Understanding the Role of Tumor-Intrinsic RIPK3 Signaling in Anti-Tumor Immunity

Abstract

Necroptosis is a form of regulated cell death that ultimately results in cell lysis. Necroptosis resistance is a common adaptation seen in tumors across many tissue types and is primarily mediated through impaired expression of the essential necroptotic kinase, receptor-interacting serine/threonine-protein kinase 3 (RIPK3), suggesting that RIPK3 and necroptosis may be detrimental for tumorigenesis. Specifically, tumor expression of RIPK3 may have important consequences for tumor immune surveillance as RIPK3 signaling in dying cells has been previously demonstrated to promote the cross-priming of tumor antigen-specific CD8+ T cells. Despite this, the role of RIPK3-dependent cell death in tumor regulation is controversial as impairing necroptosis downstream of RIPK3 has not been consistently found to abrogate tumor suppression. While RIPK3 is essential for necroptosis, it also has roles in apoptosis and cell death-independent NFkappaB signaling and cytokine production. We therefore undertook these studies to distinguish the consequences of these discrete outcomes of tumor-intrinsic RIPK3 signaling for anti-tumor immunity.To understand whether RIPK3-dependent cell death in tumor cells generates protective anti-tumor immunity, we utilized RIPK3 and OVA-expressing murine lung carcinoma (LLC-OVA) cells which undergo cell death upon proteasome inhibition. Genome editing was performed in these cells using CRISPR/Cas9 to delete caspase-8 or the necroptosis effector, mixed lineage kinase-like pseudokinase (MLKL), to allow for selective induction of necroptosis or apoptosis, respectively. Cell death in this system resulted in minimal NFkappaB-dependent cytokine production. Despite this, immunization with dying necroptotic cells (NEC) resulted in superior protection against challenge with live LLC-OVA cells compared to immunization with dying apoptotic cells (APOP). Surprisingly, NEC immunization was associated with superior tumor infiltration by CD4+ T cells and anti-tumor protection following NEC immunization was abrogated upon CD4 depletion. In contrast, neither depletion of CD8+ T cells nor impairment of CD8+ T cell cross-priming reduced the efficacy of NEC immunization. We found that the priming of tumor-specific CD4+ T cells predominantly occurred following live tumor challenge and not at the time of immunization. Consequently, immunization with necroptotic LLC-OVA cells could protect against challenge with non-OVA expressing melanoma and breast cancer cells. To gain further insight into the mechanism by which necroptotic cell immunization bolstered anti-tumor immunity in our system, we performed bulk RNAseq and found that tumors from mice that received NEC immunization exhibited enhanced expression of a type I interferon (IFN-I) gene signature. Accordingly, NEC-induced T cell infiltration and tumor suppression was abrogated with loss of host IFN-I signaling. IFN-I and interferon stimulated genes were specifically expressed in necroptotic, and not apoptotic, tumor cells in a cell death-dependent manner. Further, NEC immunization remained effective in Myd88-/-, Sting1-/-, and Mavs-/- mice, suggesting that IFN-I production, and not release of the nucleic acids, by necroptotic cells instigated the anti-tumor effects of NEC immunization. Taken together, these data suggest that necroptosis bolsters CD4+ T cell responses against tumors of distinct tissue types independent of dying cell antigen and requires host IFN-I signaling. We hope that this study will inform future efforts to understand discrete aspects of RIPK3 signaling in other disease contexts and the utilization of necroptotic cell death for cancer therapies.