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

Abstract

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.

Keywords: Immune Checkpoint Inhibitor; Immunotherapy; T cell; Tumor microenvironment - TME.

Figure 1. Loss of tumor-intrinsic MLKL impairs the efficacy of cancer immunotherapy with immune checkpoint inhibitors. (A) MLKL protein expression in gene-engineered cell lines was assessed using western blotting. (B) Treatment scheme: Mice were inoculated with either wild-type (WT) or MLKL-deficient (MLKL^−/−) tumor cells and were injected intraperitoneally with anti-CTLA-4 ± anti-PD-1 or isotype control antibodies. (C) Tumor growth and (D) overall survival of C57BL6/J mice bearing either WT or MLKL^−/− B16 melanoma tumors after treatment with anti-CTLA-4. (E) Tumor growth and (F) overall survival of mice bearing either WT or MLKL^−/− B16 melanoma tumors after treatment with anti-CTLA-4 in combination with anti-PD-1. (G) Tumor growth in BALB/c mice inoculated with either WT or MLKL^−/− CT26 colon adenocarcinomas after treatment with anti-CTLA-4 and anti-PD-1. Data show mean tumor volume±SEM or survival for n=5–10 mice per group that are either pooled from or representative of two independent experiments. CTLA-4, cytotoxic T-lymphocytes-associated protein 4; i.p., intraperitoneal; MLKL, mixed lineage kinase domain-like pseudokinase; OVA, ovalbumin; PD-1, programmed cell death protein-1.

Figure 2. Defects in the RIPK3/MLKL necroptosis machinery render tumors resistant to ICI. (A) Treatment scheme: mice were inoculated with either WT, MLKL^−/− or RIPK3-deficient (RIPK3^−/−) tumor cells and were injected intraperitoneally with anti-CTLA-4 and anti-PD-1 or isotype control antibodies. (B) Tumor growth and (C) overall survival of C57BL6/J mice bearing either WT or RIPK3^−/− B16 melanoma tumors after treatment with anti-CTLA-4 and anti-PD-1. (D–E) Some animals were sacrificed on day 14 and abundance of intratumoral phospho-MLKL⁺ (pMLKL⁺) cells was determined by immunohistochemistry (IHC). (D) Graphed data give mean pMLK⁺ cells ± SEM from n=4–5 individual mice per group. Data are representative of two independent experiments. (E) Representative microscopy images. (F) Tumor growth in BALB/c mice inoculated with either WT or RIPK3^−/− CT26 colon adenocarcinomas after treatment with anti-CTLA-4 and anti-PD-1 on days 5, 8, and 11. Data show mean tumor volume±SEM or survival for n=5–10 mice per group that are either pooled from or representative of two independent experiments. CTLA-4, cytotoxic T-lymphocytes-associated protein 4; ICI, immune checkpoint inhibitor; i.p., intraperitoneal; MLKL, mixed lineage kinase domain-like pseudokinase; OVA, ovalbumin; PD-1, programmed cell death protein-1; pMLKL, phosphorylated MLKL; RIPK3, receptor interacting protein kinase 3; WT, wild type.

Figure 3. Defects in the MLKL necroptosis machinery result in reduced cDC1 function and impaired cross-priming of tumor antigen-specific T cells. (A) Experimental setup 1: mice injected with either WT or MLKL^−/− B16.OVA melanoma cells were treated with anti-CTLA-4 and anti-PD-1 or isotype control i.p. on days 6, 9, and 12. Blood was sampled on day 13, tumors and draining lymph nodes (TdLN) were extracted on day 14 and analyzed by flow cytometry. (B) Absolute count of cDC1 and (C) MHC-I SIINFEKL^high cDC1 in TdLNs. cCD1 were defined as CD11c⁺ CD11b⁻ CD103⁺ CD8⁺ MHC-II⁺ cells. (D) Frequency of H-2Kb-SIINFEKL Tetramer⁺ CD8⁺ T cells in the peripheral blood and (E) in the tumor microenvironment (TME). Expression of (F) CD44 and (G) IFNγ in CD8⁺ T cells in the TME presented as mean fluorescence intensity (MFI). (H) Experimental setup 2: mice were injected in a bilateral B16.OVA melanoma tumor model with WT cells on the right flank and MLKL^−/− cells on the left flank. Mice were then treated with either anti-CTLA-4 and anti-PD-1 or isotype control antibodies. (I) Tumor growth in the bilateral tumor model. All data are shown as mean values±SEM for n=5–10 individual mice per group that are either pooled from or representative of two independent experiments. cDC1, conventional type 1 dendritic cell; CTLA-4, cytotoxic T-lymphocytes-associated protein 4; IFNγ, interferon-gamma; i.p., intraperitoneal; MHC-I, major histocompatibility complex-I; MLKL, mixed lineage kinase domain-like pseudokinase; ns, not significant; OVA, ovalbumin; PD-1, programmed cell death protein-1; WT, wild type.

