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 3

Genetic and pharmacologic inhibition of NLRP3 in tumors enhances NLRC5 and MHC class I expression levels.

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Genetic and pharmacologic inhibition of NLRP3 in tumors enhances NLRC5 a...
(A) Most downregulated pathways in the Nlrp3-amplified BRAFV600E PTEN–/– melanoma cell line (BRAFV600E PTEN–/– NLRP3a) versus the parental BRAFV600E PTEN–/– melanoma cell line based on KEGG pathway analysis of RNA-seq data (n = 2). KEGG, Kyoto Encyclopedia of Genes and Genomes. (B) NLRP3 and NLRC5 Western blot analysis of the BRAFV600E PTEN–/– NTC and BRAFV600E PTEN–/– NLRP3a melanoma cell lines following PBS versus IFN-γ treatment. NTC, nontarget control (n = 3). (C) Left, NLRC5 Western blot analysis of the BRAFV600E PTEN–/– melanoma cell line following PBS or IFN-γ treatment with increasing concentrations of the NLRP3 inhibitor (NLRP3i), MCC950 (n = 3). Right, NLRC5 Western blot analysis of the human WM266 melanoma cell line following PBS or IFN-γ treatment with increasing concentrations of the NLRP3 inhibitor, MCC950 (n = 3). (D) Left, pan-MHC class I flow cytometry analysis of resected in situ BRAFV600E PTEN–/– NTC and BRAFV600E PTEN–/– NLRP3a melanomas. Right, pan-MHC class I flow cytometry analysis of resected in situ BRAFV600E PTEN–/– NTC melanomas following treatment with anti–PD-1 ab versus anti–PD-1 + MCC950. ab, antibody (n = 3). (E) pan-MHC class I flow cytometry analysis of BRAFV600E PTEN–/– NTC versus Nlrp3-knockdown (KD) BRAFV600E PTEN–/– NLRP3KD cell line following PBS versus IFN-γ treatment (n = 3). (F) Left, flow cytometry analysis of pan-MHC class I expression levels of resected primary melanomas in the NLRP3–/– and ctrl transgenic melanoma mouse strains. Right, percentage MHC class I–positive tumor cells in the NLRP3–/– and ctrl transgenic melanoma mouse strains (n = 3). D and F: statistical analysis performed using unpaired t test. Data expressed as mean values ± SEM. *P < 0.05.