NLRP3 and cancer: Pathogenesis and therapeutic opportunities.

More than a decade ago IL-1 blockade was suggested as an add-on therapy for the treatment of cancer. This proposal was based on the overall safety record of anti-IL-1 biologics and the anti-tumor properties of IL-1 blockade in animal models of cancer. Today, a new frontier in IL-1 activity regulation has developed with several orally active NLRP3 inhibitors currently in clinical trials, including cancer. Despite an increasing body of evidence suggesting a role of NLRP3 and IL-1-mediated inflammation driving cancer initiation, immunosuppression, growth, and metastasis, NLRP3 activation in cancer remains controversial. In this review, we discuss the recent advances in the understanding of NLRP3 activation in cancer. Further, we discuss the current opportunities for NLRP3 inhibition in cancer intervention with novel small molecules.

Dexamethasone and OLT1177 Cooperate in the Reduction of Melanoma Growth by Inhibiting STAT3 Functions.

The NLRP3 inflammasome is a multimolecular complex that processes inactive IL-1ß and IL-18 into proinflammatory cytokines. OLT1177 is an orally active small compound that specifically inhibits NLRP3. Here, B16F10 melanoma were implanted in mice and treated with OLT1177 as well as combined with the glucocorticoid dexamethasone. At sacrifice, OLT1177 treated mice had significantly smaller tumors compared to tumor-bearing mice treated with vehicle. However, the combined treatment of OLT1177 plus dexamethasone revealed a greater suppression of tumor growth. This reduction was accompanied by a downregulation of nuclear and mitochondrial STAT3-dependent gene transcription and by a significant reduction of STAT3 Y705 and S727 phosphorylations in the tumors. In vitro, the human melanoma cell line 1205Lu, stimulated with IL-1α, exhibited significantly lower levels of STAT3 Y705 phosphorylation by the combination treatment, thus affecting the nuclear functions of STAT3. In the same cells, STAT3 serine 727 phosphorylation was also lower, affecting the mitochondrial functions of STAT3. In addition, metabolic analyses revealed a marked reduction of ATP production rate and glycolytic reserve in cells treated with the combination of OLT1177 plus dexamethasone. These findings demonstrate that the combination of OLT1177 and dexamethasone reduces tumor growth by targeting nuclear as well as mitochondrial functions of STAT3.

Activation of Host-NLRP3 Inflammasome in Myeloid Cells Dictates Response to Anti-PD-1 Therapy in Metastatic Breast Cancers.

Tumor-associated inflammation leads to dysregulated cytokine production that promotes tumor immune evasion and anti-tumor immunity dysfunction. In advanced stage breast cancer, the proinflammatory cytokine IL-1ß is overexpressed due to large proportions of activated myeloid cells in the tumor microenvironment (TME). Here, we demonstrate the role of the host nucleotide-binding domain, leucine-rich containing family, pyrin domain-containing 3 (NLRP3) inflammasome in metastatic breast cancer. In vitro, we show that stimulation of THP-1 cells with conditioned media collected from MDA-MB-468 cells induced NLRP3 activation and increased Pdcd1l1 expression. In vivo, mice deficient in NLRP3 orthotopically implanted with metastatic breast cancer cell line (E0771) showed significant reduction in tumor growth (p < 0.05) and increased survival (p < 0.01). Inhibition of NLRP3 with the small molecule OLT1177® reduced expression of Pdcd1l1 (p < 0.001), Casp1 (p < 0.01) and Il1b (p < 0.01) in primary tumors. Furthermore, tumor-bearing mice receiving OLT1177® showed reduced infiltration of myeloid-derived suppressor cells (MDSCs) (p < 0.001) and increased CD8+ T cells (p < 0.05) and NK cells (p < 0.05) in the TME. NLRP3 inhibition in addition to anti-PD-1 treatment significantly reduced tumor growth from the monotherapies (p < 0.05). These data define NLRP3 activation as a key driver of immune suppression in metastatic breast cancers. Furthermore, this study suggests NLRP3 as a valid target to increase efficacy of immunotherapy with checkpoint inhibitor in metastatic breast cancers.

Tumor NLRP3-Derived IL-1ß Drives the IL-6/STAT3 Axis Resulting in Sustained MDSC-Mediated Immunosuppression.

