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 5

Inhibiting NLRP3 overcomes resistance to anti–PD-1 Immunotherapy in an orthotopic model of CIN gastric adenocarcinoma.

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Inhibiting NLRP3 overcomes resistance to anti–PD-1 Immunotherapy in an o...
(A) p-STAT1 and t-STAT1 Western blot of whole tumor tissue lysate following treatment of orthotopic KPS CIN gastric tumors with vehicle control (ctrl) versus MCC950 (NLRP3i) (n = 3). (B) Pan-MHC class I IHC analysis of tumor tissues derived from orthotopic KPS CIN gastric tumors following treatment with either NLRP3i versus vehicle ctrl (n = 3). Representative images shown. Scale bar = 20 μm. (C) Pan-MHC class I flow cytometry analysis of FACS-sorted CD90.2–CD31–CD45– ctrl tumor cells following treatment of orthotopic KPS CIN gastric tumors with either NLRP3i versus vehicle ctrl. Left, representative pan-MHC class I flow histograms. Statistical analysis based on unpaired t test (n = 3). (D) Gross image of an orthotopic KPS CIN gastric tumor. Red arrow, implanted tumor cells in the gastric mucosa. Right, representative H&E microscopy of an orthotopic KPS CIN gastric tumor. Scale bar = 50 μm. (E) Schematic describing the anti–PD-1/NLRP3i treatment schedule for the orthotopic KPS CIN gastric cancer model. (F) Survival analysis of mice harboring orthotopic KPS CIN gastric tumors following treatment with IgG isotype control, anti–PD-1 alone, NLRP3i alone, versus combination anti–PD-1/NLRP3i (n = 10 mice/group). Statistical analysis based on log-rank test. (G) IVIS monitoring of orthotopic tdTomato-expressing KPS gastric tumors in syngeneic mice while undergoing IgG isotype control, anti–PD-1 alone, versus combination anti–PD-1/NLRP3i therapy. (H) IVIS quantification of tdTomato-expressing KPS gastric tumors while undergoing IgG isotype control, anti–PD-1 alone, versus combination anti–PD-1/NLRP3i therapy over time (n = 8 mice/group). (I) Left, NLRP3-ASC PLA of orthotopic KPS CIN gastric tumors following treatment with IgG isotype ctrl, anti–PD-1 ab, and anti–PD-1/NLRP3i. Right, plasma HSP70 ELISA performed on day 25 following treatment with IgG isotype ctrl, anti–PD-1 ab, and anti–PD-1/NLRP3i (n = 5 mice/group). (J) Left, flow cytometry analysis of tumor-infiltrating CD11b+Ly6G+F4/80– PMN-MDSCs following treatment with IgG isotype ctrl, anti–PD-1 ab, and anti–PD-1/NLRP3i. Right, flow cytometry analysis of tumor-infiltrating CD8+ T cells following treatment with IgG isotype ctrl, anti–PD-1 ab, and anti–PD-1/NLRP3i (n = 5 mice/group). (H) Statistical analysis performed using 2-way ANOVA followed by Tukey’s multiple-comparison test. (I and J) Statistical analysis performed using a 1-way ANOVA followed by Tukey’s multiple-comparison test. All data expressed as mean values ± SEM. *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.00005.