Figure 4. RIPK3/MLKL-mediated tumor cell necroptosis fosters uptake of tumor cell debris by and maturation of bystander APCs. (A) Mice inoculated with either WT or MLKL^–/– B16.OVA melanoma cells were treated with anti-CTLA-4 and anti-PD-1 or isotype control i.p. on days 6, 9, and 12 as described for figure 3. Tumor tissue was extracted on day 14 and was pooled from three individual mice per group for subsequent ex vivo cytokine and chemokine profiling. Heatmap showing the relative abundance of cytokines and chemokines in the TME. (B) Regulated tumor cell death in MOC1 oral squamous carcinoma cells after exposure to TNF-α and a SMAC-mimetic (TS, apoptosis induction), combination of TS and the pan-caspase inhibitor zVAD (TSZ, necroptosis induction), or additional necrostatin-1 (TSZ Nec-1) to inhibit necroptosis. (C–D) Bone marrow-derived DCs or macrophages were co-cultured with either WT, MLKL^–/– or RIPK3^–/– CFSE-labeled MOC1 tumor cells undergoing different forms of cell death and were then analyzed by flow cytometry. (C) Uptake of CFSE⁺ tumor cell debris by macrophages (defined as CD11b⁺ F480⁺). (D) CD86 expression on dendritic cells (defined as CD11c⁺) after co-culture. APCs, antigen-presenting cells; CTLA-4, cytotoxic T-lymphocytes-associated protein 4; DC, dendritic cell; GM-CSF, granulocyte-macrophage colony-stimulating factor; IFNγ, interferon-gamma; IL, interleukin; i.p., intraperitoneal; MLKL, mixed lineage kinase domain-like pseudokinase; OVA, ovalbumin; PD-1, programmed cell death protein-1; RIPK3, receptor interacting protein kinase 3; TME, tumor microenvironment; WT, wild type.

Figure 5. Epigenetic upregulation of the necroptosis machinery by hypomethylating agents can augment tumor susceptibility to ICI immunotherapy. Mice were inoculated with B16.OVA WT cells and treated with two injections of intratumoral 5-azacytidine (AZA) on days 8 and 11. RNA was extracted from the tumor tissue on day 12 and analyzed via qPCR. (A) Relative expression of Mlkl and Ripk3 mRNA normalized to untreated tumors. Data were pooled from two separate experiments. (B) Treatment scheme: WT mice were inoculated with either WT or MLKL^−/− B16.OVA cells. Recipients were injected intraperitoneally with anti-CTLA-4/anti-PD-1 or isotype control antibodies in combination with intratumoral application of AZA. (C) Mean tumor volume±SEM of n=5–10 individual mice per group that were pooled from two independent experiments. CTLA-4, cytotoxic T-lymphocytes-associated protein 4; ICI, immune checkpoint inhibitor; i.p., intraperitoneal; i.t., intratumoral; MLKL, mixed lineage kinase domain-like pseudokinase; mRNA, messenger RNA; ns, not significant; OVA, ovalbumin; PBS, phosphate-buffered saline; PD-1, programmed cell death protein-1; qPCR, quantitative PCR; RIPK3, receptor interacting protein kinase 3; WT, wild type.

Figure 6. High transcriptional activity of MLKL in human melanoma correlates with prolonged survival and durable responses to ICI immunotherapy. (A) Overall survival in 458 patients with advanced malignant melanoma from TCGA by expression of MLKL in RNA-sequencing of bulk tumor tissue. (B) Progression-free survival in n=73 patients with malignant melanoma undergoing anti-PD-1±anti-CTLA-4 ICI immunotherapy by expression of MLKL in tumor samples. CTLA-4, cytotoxic T-lymphocytes-associated protein 4; ICI, immune checkpoint inhibitor; MLKL, mixed lineage kinase domain-like pseudokinase; PD-1, programmed cell death protein-1; TCGA, The Cancer Genome Atlas.