An inducible RIPK3-driven necroptotic system enhances cancer cell–based immunotherapy and ensures safety
Kok-Siong Chen, … , Natalia Claire Mendonca, Khalid Shah
Kok-Siong Chen, … , Natalia Claire Mendonca, Khalid Shah
Published November 19, 2024
Citation Information: J Clin Invest. 2025;135(2):e181143. https://doi.org/10.1172/JCI181143.
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Research Article Immunology Oncology

An inducible RIPK3-driven necroptotic system enhances cancer cell–based immunotherapy and ensures safety

Abstract

Recent progress in cancer cell–based therapies has led to effective targeting and robust immune responses against cancer. However, the inherent safety risks of using live cancer cells necessitate the creation of an optimized safety switch without hindering the efficacy of immunotherapy. The existing safety switches typically induce tolerogenic cell death, potentially leading to an immunosuppressive tumor immune microenvironment (TIME), which is counterproductive to the goals of immunotherapy. Here, we developed and characterized an inducible receptor-interacting protein kinase 3–driven (RIPK3-driven) necroptotic system that serves a dual function of safety switch as well as inducer of immunogenic cell death, which in turn stimulates antitumor immune responses. We show that activation of the RIPK3 safety switch triggered immunogenic responses marked by an increased release of ATP and damage-associated molecular patterns (DAMPs). Compared with other existing safety switches, incorporating the RIPK3 system inhibited tumor growth, improved survival outcomes in tumor-bearing mice, and fostered long-term antitumor immunity. Moreover, the RIPK3 system reinvigorated the TIME by promoting DC maturation, polarizing the macrophages toward a M1 phenotype, and reducing the exhaustion of CD4+ and CD8+ T lymphocytes. Our study highlights the dual role of the RIPK3-driven necroptotic system in improving the safety and efficacy of cancer cell–based therapy, with broader implications for cellular therapies.

Authors

Kok-Siong Chen, Sarah Manoury-Battais, Nobuhiko Kanaya, Ioulia Vogiatzi, Paulo Borges, Sterre J. Kruize, Yi-Ching Chen, Laura Y. Lin, Filippo Rossignoli, Natalia Claire Mendonca, Khalid Shah

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Figure 1

Establishing an inducible RIPK3-driven necroptotic safety switch.

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Establishing an inducible RIPK3-driven necroptotic safety switch. (A) Co...
(A) Coclustering of primary tumor samples (n = 2,785) in TCGA based on apoptotic (GO: 0097190) and necroptotic (GO: 0097527) signaling pathway gene signatures. (B) Survival curves showing survival probability for C1 and C2 clusters from A. The survival curves between 2 groups were compared using a log-rank (Mantel-Cox) test with Bonferroni correction. (C) Expression of HMGB1 in C1 and C2 clusters from A. (D) Coclustering of primary tumor samples (n = 222) in the CCGA based on apoptotic (GO: 0097190) and necroptotic (GO: 0097527) signaling pathway gene signatures. (E) Survival curves showing overall survival probability for C2 and C3 clusters from D. The survival curves between 2 groups were compared using a log-rank (Mantel-Cox) test with Bonferroni correction. (F) Expression of HMGB1 in C2 and C3 clusters from D. (G) Schematic representation of the RIPK3 B/B-inducible safety switch system. (H) CT2A and CT2A-RIPK3 cell viability assay following treatment with B/B in a dose-dependent manner for 6 hours and time-lapse imaging of the cells before and after treatment with 10 nM B/B for 6 hours. (I) Western blot analysis of p-MLKL, p-RIPK3, and cleaved caspase 3 levels upon B/B treatment in CT2A and CT2A-RIPK3 cells with different treatment durations. (J) Schematic of the experimental timeline for intracranial GBM cell implantation and the treatment schedule for B/B. (K) Graph of Fluc signal in C57BL6 mice after intracranial implantation of CT2A cells with B/B treatment (n = 3) and CT2A-RIPK3 cells with (n = 3) and without (n = 3) B/B treatment. Dotted line represents the B/B treatment time point. Data represent the mean ± SEM. (L) Kaplan-Meier curves showing the survival probability for C57BL6 mice after intracranial implantation with CT2A with B/B treatment (n = 3) and CT2A-RIPK3 with (n = 3) and without (n = 3) B/B treatment. *P < 0.05, ***P < 0.001, and ****P < 0.0001, by log-rank (Mantel-Cox) test with Bonferroni correction (B, E, and L) and unpaired, 2-tailed Student’s t test (C and F). RPKM, reads per kilobase per million mapped reads.