Synergy of HSP90 Inhibition and Radiation Therapy for cGAS/STING Pathway Activation

Abstract

Even though immunotherapy is a common part in cancer treatment, only a small subset of patients profits long term. Since radiation therapy (RT) has been shown to improve tumor immunogenicity, treatment strategies combining RT and immunotherapy are an emerging field in cancer research. The inhibition of DNA damage repair (DDR) mechanisms is a potential target, because its impairment enhances, like RT alone, type-I interferon induction by activating the cGAS/STING pathway. Type-I interferons mediate the immunomodulatory effects following RT. Therefore, combination of RT and DDR inhibition appears to be a promising approach to enhance tumor immunogenicity. To induce broad DDR impairment, we here employ TAS-116 (Taiho Pharmaceutical Co., Ltd.), an inhibitor of Heat Shock Protein 90 (HSP90). First, we determined the tumor type-dependent toxicity by titering TAS-116 in six murine tumor cell lines and measured cell death via flow cytometry after 24h. Treatment with subtoxic concentrations of TAS-116 one hour prior to RT (up to 8Gy) resulted in increased γH2AX foci, indicating DNA damage, both in RP157.8 SCLC and B16F10 melanoma cells. Declining γH2AX foci were seen upon 12Gy in vitro. Our data suggest that irreparable DNA damage may occur which leads to cell death. The induction of DNA damage following TAS-116 treatment in combination with 8Gy RT was confirmed in vivo using a transplantable mouse tumor model. To confirm our hypothesis that the observed DNA damage mediates STING pathway activation in vitro, we performed Western blot analysis for pSTING at timepoints up to 72h after RT. While TAS-116 alone led to an increase up to 72h, highest pSTING levels were observed after 3h in combination with 8Gy RT, demonstrating synergistic effects for STING activation by accelerating and enhancing STING phosphorylation. While our results display an enhanced specific CD8+ T-cell-mediated killing in vitro using the transfected lymphoma cell line E.G7 after either treatment alone, combination of TAS-116 and RT up to 8Gy led to a significant reduction of this effect. We hypothesize that an observed upregulation of PD-L1 on the tumor cell surface, a known adverse effect of STING activation and suppressor of CD8+ T-cell-mediated killing, causes this phenomenon. Here, we demonstrate a synergy of HSP90 inhibition and RT to activate the immunogenic cGAS/STING pathway. However, to harness this effect therapeutically, anti-PD-1 therapy seems to be required.