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 6

Elevated tumor-intrinsic NLRP3 activity and NLRP3 amplification correlate with anti–PD-1 resistance in patients with advanced gastroesophageal adenocarcinoma.

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Elevated tumor-intrinsic NLRP3 activity and NLRP3 amplification correlat...
(A) KeyLARGO clinical protocol included a pembrolizumab-only lead-in phase. Data collected from 5 responding (R) and 5 non-responding (NR) patients. (B) H&E, IF, and spatial transcriptomics workflow on GE adenocarcinoma tissue specimens. (C) Left, mean CD8+ T cells in ROIs day 0 and day 8. Original magnification, 40×. ROIs, regions of interest. Right, representative ROIs in a responder on day 0 and day 8. Scale bar = 200 μm. Green, CD8. Cyan, pan-cytokeratin (pan-CK). Red, elastase. Dark blue, nuclei. (D) Baseline mean CD8+ T cells in ROIs based on CD8 IF collected from R and NR. (E) Mean CD8+ T cells in ROIs based on CD8 IF on day 0 and day 8 from R (left) and NR (right). (F) Left, mean neutrophils in ROIs based on elastase IF collected on day 0 and day 8. Right, representative ROIs in an NR patient on day 0 and day 8. Scale bar = 200 μm. (G) Baseline mean neutrophils in ROIs based on elastase IF collected from R and NR. (H) Mean neutrophils in ROIs based on elastase IF collected on day 0 and day 8 from R (left) and NR (right). (I) Left, baseline NLRP3-ASC PLA dots per hpf collected from R and NR. Right, representative PLA IF field in R and NR. Scale bar = 5 μm. (J) Baseline plasma HSP70 levels categorized according to best objective response. CR, complete response; PR, partial response; SD, stable disease; PD, progressive disease. One-way ANOVA followed by Tukey’s multiple comparisons test. (K) Baseline HSP70 levels collected on day 0 and day 8 from R (left) and NR (right). (L) NLRP3/CEP1 copy number ratio based on FISH collected from R and NR. Right, representative IF field in R and NR. Red, NLRP3 probe. Green, chromosome enumeration probe for chromosome 1 (CEP1). Original magnification, 100×. Scale bar = 5 μm. (M) PFS (left) and OS (right) stratified based on NLRP3 amplification status. Log-rank test. (N) ssGSEA used to correlate PMN-MDSC-sig with NLRP3-tsig in the gastric cancer (STAD) TCGA. (O) Disease-free survival of patients with gastric cancer in TCGA (STAD) based on the NLRP3-tsig transcriptional signature (High, above the median; Low, below the median). Log-rank test. All other statistical analyses performed using unpaired t tests. Data expressed as mean values ± SEM. *P < 0.05, **P < 0.005.