Defects in the necroptosis machinery are a cancer resistance mechanism to checkpoint inhibitor immunotherapy.

Background: Immune checkpoint inhibitors (ICIs) of programmed cell death protein-1 (PD-1) or cytotoxic T-lymphocytes-associated protein 4 (CTLA-4) reinvigorate strong polyclonal T-cell immune responses against tumor cells. For many patients, these therapies fail because the development of spontaneous immune responses is often compromised, as the tumor microenvironment (TME) lacks proinflammatory signals resulting in suboptimal activation of antigen-presenting cells (APCs). Necroptosis is a special form of programmed cell death associated with leakage of inflammatory factors that can lead to APC maturation. However, it is unclear to which extent functional necroptosis in tumor cells contributes to ICI immunotherapy.

Methods: With genetically engineered tumor cell lines that lack specific components of the necroptosis machinery (mixed lineage kinase domain-like pseudokinase (MLKL), receptor interacting protein kinase 3 (RIPK3)), we addressed the importance of necroptotic tumor cell death for the efficacy of ICI immunotherapy in murine models. Preclinical data were aligned with genome-wide transcriptional programs in patient tumor samples at diagnosis and during ICI treatment for the activity of these pathways and association with treatment outcome.

Results: Mice bearing MLKL-deficient or RIPK3-deficient tumors failed to control tumor growth in response to anti-PD-1/anti-CTLA-4 immunotherapy. Mechanistically, defects in the necroptosis pathway resulted in reduced tumor antigen cross-presentation by type 1 conventional dendritic cells (DCs) in tumor-draining lymph nodes, and subsequently impaired immunotherapy-induced expansion of circulating tumor antigen-specific CD8⁺ T cells and their accumulation and activation in the TME. In vitro, co-culture of tumor cells undergoing necroptotic but not apoptotic programmed cell death resulted in increased uptake by phagocytic cells, associated with maturation and activation of DCs. Treatment of tumors with the epigenetic modulator azacytidine enhanced intrinsic transcriptional activity of the necroptosis machinery, and hence their susceptibility to ICI immunotherapy. In humans, transcriptome analysis of melanoma samples revealed a strong association between high expression of MLKL and prolonged overall survival and durable clinical response to immunotherapy with anti-PD-1 and/or anti-CTLA-4 checkpoint inhibitors.

Conclusions: Defective necroptosis signaling in tumor cells is a cancer resistance mechanism to ICI immunotherapy. Reversion of epigenetic silencing of the necroptosis pathway can render tumors susceptible to checkpoint inhibition.