Inhibition Potentiates Immunogenicity in Merkel Cell Carcinoma: a Promising Approach to Overcome Resistance to Anti-Pd-1 Immunotherapy

Background and aims: ∼50% of patients with advanced Merkel cell carcinoma (MCC), an aggressive immunogenic tumor, demonstrate a striking response rate to anti-PD-1 immunotherapy. However, novel treatment strategies are required for the remaining patients with primary or acquired resistance who do not achieve durable benefit from PD-1 blockade. ATR (ataxia telangiectasia and Rad3-related kinase) mediates a key survival mechanism for cells with high replication stress (e.g., Ki-67+ tumors) by ensuring completion of DNA replication prior to mitosis. Surprisingly, preclinical and clinical studies have shown that targeting ATR, after the recent development of specific and potent ATR inhibitors (ATRi), may also stimulate anti-tumor immune response and augment the efficacy of immune-checkpoint inhibitors. Although the mechanisms determining how an ATRi may promote anti-tumor immunity are not well understood. MCC, which has a ∼70% Ki-67 positivity, could serve as an excellent tumor to test whether ATR inhibition can overcome resistance to anti-PD-1 therapy. We hypothesize that ATRi may potentiate immunogenic cell death (ICD) to recruit and activate antigen presenting cells. Methods: To test our hypothesis, representative Merkel cell polyomavirus-positive (VP) and virus-negative (VN) MCC cell lines were treated with an ATRi, alone or in combination with radiation (RT), were assessed for the induction of intrinsic immune markers (PD- L1, MHC-I, calreticulin, interferon-β and extracellular ATP release) via flow cytometry and ELISA. Next, ATRi ± RT treated VP-MCC cells were co-cultured with human monocyte-derived dendritic cells (moDCs) post-ATRi removal, and assayed for DC activation (CD86, CD80) and immune markers (PD-L1, MHC-I) via flow cytometry. Results: ATRi, alone or in combination with radiation, induced canonical markers of ICD and innate immunity in both VP- and VN-MCC cells. ATRi synergized with radiation to enhance activation and immunogenic markers on moDCs when compared to ATRi or RT treatment alone; enhancing MCC tumor cells’ ability to activate dendritic cells in vitro. Conclusion: ATRi augments tumor-intrinsic immune markers, and tumor cells treated with ATRi plus radiation activate antigen presenting cells in vitro. In summary, these data provide encouraging evidence for a clinical trial (in development) of ATR inhibition for patients with PD-1-refractory MCC.