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BackgroundImmunotherapy resistance has been correlated with epithelial-to-mesenchymal transition (EMT),1 2 however our understanding of tumor-intrinsic mechanisms driving this immune evasive phenotype is lacking. We have previously shown that Wnt ligands are upregulated in anti-PD-1 resistant melanomas,3 and postulated that upstream transcriptional regulation of select EMT pathways may underpin these findings. The hedgehog signaling (HH) transcription factor Gli2 promotes EMT.MethodsGli2 was constitutively activated (Gli2CA ) in a BRAFV600EPTEN-/- murine cell line via an N-terminal truncating mutation and silenced using CRISPR-Cas9. Multi-parameter flow cytometry and RNAseq was utilized to evaluate the impact of Gli2 on the tumor immune microenvironment. Anti-PD-1 resistance studies were performed in Gli2CA and control tumors. Bioinformatics studies were conducted using the melanoma TCGA and Hugo et al databases.2ResultsWe found upregulation of Gli2 targets in patients with anti-PD-1-refractory metastatic melanoma as well as in an autochthonous BRAFV600EPTEN-/- melanoma model after escape from anti-PD-1. RNAseq and Western blot studies demonstrated Gli2CA to promote EMT and Wnt ligand production in addition to upregulated COX2 in BRAFV600EPTEN-/- melanoma. This finding was reversed by genetic ablation and pharmacologic inhibition of Gli2, implicating a previously undescribed role for Gli2 in modulating COX2. These data were consistent with a notable correlation between a Gli2 signature and a prostaglandin synthesis signature in human melanoma TCGA database. Flow cytometry analysis showed exclusion of cytolytic T and NK cells, a shift from cDC1s to cDC2s, and enhanced MDSC recruitment in Gli2CA tumors. Consistent with these findings, whole tumor RNAseq of Gli2CA tumors demonstrated a decrease in Cd3e, Prf1, and Xcr1 with a concomitant increase in Cxcl1, Cxcl2, Ccl2, Ptgs2, and Arg1 relative to control tumors. RNAseq of FACS-sorted DCs from Gli2CA tumors demonstrated a loss of cDC1-associated genes including Xcr1, Wdfy4, and Clec9a compared to DCs derived from control tumors. In-line with our previous results showing that Wnt5a promotes MDSC recruitment in a Yap-dependent manner,4 we found that Yap inhibition or Wnt5a deletion in the BRAFV600EPTEN-/-Gli2CA cell line diminished MDSC-recruiting chemokines. Further consistent with these findings, Gli2CA tumors resist anti-PD-1 antibody therapy.Abstract 923 Figure 1Gli2 in tumors promotes Wnt and prostaglandin signaling, generating an immunosuppressive microenvironmentConclusionsOur data demonstrates that the HH transcription factor Gli2 drives the development of a tolerogenic tumor microenvironment unfavorable to anti-PD-1 immunotherapy by coordinating the upregulation of Wnt ligand expression and prostaglandin synthesis (figure 1). We propose that HH gene signatures are worthy of further study as a guide for selecting Wnt ligand and prostaglandin inhibitors in future immunotherapy studies.AcknowledgementsThe authors would like to acknowledge the Duke Cancer Institute Flow Cytometry Core.ReferencesBagaev A, et al. Conserved pan-cancer microenvironment subtypes predict response to immunotherapy. Cancer Cell 2021.Hugo W, et al. Genomic and transcriptomic features of response to anti-PD-1 therapy in metastatic melanoma. Cell 2016;165(1):35–44.DeVito NC, et al. Pharmacological Wnt ligand inhibition overcomes key tumor-mediated resistance pathways to anti-PD-1 immunotherapy. Cell Rep 2021;35(5):109071.Theivanthiran B, et al. A tumor-intrinsic PD-L1/NLRP3 inflammasome signaling pathway drives resistance to anti-PD-1 immunotherapy. J Clin Invest 2020;130(5):2570–2586.

