Inhibition of ATR as a therapeutic strategy to enhance immunotherapy in head and neck cancer

Abstract Background: Immunotherapy has become a new standard in the care of patients with head and neck squamous cell carcinoma (HNSCC). A number of patients’ tumors do not respond to this treatment, and recent studies have established an immunomodulatory role for the ATR-CHK1 signaling axis in the tumor microenvironment and the specific effects of ATR inhibitors in stimulating an innate immune response. Therefore, we have assessed the efficacy of the novel ATR inhibitor (AZD6738) as monotherapy and in combination with anti-PD-1 antibody in a syngeneic mouse model of HNSCC. Methods: In vitro clonogenic survival assays were used to evaluate the IC50 values of the ATR inhibitor AZD6738 in syngeneic MOC1, MOC2 and ROC1 mouse HNSCC cell lines. Western blotting and ATR shRNA knock-down strategies were performed to examine drug specificity and dissect molecular mechanisms. An orthotopic syngeneic mouse model of head and neck cancer was used to explore in vivo efficacy of the combination treatment. Immune profiling on tumor tissues obtained following drug treatment was assessed by immunohistochemistry and multiplex immunofluorescence analyses. Conditioned medium (CM) and bone marrow (BM) cells were obtained and subjected to flow cytometric analyses to assess myeloid cell phenotypic and functional markers. Coculture of the BM cells with mouse splenic T cells was also conducted to measure suppression of T cell activation and proliferation. Results: MOC1, MOC2 and ROC1 cell lines treated in vitro with various concentrations of single agent AZD6738 showed an IC50 value of 0.18-0.625 µmol/L. Combination of AZD3867 and anti-PD-1 antibody significantly reduced tumor growth and prolonged survival in mice injected with MOC1 cells orthotopically in the oral tongue compared to control and other treatment groups (P <0.0001 and P = 0.001, respectively). Combination therapy was well-tolerated and no weight loss was observed in mice. The CD4+T, CD8+ T lymphocytes and NK cells were elevated in mice tumors treated with combination of the drug compared to other treatment groups. Additionally, the inhibitory FoxP3+ Treg cells were significantly reduced (P = 0007) in mice tumors following the combination treatment. The BM cells exposed to CM collected from ATR shRNA MOC1/2 cell lines, and those treated ex-vivo with AZD6738 alone demonstrated decreased arginase-1 expression and a reduced capacity to inhibit T cell activation and proliferation relative to control groups. These findings suggest a role for ATR in modulating myeloid derived suppressive cell (MDSC) function. Mechanistically, we demonstrated that the ATR inhibition caused DNA damage response leading to cGAS/STING pathway activation associated with decreased STAT3 phosphorylation in vitro and in vivo in syngeneic head and neck cancer cells. Conclusion: In this study, we demonstrate that the immunogenic effects of AZD6738 are mediated by cytotoxic T cells as well as innate immunity. The combination of AZD6738 and anti-PD-1 warrants further exploration as a novel combination therapy for HNSCC. Citation Format: Abdullah A. Osman, Fabio H. B. da Costa, Adewale A. Adebayo, Mason D. Bartels, Tongxin Xie, Moran Amit, Mutsuki Kawabe, Roberto Rangel, Mitchell J. Frederick, Vlad Sandulache, Andrew G. Sikora, Jeffrey N. Myers. Inhibition of ATR as a therapeutic strategy to enhance immunotherapy in head and neck cancer [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 PO-085.