Immunotherapy response and resistance: Dominant mechanisms in Merkel cell carcinoma

Abstract

Merkel cell carcinoma (MCC) is a rare, but deadly skin cancer that is rapidly increasing in incidence. Programed death-1 (PD-1) pathway blockade has been successful in treating MCC, but it is not effective for all patients. PD-1 therapy works by reinvigorating exhausted T cells, but major resistance mechanisms in MCC are currently unknown. Data from other cancers and murine models have described many mechanisms of immunotherapy resistance including terminal exhaustion in T cells, an immunosuppressive environment tumor microenvironment, T cell exclusion from the tumor, downregulation of antigen presentation and a lack of immunogenic antigens. To overcome PD-1 blockade resistance, several strategies are being pursued that target these different mechanisms. However, prioritization of clinical trials is challenging in rare cancers like MCC where only a few trials can be carried out simultaneously. In Chapter 1 and 2, we describe the current state of immunotherapy for MCC and challenges in the treatment of this cancer. In Chapter 3, we characterized cancer-specific T cells in 35 MCC patients and found that higher MCPyV-specific CD8 T-cell frequency in pre-treatment blood correlated with response to immunotherapy. Single cell RNA sequencing revealed that MCPyV-specific CD8 T cells in blood had increased stem/memory signatures and decreased exhaustion signatures compared to those in tumors. This suggests that the blood acts a reservoir of cancer-specific T cells at earlier stages of exhaustion. However, longitudinal samples showed emergence of immunotherapy resistance via downregulation of MHC-I despite abundant circulating cancer-specific CD8 T cells. This was studied in greater detail in Chapter 4. There we detail a second patient with secondary resistance to PD-1 pathway blockade despite the presence of cancer-specific CD8 T cells in the patient’s tumor and blood. This patient’s tumor lacked MHC-I expression, which was partially restored with an intralesional STING agonist. The patient experienced a durable partial response to treatment. Chapters 5, 6 and 7 further describe antigen-specific T cells and the development of exhaustion in these cells. In Chapter 5, we detail neoantigen-specific T cells and show that these cells were predominantly CD4 restricted. These cells were prevalent in a patient with a profound response to anti-PD-L1 and exhibited a TH1 phenotype that could support anti-tumor immunity. Chapter 6 describes how the development of terminal exhaustion in T cells is potentiated by IL-2 signaling. Chapter 7 describes the tumor microenvironment in human tumors using a novel bioinformatic method and spatially linked RNA sequencing. The next three chapters describe new clinical trials based in part on the data presented in the previous chapters: Triple immune checkpoint blockade targeting PD-1, TIM3 and LAG3 (Chapter 8), therapeutic vaccination (Chapter 9) or inhibition of ATR (Chapter 10). Finally, Chapters 11 and 12 describe advancements in clinical management in MCC and how more personal minimal management can lead to improved patient care. We show that anti-PD-1 therapy dosing can be reduced in patients who respond to PD-1 blockade with compromising response (Chapter 11). We also investigate the importance of surgical margins and radiotherapy in MCC and demonstrate that tissue sparing surgery and adjuvant radiation can achieve protect against local recurrences when used in combination (Chapter 12). We have used our extensive and unique repository of clinically annotated MCC blood and tumor tissues, along with clinical trial specimens, to characterize cancer-specific T cells and elucidate mechanisms of immunotherapy response and resistance in MCC, particularly an absence of circulating MCPyV-specific CD8 T cells. These studies have helped us define the mechanisms by which some patients mediate superior disease control and contributed to our goal of improving therapeutic options for patients with advanced MCC.