Tumor NLRP3 amplification promotes immunotherapy resistance by inhibiting STAT1 signaling and MHC class I expression
Balamayroon Theivanthiran, Nagendra Yarla, Kaylee Villarreal, Y-Van Nguyen, Mahere Rezazade Bazaz, Ernesto Pena Calderin, Linda Cao, Kyra Majors, Michael P. Plebanek, Alisha Holtzhausen, Emily Bolch, Douglas B. Johnson, Hope Uronis, John H. Strickler, Nicholas C. DeVito, Brent A. Hanks
Balamayroon Theivanthiran, Nagendra Yarla, Kaylee Villarreal, Y-Van Nguyen, Mahere Rezazade Bazaz, Ernesto Pena Calderin, Linda Cao, Kyra Majors, Michael P. Plebanek, Alisha Holtzhausen, Emily Bolch, Douglas B. Johnson, Hope Uronis, John H. Strickler, Nicholas C. DeVito, Brent A. Hanks
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Research Article Immunology Oncology

Tumor NLRP3 amplification promotes immunotherapy resistance by inhibiting STAT1 signaling and MHC class I expression

Abstract

Immunotherapy resistance remains a challenge in immuno-oncology, and predictive biomarkers are needed to guide combination immunotherapy selection for the individual patient. We showed that elevated tumor-intrinsic NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) signaling activity correlates with checkpoint inhibitor resistance in several independent cohorts of stage III/IV melanoma and gastroesophageal (GE) adenocarcinoma. In situ hybridization demonstrated tumor NLRP3 copy number gain in immunotherapy-resistant melanomas and GE adenocarcinomas harboring enhanced NLRP3 signaling activity. Nlrp3 amplification suppressed NOD-, LRR-, and CARD-containing 5–mediated (NLRC5-mediated) MHC class I upregulation, while spatial transcriptomic analysis of patient-derived GE adenocarcinomas confirmed that NLRP3 signaling activity inversely correlated with NLRC5 and MHC class I–associated gene expression. Mechanistically, NLRP3 binds to and inhibits signal transducer and activator of transcription 1 (STAT1) dimerization, nuclear translocation, and NLRC5 transcription. Consistent with these findings, pharmacologic inhibition of the NLRP3 inflammasome augmented tumor STAT1/NLRC5 signaling, enhanced MHC class I surface expression, and overcame anti–programmed cell death 1 resistance in an orthotopic model of gastric adenocarcinoma. This work reveals a fundamental link between cellular stress and tumor-mediated immune evasion and indicates that the tumor NLRP3 signaling pathway merits further clinical study as a therapeutic target and a source of companion biomarkers for overcoming checkpoint inhibitor resistance in patients with cancer.

Authors

Balamayroon Theivanthiran, Nagendra Yarla, Kaylee Villarreal, Y-Van Nguyen, Mahere Rezazade Bazaz, Ernesto Pena Calderin, Linda Cao, Kyra Majors, Michael P. Plebanek, Alisha Holtzhausen, Emily Bolch, Douglas B. Johnson, Hope Uronis, John H. Strickler, Nicholas C. DeVito, Brent A. Hanks

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Figure 2

Tumor NLRP3 inflammasome transcriptional signature predicts for anti–PD-1 resistance in multiple cohorts of patients with stage IV melanoma.

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Tumor NLRP3 inflammasome transcriptional signature predicts for anti–PD-...
(A) Schematic describing the generation of a tumor NLRP3 transcriptional signature (NLRP3-tsig). BRAFV600E PTEN–/–NLRP3a cell line was generated using a CRISPR activation (CRISPRa) protocol and implanted into syngeneic mice by subcutaneous injection. The Tyr-cre BRAFV600E PTEN–/– NLRP3fl/fl tumor model is a transgenic melanoma model with melanoma tissue–restricted silencing of Nlrp3. Each of these tumor models were resected for RNA-seq. Generated using BioRender. NLRP3a, Nlrp3 amplification. (B) Top, schematic illustrating the generation of the melanoma-specific Nlrp3–/– transgenic mouse model: Tyr-cre BRAFV600E PTENfl/fl NLRP3fl/fl (NLRP3–/–). Generated using BioRender. Bottom left, induced primary melanoma progression in the NLRP3–/– and ctrl transgenic melanoma mouse strains. Bottom right, end tumor volumes in the NLRP3–/– and ctrl transgenic melanoma models. Statistical analysis performed using unpaired t tests. (C) Single-sample gene set enrichment analysis (ssGSEA) shows a PMN-MDSC transcriptional signature (PMN-MDSC-sig) correlates with NLRP3-tsig in the melanoma (SKCM) TCGA. (D) Receiver operating characteristic (ROC) curve analysis of the NLRP3-tsig signature in the Hugo et al. and Gide et al. datasets of patients with stage IV melanoma treated with anti–PD-1 immunotherapy. AUC, area under the curve. (E) ROC curve analysis comparing the NLRP3-tsig signature with PMN-MDSC-sig and M-MDSC-sig transcriptional signatures in the Hugo et al. dataset. (F) ROC curve analysis comparing the NLRP3-tsig signature with previously published transcriptional signatures in the Hugo et al. dataset. (G) ROC curve analysis comparing the NLRP3-tsig signature with previously published anti–PD-1 resistance transcriptional signatures in the Lee et al. dataset of patients with stage IV melanoma treated with anti–PD-1 immunotherapy. (H) Gene Expression Profiling Interactive Analysis (GEPIA) utilized to analyze the disease-free survival of patients with melanoma in TCGA (SKCM) based on the NLRP3-tsig transcriptional signature (high, above the median; low, below the median). Statistical analysis performed by log-rank test. ***P < 0.0005.