BackgroundConventional dendritic cells (DCs) are essential mediators of anti-tumor immunity and the efficacy of anti-PD-1 checkpoint immunotherapies.1 Recent studies suggest that tumor-mediated development of a sub-population of tolerogenic DCs plays an important role in immune evasion.2 3 Metabolic reprogramming regulates tolerogenic DCs in the tumor microenvironment (TME).4 5 Activation of DCs leads to rewiring of cDC metabolism towards glycolysis to support T cell activation while tolerogenic DCs display enhanced fatty acid oxidation.6 Related to DC metabolic alterations, tumor-associated DCs (TADCs) are enriched in lipids and have a reduced capacity to present antigen to T cells. Lipid homeostasis is maintained through a complex network of transcription factors including sterol regulatory element-binding protein-2 (SREBP2) which drives the expression of mevalonate pathway genes.7 The identification of those tumor-controlled pathways that regulate tolerogenic DCs in the TME is expected to lead to the discovery of a novel family of immunotherapeutic targets.MethodsWe use transgenic mouse models of melanoma, sentinel lymph node (LN) tissue specimens derived from melanoma patients, single-cell RNA sequencing (scRNAseq), and flow cytometry-based metabolic assays to identify novel tumor-associated regulatory programs amongst different sub-populations of conventional DCs in the TME.Results scRNAseq of DCs isolated from the tumor-draining LN (TDLN) of a BRAFV600EPTEN-/- transgenic melanoma model revealed critical genetic differences in distinct DC sub-populations. We observed a migratory DC subset enriched in the expression of numerous immunoregulatory genes and identified CD63 as a surface marker to distinguish this DC subset from other conventional cDC1s and cDC2s. Further studies demonstrated CD63+ DCs to suppress T cell activation and promote CD4+FOXP3+ regulatory T cell (Treg) differentiation. Relative to other cDC subsets, CD63+ DCs overexpress genes of the mevalonate pathway leading to increased lipid content. Treatment of melanoma-bearing mice with a pharmacologic inhibitor of SREBP2 leads to a significant reduction in CD63+ DCs in the TDLN and reduced Tregs, resulting in suppressed tumor growth. Importantly, scRNAseq of DCs isolated from sentinel LNs of melanoma patients reveal that this population is conserved in humans.ConclusionsLipid homeostasis in TADCs is a major determinant of their metabolic state, but despite significant advances, the molecular pathways regulating tolerogenic DCs have remained poorly understood. Collectively, this data demonstrates an important role of the mevalonate pathway in driving a tolerogenic DC program and highlights the therapeutic targeting of SREBP2 and DC lipid metabolism as a promising approach to overcoming immune tolerance in the TME and boosting immunotherapy responses.ReferencesGardner A, Ruffell B. Dendritic cells and cancer immunity. Trends Immunol 2016;37:855–865. doi:10.1016/j.it.2016.09.006DeVito NC, Plebanek MP, Theivanthiran B, Hanks BA. Role of tumor-mediated dendritic cell tolerization in immune evasion. Front Immunol 2019;10:2876. doi:10.3389/fimmu.2019.02876Gerhard GM, Bill R, Messemaker M, Klein AM, Pittet MJ. Tumor-infiltrating dendritic cell states are conserved across solid human cancers. J Exp Med 2021;218. doi:10.1084/jem.20200264Plebanek MP, Sturdivant M, DeVito NC, Hanks BA. Role of dendritic cell metabolic reprogramming in tumor immune evasion. Int Immunol 2020;32:485–491. doi:10.1093/intimm/dxaa036Wculek SK, Khouili SC, Priego E, Heras-Murillo I, Sancho D. Metabolic control of dendritic cell functions: digesting information. Front Immunol 2019;10:775. doi:10.3389/fimmu.2019.00775Zhao F. et al. Paracrine Wnt5a-beta-Catenin signaling triggers a metabolic program that drives dendritic cell tolerization. Immunity 2018;48:147-+, doi:10.1016/j.immuni.2017.12.004Xue L. et al. Targeting SREBP-2-Regulated mevalonate metabolism for cancer therapy. Front Oncol 2020;10:1510, doi:10.3389/fonc.2020.01510Ethics ApprovalCollection of human tissue specimens was approved by the Duke Institutional Review Board under the title Immune Markers of Sentinel Nodes in Melanoma and the protocol number Pro00090678. All patients gave informed consent prior to participating in the study. All experiments involving animals were approved by the Duke University Institutional Animal Care and Use Committee (IACUC) under protocol number A174-18-07

Simple Summary The tumor-intrinsic NLRP3 inflammasome is a newly recognized player in the regulation of tumor-directed immune responses and promises to provide fresh insight into how tumors respond to immunotherapy. This brief review discusses recent data describing how activation of the tumor-intrinsic NLRP3 inflammasome contributes to immune evasion and what this pathway may provide to the field of immuno-oncology both in terms of pharmacologic targets capable of boosting responses to checkpoint inhibitor therapies and predictive biomarkers indicating which tumors may be most susceptible to these new therapeutic strategies. Abstract The tumor-intrinsic NOD-like receptor family, pyrin-domain-containing-3 (NLRP3) inflammasome, plays an important role in regulating immunosuppressive myeloid cell populations in the tumor microenvironment (TME). While prior studies have described the activation of this inflammasome in driving pro-tumorigenic mechanisms, emerging data is now revealing the tumor NLRP3 inflammasome and the downstream release of heat shock protein-70 (HSP70) to regulate anti-tumor immunity and contribute to the development of adaptive resistance to anti-PD-1 immunotherapy. Genetic alterations that influence the activity of the NLRP3 signaling axis are likely to impact T cell-mediated tumor cell killing and may indicate which tumors rely on this pathway for immune escape. These studies suggest that the NLRP3 inflammasome and its secreted product, HSP70, represent promising pharmacologic targets for manipulating innate immune cell populations in the TME while enhancing responses to anti-PD-1 immunotherapy. Additional studies are needed to better understand tumor-specific regulatory mechanisms of NLRP3 to enable the development of tumor-selective pharmacologic strategies capable of augmenting responses to checkpoint inhibitor immunotherapy while minimizing unwanted off-target effects. The execution of upcoming clinical trials investigating this strategy to overcome anti-PD-1 resistance promises to provide novel insight into the role of this pathway in immuno-oncology.