Tumors evade the immune system by inducing inflammation. In melanoma, tumor-derived IL-1ß drives inflammation and the expansion of highly immunosuppressive myeloid-derived suppressor cells (MDSCs). Similar in many tumors, melanoma is also linked to the downstream IL-6/STAT3 axis. In this study, we observed that both recombinant and tumor-derived IL-1ß specifically induce pSTAT3(Y705), creating a tumor-autoinflammatory loop, which amplifies IL-6 signaling in the human melanoma cell line 1205Lu. To disrupt IL-1ß/IL-6/STAT3 axis, we suppressed IL-1ß-mediated inflammation by inhibiting the NOD-like receptor protein 3 (NLRP3) using OLT1177, a safe-in-humans specific NLRP3 oral inhibitor. In vivo, using B16F10 melanoma, OLT1177 effectively reduced tumor progression (p< 0.01); in primary tumors, OLT1177 decreased pSTAT3(Y705) by 82% (p<0.01) and II6 expression by 53% (p<0.05). Disruption of tumor-derived NLRP3, either pharmacologically or genetically, reduced STAT3 signaling in bone marrow cells. In PMN-MDSCs isolated from tumor-bearing mice treated with OLT1177, we observed significant reductions in immunosuppressive genes such as Pdcd1l1, Arg1, Il10 and Tgfb1. In conclusion, the data presented here show that the inhibition of NLRP3 reduces IL-1ß induction of pSTAT3(Y705) preventing expression of immunosuppressive genes as well as activity in PMN-MDSCs.

Oncogene-induced maladaptive activation of trained immunity in the pathogenesis and treatment of Erdheim-Chester disease.

Trained immunity (TI) is a proinflammatory program induced in monocyte/macrophages upon sensing of specific pathogens and is characterized by immunometabolic and epigenetic changes that enhance cytokine production. Maladaptive activation of TI (ie, in the absence of infection) may result in detrimental inflammation and development of disease; however, the exact role and extent of inappropriate activation of TI in the pathogenesis of human diseases is undetermined. In this study, we uncovered the oncogene-induced, maladaptive induction of TI in the pathogenesis of a human inflammatory myeloid neoplasm (Erdheim-Chester disease, [ECD]), characterized by the BRAFV600E oncogenic mutation in monocyte/macrophages and excess cytokine production. Mechanistically, myeloid cells expressing BRAFV600E exhibit all molecular features of TI: activation of the AKT/mammalian target of rapamycin signaling axis; increased glycolysis, glutaminolysis, and cholesterol synthesis; epigenetic changes on promoters of genes encoding cytokines; and enhanced cytokine production leading to hyperinflammatory responses. In patients with ECD, effective therapeutic strategies combat this maladaptive TI phenotype; in addition, pharmacologic inhibition of immunometabolic changes underlying TI (ie, glycolysis) effectively dampens cytokine production by myeloid cells. This study revealed the deleterious potential of inappropriate activation of TI in the pathogenesis of human inflammatory myeloid neoplasms and the opportunity for inhibition of TI in conditions characterized by maladaptive myeloid-driven inflammation.

Targeting tumor-derived NLRP3 reduces melanoma progression by limiting MDSCs expansion.

Interleukin-1ß (IL-1ß)-mediated inflammation suppresses antitumor immunity, leading to the generation of a tumor-permissive environment, tumor growth, and progression. Here, we demonstrate that nucleotide-binding domain, leucine-rich containing family, pyrin domain-containing-3 (NLRP3) inflammasome activation in melanoma is linked to IL-1ß production, inflammation, and immunosuppression. Analysis of cancer genome datasets (TCGA and GTEx) revealed greater NLRP3 and IL-1ß expression in cutaneous melanoma samples (n = 469) compared to normal skin (n = 324), with a highly significant correlation between NLRP3 and IL-1ß (P < 0.0001). We show the formation of the NLRP3 inflammasome in biopsies of metastatic melanoma using fluorescent resonance energy transfer analysis for NLRP3 and apoptosis-associated speck-like protein containing a CARD. In vivo, tumor-associated NLRP3/IL-1 signaling induced expansion of myeloid-derived suppressor cells (MDSCs), leading to reduced natural killer and CD8+ T cell activity concomitant with an increased presence of regulatory T (Treg) cells in the primary tumors. Either genetic or pharmacological inhibition of tumor-derived NLRP3 by dapansutrile (OLT1177) was sufficient to reduce MDSCs expansion and to enhance antitumor immunity, resulting in reduced tumor growth. Additionally, we observed that the combination of NLRP3 inhibition and anti-PD-1 treatment significantly increased the antitumor efficacy of the monotherapy by limiting MDSC-mediated T cell suppression and tumor progression. These data show that NLRP3 activation in melanoma cells is a protumor mechanism, which induces MDSCs expansion and immune evasion. We conclude that inhibition of NLRP3 can augment the efficacy of anti-PD-1 therapy.

