Abstract LB-116: Combination of a cell cycle checkpoint kinase inhibitor and ionizing radiation leads to abscopal tumor response through increased CD8+ infiltration

Abstract

Abstract Purpose/Objectives: The use of Immune checkpoint blockade across disease sites has been successful but many patients still remain unresponsive to immunotherapy. Therefore, it is imperative to Identify regimens to augment the efficacy of immunomodulatory agents. Recently it has been shown that mitotic cell cycle progression following ionizing radiation (IR)-induced double-stranded DNA breaks triggers micronuclei (MN) formation. By promoting inflammatory signaling through their repository role of cGAS that stimulates interferon-induced gene (STING) expression, MN can activate cell-intrinsic immune surveillance mechanisms. Our hypothesis was based on the rational that checkpoint kinase inhibitors abrogating the G2/M cell cycle checkpoint in the presence of IR-induced DNA damage will result in increased formation of MN and an augmented immune response. Materials/Methods: MCF10A and B16F10 cells were treated using IR ± AZD7762, a CHK1/2 inhibitor. Both cell lines were treated with 25nM of AZD7762 and received 17 and 8 Gy IR, respectively. Immunofluorescent studies were performed to assess the MN formation and cGAS co-localization. RT-qPCR was carried out to measure the IFN-β and CCL5 mRNA levels and Western blot evaluated phosphorylation of STAT1. Knockdown of TMEM173 (STING) in MCF10A - cells was used to assess IFN-β and CCL5 expression as a rescue experiment. Finally, a B16F10 melanoma tumor model was analyzed to evaluate the in vivo effects of the combinatorial treatment. Cells were injected in both right and left flank of the mice but only the right flank tumors irradiated with a single dose of 17Gy. Tumor tissues were collected and CD8+ T-cell infiltration was assessed. Results: MCF10A cells showed a statistical significant increase in MN formation (16%) in both 24 and 48 hours when treated with AZD7762+IR compared with IR (5%) or AZD7762 (8%) alone. IFN-β, CCL5 and p-STAT1 levels showed a similar trend as MN. When MCF10A cells with TMEM173 downregulation (siRNA) were treated with AZD7762+IR the expression of CCL5 was reversed confirming that the activation of the immune response is through the cGAS-STING pathway. The in vivo studies revealed that the combination of AZD7762+IR not only led to a significant impairment of the treated tumor growth but also and most importantly to an increased systemic immune response and a significant improved tumor control at the abscopal site. CD8+ T-cell tumor infiltration was found to be significantly higher in the combination group compared to single treatment in both treated and abscopal tumors. Conclusion: The combination of IR with agents targeting the G2/M checkpoint to drive mitotic progression was able to induce increased MN formation and interferon signaling in vitro and led to significant tumor growth delay in both irradiated and abscopal tumor sites. Citation Format: Ilias V. Karagounis, Hann-Hsiang Chao, Christoforos Tomas, Constantinos Koumenis, Amit Maity. Combination of a cell cycle checkpoint kinase inhibitor and ionizing radiation leads to abscopal tumor response through increased CD8+ infiltration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-116.