU-012, and FaDu. We confirmed dose-dependent inhibition of ATR signaling by BAY using Western blot analysis of downstream phosphorylated CHK1 in cells treated with RT+vehicle versus RT+BAY. Additionally, dose-dependent increase in yH2AX (DNA damage marker) was observed at 18 hours post-RT in cells treated with serial dilutions of BAY vs vehicle. In clonogenic assays, cells treated with 100nM BAY (4h) and 0-8Gy RT demonstrated radiosensitization compared to vehicle. Cell cycle analysis showed that BAY pre-treatment significantly abrogated RT-induced G2/M cell cycle arrest (p<0.01, unpaired t-test). Cells treated with BAY before RT vs vehicle + RT or BAY alone exhibited increased co-staining for yH2AX and pHH3 (mitosis marker) by flow cytometry and immunofluorescence, indicative of mitotic catastrophe. Additionally, MOC2 cells treated with BAY alone or BAY + RT exhibited significantly increased micronuclei and multinucleation events at 24 hours compared to vehicle alone (p=0.0009, unpaired t test) or vehicle + RT (p=0.01), respectively. For in vivo evaluation of BAY in a radioresistant syngeneic mouse model, intramuscular MOC2 tumors were induced in the gastrocnemius of C57BL/6J mice. When tumors reached 70-280 mm3, mice were randomized (n=10/group) to receive vehicle, BAY (40mg/kg), RT (8Gy x 3), or BAY + RT. Image-guided RT was administered every 4 days. Vehicle or BAY was given orally twice on the day of RT or sham RT (morning/evening) and once the morning after RT or sham RT. Median survival was significantly increased in treated groups: 39.5d for BAY + RT vs. 14d for vehicle + sham RT (p=0.0002, Log-rank test), 22d for BAY + sham RT (p=0.0003), and 23d for vehicle + RT (p=0.0007). Thus, the small molecule ATR inhibitor BAY 1895344 radiosensitizes HNSCC in vitro and increases survival combined with RT in vivo, indicating therapeutic potential for targeting ATR in head and neck cancer. The combination of BAY 1895344 (elimusertib), pembrolizumab, and stereotactic body radiation therapy is currently being tested in a phase I clinical trial for patients with unresectable or metastatic HNSCC within a previously irradiated field (NCT04576091).
Citation Format: Allison N. Pittman, Diana A. Odhiambo, Rico J. Castillo, Ashlyn G. Rickard, Joshua Chen, Joshua E. Himes, Alex M. Bassil, Nerissa T. Williams, David G. Kirsch, Yvonne M. Mowery. Evaluating the therapeutic potential of ATR inhibitor BAY 1895344 and radiation therapy for head and neck squamous cell carcinoma [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr IA04.
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