Targeting DNA Damage Response Modulates "don't eat me" signals to convert radiotherapy into a systemic immunotherapy to treat metastatic Colorectal Cancer
Introduction
Colorectal cancers (CRC) are the second leading cause of cancer-related death. In rare clinical cases, radiotherapy (RT) has been shown to elicit the “abscopal effect” in which not only the targeted tumor shrinks, but other untreated sites of cancer also regress. Radiotherapy can generate complete responses when targeting early stage primary colorectal tumors but has very limited therapeutic benefit for metastatic colorectal cancer (mCRC) with abscopal effects occurring in <5% of patients. The abscopal effect is heavily linked to activation of systemic anti-tumor immune activation but, the mechanisms that determine whether abscopal effects occur and how to enhance them are not well understood. Recent data shows that RT induces the “don’t eat me signals” CD47 and PD-L1 on the surface of tumor cells. When bound to their cognate receptors SIRPα and PD-1 on myeloid cells, these signals block phagocytosis of dying tumors cells which prevents the activating of tumor-specific T cell responses necessary to generate abscopal effects. Previously, we showed that treatment of tumor cells with a DNA-damage repair inhibitor of ATR prevents the induction of CD47 and PD-L1 signals, resulting in increased antitumor abscopal activity in vivo. Our ongoing studies suggest that inhibition of critical DNA damage repair proteins such as WEE1 and PARP elicits a similar effect. Therefore our hypothesis is that selective targeting of specific nodes of the DDR following local radiotherapy, prevents induction of anti-phagocytic ligands (Don’t eat me signals), enhances immunogenic tumor cell phagocytosis, antigen cross-presentation, and regression of abscopal lesions.
Methods
Flow cytometry was performed on several colorectal cancer cell lines including MC38, HCT116, CT26, and HT29 post-radiation with 0, 5, or 10gy in combination with/without small molecule inhibitors of DNA damage repair proteins. Zn-c3 (WEE1i), Ceralasertib (ATRi) and Olaparib (PARPi) were used and cells were stained after 24hr with antibodies against CD47, MHC-I, and PD-L1. In order to investigate phagocytosis, we used J774, RAW 264.7, and THP-1 dual reporter macrophage lines, which were added to MC38 and HCT116 cells that were pre-treated for 48hrs. The macrophages were cocultured with the treated tumor cells for 4hrs and flow cytometry was used to quantity the proportion of macrophages that consumed tumor cells.
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, HCT116, and HT29 cells. Addition of DDRi against WEE1(p=<0.05) or ATR(p=<0.05) prevented “Don’t Eat Me” induction following RT. In this context, PARP inhibitors were also able to reduce CD47 and MHC-I induction but had little effect on PD-L1. We also observed an increase in phagocytosis by J774 macrophages of MC38 tumor cells post-RT + DDRi after 48hrs of treatment with the most effective inhibitor being Zn-C3(p=<0.05) followed by Ceralasertib(p=<0.05) and Olaparib.
Conclusion
The goal of our research is to describe the relationship between RT, the resulting DNA damage response, and the efficiency of subsequent phagocytosis of dying tumor, so that the benefits of RT for CRC patients can be improved. Our results show treatment of colorectal cancer cell lines MC38, HCT116, and HT29 with RT alone induces the expression of multiple “Don’t eat me” signals. Blocking key elements of DDR pathways, however, was able to prevent the induction of these signals and enhance downstream anti-tumor immunity. This outcome occurred independent of the cell lines’ TP53 status, indicating that this phenomenon is not regulated by TP53. Preventing “Don’t Eat Me” signal induction using DDRi significantly increased phagocytosis of irradiated tumor cells by macrophages. Our findings show that blockade of key DDR proteins in concert with RT prevents anti-phagocytic checkpoint induction and improves anti-tumor immune priming.