The selective ROCK2 inhibitor KD025 reduces IL-17 secretion in human peripheral blood mononuclear cells independent of IL-1 and IL-6.

Reducing the activities of the pro-inflammatory cytokine IL-17 is an effective treatment strategy for several chronic autoimmune disorders. Rho-associated coiled-coil containing kinase 2 (ROCK2) is a member of the serine-threonine protein kinase family that regulates IL-17 secretion in T cells via signal transducer and activator of transcription 3 (STAT3)-dependent mechanism. We reported here that the selective ROCK2 inhibitor KD025 significantly reduced in vitro production of IL-17 in unfractionated human peripheral blood mononuclear cells (PBMCs) stimulated with the dectin-1 agonist Candida albicans. C. albicans induced IL-17 was reduced by 70% (p < 0.0001); a similar reduction (80%) was observed in PBMC stimulated with the Toll-like receptor 2 agonist Staphylococcus epidermidis (p < 0.0001). Treatment of PBMC with KD025 was not associated with a reduction in IL-1ß, IL-6 or IL-1α levels; in contrast, a 1.5 fold increase in the level of IL-1 receptor antagonist (IL-1Ra) was observed (p < 0.001). KD025 down-regulated C. albicans-induced Myosin Light Chain and STAT3, whereas STAT5 phosphorylation increased. Using anti-CD3/CD28 activation of the TCR, KD025 similarly suppressed IL-17 independent of a reduction in IL-1ß. Thus, ROCK2 directly regulates IL-17 secretion independent of endogenous IL-1 and IL-6 supporting development of selective ROCK2 inhibitors for treatment of IL-17-driven inflammatory diseases.

Interleukin-37 treatment of mice with metabolic syndrome improves insulin sensitivity and reduces pro-inflammatory cytokine production in adipose tissue.

Obesity and the metabolic syndrome are characterized by chronic, low-grade inflammation mainly originating from expanding adipose tissue and resulting in inhibition of insulin signaling and disruption of glycemic control. Transgenic mice expressing human interleukin 37 (IL-37), an anti-inflammatory cytokine of the IL-1 family, are protected against metabolic syndrome when fed a high-fat diet (HFD) containing 45% fat. Here, we examined whether treatment with recombinant IL-37 ameliorates established insulin resistance and obesity-induced inflammation. WT mice were fed a HFD for 22 weeks and then treated daily with IL-37 (1 µg/mouse) during the last 2 weeks. Compared with vehicle only-treated mice, IL-37-treated mice exhibited reduced insulin in the plasma and had significant improvements in glucose tolerance and in insulin content of the islets. The IL-37 treatment also increased the levels of circulating IL-1 receptor antagonist. Cultured adipose tissues revealed that IL-37 treatment significantly decreases spontaneous secretions of IL-1ß, tumor necrosis factor α (TNFα), and CXC motif chemokine ligand 1 (CXCL-1). We also fed mice a 60% fat diet with concomitant daily IL-37 for 2 weeks and observed decreased secretion of IL-1ß, TNFα, and IL-6 and reduced intracellular levels of IL-1α in the liver and adipose tissue, along with improved plasma glucose clearance. Compared with vehicle treatment, these IL-37-treated mice had no apparent weight gain. In human adipose tissue cultures, the presence of 50 pm IL-37 reduced spontaneous release of TNFα and 50% of lipopolysaccharide-induced TNFα. These findings indicate that IL-37's anti-inflammatory effects can ameliorate established metabolic disturbances during obesity.