Inhibiting ATR Modulates Response to Radiation and Stimulates Immune Responses

Abstract

Radiotherapy (RT) has been shown, albeit rarely, to stimulate anti-tumor immune responses. Recent evidence suggests this can, in part, be attributed to the accumulation of cytoplasmic DNA in the form of micronuclei (MN) following RT, which can stimulate IRF3 and NF-κB immune signaling that recruits various immune components to the tumor microenvironment. We hypothesized this effect is increased by combining RT with AZD6738, an inhibitor of ATR, which is a key protein in DNA repair and cell cycle regulation. Furthermore, we propose that the more clustered DNA damage caused by protons results in a higher level of MN than photons when these radiation types are combined with AZD6738.H1299 and PANC-1 cells were treated with 6 MV x-rays and 9.9 keV/mm (dose-weighted LET in water) protons alone or with AZD6738 (1 µM) to assess DNA repair, cell cycle distribution, MN and c-GAS positive MN (MN-cGAS+) 1 to 72 h after RT+AZD6738. We then irradiated a syngeneic 4T1 tumor model in BALB/c (hind leg tumors) with 6 MV x-rays to 3 × 6 Gy fractions with and without AZD6738 to assess tumor growth delay and the status of tumor infiltrating immune cells and immune cells in the draining lymph nodes (DLN).Protons+AZD6738 and photons+AZD6738 increased gH2AX foci, abrogated G2 arrest and amplified MN and MN-cGAS+ compared to photons or protons alone for both cell lines (H1299 and PANC-1). Compared to photons or AZD6738 alone, photons+AZD6738 delayed tumor growth, increased survival time and decreased the number of metastatic lung nodules. Photons+AZD6738 and photons alone increased T-regs and decreased T helper 1 cells in the tumor compared to vehicle and ATRi alone. Photons+AZD6738 increased and decreased CD8+ T cells in the tumor and DLN compared to the other treatments, respectively. Photons alone increased CD8+ PD-1+ T cells and photons+AZD6738 decreased that population back to baseline for both tumor and DLN. CD8+ GranzB+ T cells increased for photons+AZD6738 and photons alone in the tumor. Photons+AZD6738 and AZD6738 alone increased type 1 macrophages (M1) and decreased M2. Photons+AZD6738 increased the total population of dendritic cells (DCs) and non-significantly increased type 1 and 2 classical DCs compared to other treatments.Our data show that RT+AZD6738 initiates intracellular immune signaling, likely attributable to disruption of DNA repair and abrogation of cell cycle arrest. We also observed this effect was amplified with proton RT. Our findings of increased immune signaling were corroborated in vivo with the observation that RT+AZD6738 increased CD4+, CD8+ and macrophage populations within the tumor, however after an initial delay, tumors continued to progress. Additional investigation is warranted to determine the balance of regulatory elements such as T-regs, immune checkpoints, and antitumor immune signaling.