787 Targeting DNA damage response modulates antiphagocytic signals to convert radiotherapy into a systemic immunotherapy to treat metastatic colorectal cancer

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Background In rare clinical cases, radiotherapy (RT) has been shown to exhibit the abscopal effect – shrinking not only the targeted tumor but also untargeted sites of disease. The abscopal effect is heavily linked to activation of systemic anti-tumor immunity; however, mechanistic understanding of how to meaningfully enhance the frequency of these responses in patients has been lacking. RT induces the ‘don’t eat me signals’ CD47 and PD-1 on the surface of colorectal tumor cells, limiting tumor cell phagocytosis decreasing the likelihood of abscopal effects. We showed that treatment of tumor cells with a DNA-damage response (DDR) inhibitor targeting the ATR kinase limits the induction of CD47 and PD-L1 signals thus promoting increased antitumor abscopal activity in vivo. Here, we aim to show that selective targeting of the DDR following local radiotherapy can prevent induction of anti-phagocytic ligands thereby enhancing both immunogenic tumor cell phagocytosis and regression of abscopal lesions. Methods Flow cytometry was performed on the MC38 and HCT116 colorectal cancer models post-radiation with 0, 5, or 10gy in combination with/without small molecule inhibitors of various DNA damage repair proteins. Azenosertib (WEE1i), Ceralasertib (ATRi) and Olaparib (PARPi) were used, and cells were stained after 24hr with antibodies against CD47, MHC-I, and PD-L1. For phagocytosis experiments, J774 and RAW 264.7 dual reporting macrophage lines were added into MC38 and HCT116 cells that were treated for 48hrs and analyzed using flow cytometry. Results We observed a dose-dependent increase in the expression of the ‘Don’t eat me signals’ CD47, PD-L1, and MHC-I from 0, 5, to 10Gy after 24hrs in MC38 and HCT116 cells. When cells were treated with the inhibitors of the DDR nodes WEE1(p=<0.05) and ATR(p=<0.05) in combination with RT, however, we no longer observed this induction. There was also an increased frequency and expression of pro-phagocytic ‘Eat Me’ signals Calreticulin and HSP90 post-RT in MC38. There was also an increase in phagocytosis by the J774 dual of MC38 post-RT + DDRi after 48hrs of treatment. In this case, the most effective inhibitors used in combination with RT were Azenosertib(p=<0.05) and Ceralasertib(p=<0.05). Conclusions Our results show treatment of colorectal cancer cell lines MC38, HCT116, and HT29 with RT alone induces the expression of antiphagocytic signals, however, in cells concurrently treated with DDR inhibitors the induction of these signals was attenuated. This decrease in antiphagocytic and increase in prophagocytic signals in combination groups was associated with increased phagocytosis of tumor cells by macrophages.

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