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 1

Elevated tumor-intrinsic NLRP3 activity and NLRP3 amplification correlate with anti–PD-1 resistance in multiple cohorts of patients with advanced melanoma.

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Elevated tumor-intrinsic NLRP3 activity and NLRP3 amplification correlat...
(A) Tumor NLRP3 inflammasome activity levels measured based on NLRP3-ASC proximity ligation assays (PLAs) in responder (R) and nonresponder (NR) patients with stage IV melanoma treated with anti–PD-1 monotherapy. Quantified as dots per high-power field (hpf). Original magnification, 40×. Right, representative PLA images in R and NR patients. Scale bar = 10 μm. Vanderbilt patient cohort. (B) Progression-free survival (PFS) of patients with naive stage IV melanoma treated with anti–PD-1 monotherapy stratified based on baseline NLRP3-ASC PLA scores below and above the median. Multivariate analysis performed using the Cox proportional hazards model. (C) Baseline tumor NLRP3/CEP1 copy number ratios based on FISH in R and NR patients with stage IV melanoma undergoing anti–PD-1 monotherapy. Right, representative FISH images in R and NR patients. Original magnification, 100×. Scale bar = 5 μm. Duke patient cohort. CEP1, chromosome enumeration probe 1. (D) Spearman’s correlation analysis between NLRP3 activity levels based on NLRP3-ASC PLAs and NLRP3 copy number based on FISH in stage IV melanoma tissues. (E) Tumor NLRP3/CEP1 copy number ratios based on FISH in R and NR patients with stage IV melanoma. Vanderbilt patient cohort. (F) Baseline tumor NLRP3 inflammasome activity levels measured based on NLRP3-ASC PLAs in nonrelapsed and relapsed patients with stage III melanoma treated with adjuvant anti–PD-1 immunotherapy. (G) Plasma HSP70 levels based on ELISA in nonrelapsed and relapsed patients with stage III melanoma treated with adjuvant anti–PD-1 immunotherapy. (H) Tumor NLRP3/CEP1 copy number ratios in nonrelapsed and relapsed patients with stage III melanoma treated with adjuvant anti–PD-1 immunotherapy. (I and J) Spearman’s correlation analysis between NLRP3 activity levels based on NLRP3-ASC PLAs and HSP70 plasma levels based on ELISA (I) and NLRP3-ASC PLAs and NLRP3 copy number based on FISH (J) in stage III melanoma tissues. (K) Recurrence-free survival (RFS) of patients with stage III melanoma treated with adjuvant anti–PD-1 immunotherapy stratified based on baseline NLRP3-ASC PLA scores below and above the median. Statistical analysis performed by log-rank test. A, C, and E–H: statistical analysis performed using unpaired t tests. Data expressed as mean values ± SEM. **P < 0.005, ***P < 0.0005.