DNA Damage Response Inhibitors as Potentiators of Radiation and Immunogenicity in Merkel Cell Carcinoma

Abstract

<h3>Purpose/Objective(s)</h3> Both Merkel cell polyomavirus positive (VP) and negative (VN) Merkel cell carcinoma (MCC) are highly immunogenic with the highest response rates to immune checkpoint inhibitors (ICI) among solid tumors. Approximately 50% of patients with advanced VP or VN MCC attain durable disease control with ICI; but, patients with MCC (or other cancers) refractory to ICI have limited options necessitating new immune stimulating therapies. Cell cycle checkpoints and the DNA damage response (DDR) are promising targets, and selective inhibitors of key DDR components, including ATR, ATM & DNA-PK, are in early clinical trials. Deregulation of early cell cycle components (e.g., p53 & Rb inactivation paired with Myc protein upregulation) promotes rapid MCC cell division, suggesting high replication stress and susceptibility to DDR inhibitors (DDRi) or DNA damaging agents. Indeed, MCC is very radiosensitive. Animal studies show DDRi + radiation synergize to induce antitumor immunity. The mechanisms for how DDRi promote antitumor immunity are not well understood; however, they may drive immunogenic tumor cell death. Here, we assess potential synergy of DDRi + radiation to upregulate MCC immune markers. <h3>Materials/Methods</h3> MCC-VP (WaGa & MKL2) and VN (MCC13-HL) MCC cell lines were treated with various doses of single fraction radiation (0, 2, 4, 8, 16, 24 Gray; 160 kV x-rays) +/- inhibitors of ATR, ATM or DNA-PK. Cells were assessed for viability, expression of IFNβ (ELISA), and MHC-I expression (flow cytometry at 3 days post-radiation). <h3>Results</h3> In VN-MCC cells, ATRi and ATMi potentiated low-dose radiation (2-4 Gy) to induce IFNβ release and MHC-I upregulation compared to drug or radiation treatment alone at equivalent doses. DNA-PKi was the most potent radiosensitizer inducing rapid cell death when combined with low-dose radiation (2-4 Gy), but early cell death limited IFNβ production relative to VN-MCC treated with radiation alone. DNA-PKi + low-dose radiation to upregulated MHC-I in VN-MCC. All DDRi tested abrogated peak IFNβ production in VN-MCC at radiation doses ≥8 Gy. The Merkel cell polyomavirus inhibits cGAS/STING mediated IFNβ production, and radiation + DDRi did not induce detectable IFNβ release in two VP cell lines. All MCC cell lines were viable when treated with DDRi alone. <h3>Conclusion</h3> Given inherent immunogenicity, cell cycle checkpoint deficiencies and radiosensitivity, MCC is an ideal tumor to test the potential of DDRi to overcome ICI-resistance. Our preliminary data suggest that ATRi and ATMi may effectively potentiate immunogenic cell death in a VN-MCC cell line when paired with 2-4 Gy radiation, yet abrogate IFNβ production with higher radiation doses <b>in vitro</b> suggesting higher doses coupled with DDRi induce non-immunogenic cell death. DNA-PKi was the most potent radiosensitizer in 3 MCC cell lines by cell viability assays but less effective at inducing IFNβ. <b>In vitro</b> studies are ongoing to support future animal studies as we explore a potential trial for DDRi in ICI-resistant MCC.