SUMMARY While immune checkpoint blockade is associated with prolonged responses in multiple cancers, most patients still do not benefit from this therapeutic strategy. The Wnt-β-catenin pathway is associated with diminished T cell infiltration; however, activating mutations are rare, implicating a role for autocrine/paracrine Wnt ligand-driven signaling in immune evasion. In this study, we show that proximal mediators of the Wnt signaling pathway are associated with anti-PD-1 resistance, and pharmacologic inhibition of Wnt ligand signaling supports anti-PD-1 efficacy by reversing dendritic cell tolerization and the recruitment of granulocytic myeloid-derived suppressor cells in autochthonous tumor models. We further demonstrate that the inhibition of Wnt signaling promotes the development of a tumor microenvironment that is more conducive to favorable responses to checkpoint blockade in cancer patients. These findings support a rationale for Wnt ligand-focused treatment approaches in future immunotherapy clinical trials and suggest a strategy for selecting those tumors more responsive to Wnt inhibition., Graphical abstract, In brief Anti-PD-1-refractory melanoma exhibits elevated Wnt ligand signaling activity. DeVito et al. demonstrate that pharmacologic inhibition of proximal Wnt ligand signaling sensitizes transgenic models of melanoma and lung cancer to anti-PD-1 checkpoint inhibitor immunotherapy by reversing dendritic cell tolerization and suppressing recruitment of granulocytic myeloid-derived suppressor cells to the tumor bed.

The dendritic cell (DC) is recognized as a vital mediator of anti-tumor immunity. More recent studies have also demonstrated the important role of DCs in the generation of effective responses to checkpoint inhibitor immunotherapy. Metabolic programming of DCs dictates their functionality and can determine which DCs become immunostimulatory versus those that develop a tolerized phenotype capable of actively suppressing effector T-cell responses to cancers. As a result, there is great interest in understanding what mechanisms have evolved in cancers to alter these metabolic pathways, thereby allowing for their continued progression and metastasis. The therapeutic strategies developed to reverse these processes of DC tolerization in the tumor microenvironment represent promising candidates for future testing in combination immunotherapy clinical trials.

BackgroundOur understanding of those underlying mechanisms that contribute to metastatic progression in melanoma remains limited. While uncommon, melanoma hyperprogression in response to immunotherapy is likely to be an extreme form of acquired resistance. Therefore, studies that define the underlying mechanisms of these processes are expected to provide insight into the discovery of novel therapeutic targets and predictive biomarkers. We previously demonstrated that tumor-intrinsic NLRP3 drives adaptive resistance to anti-PD-1 immunotherapy (anti-PD-1) by inducing the recruitment of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) via the upregulation of CXCL5. Gain-of-function polymorphisms in the TLR4 gene have been associated with pulmonary metastases in melanoma patients. We have shown HSP70 to promote PMN-MDSC chemotaxis via the stimulation of TLR4 signaling. As a result, we hypothesized that the tumor NLRP3 inflammasome may also contribute to distant metastatic progression and disease hyperprogression during immunotherapy by establishing a long-distance signaling axis mediated by HSP70-TLR4 signaling in the lung.MethodsWe pharmacologically and genetically inhibited the NLRP3 inflammasome and HSP70 in a transgenic BRAFV600E mouse model to examine their role in distant metastatic progression before and during anti-PD-1. An inducible type II pulmonary epithelial cell-specific TLR4 knock-out mouse model was engineered to examine the role of distant HSP70-TLR4 signaling in the recruitment of PMN-MDSCs and subsequent metastatic progression to the lung. Plasma HSP70 levels were monitored in melanoma patients undergoing anti-PD-1ResultsAnti-PD-1 significantly increases CXCL2/CXCL5 expression and PMN-MDSC accumulation in the lungs of the transgenic BRAFV600E model. This effect is reversed by 1) tumor-targeted ablation and pharmacologic inhibition of NLRP3 but not systemic host knock-out of NLRP3, 2) Pharmacologic Wnt5a ligand inhibition, 3) tumor-specific ablation and inhibition of HSP70. Inducible knock-out of TLR4 in type II epithelial cells suppressed Wnt5a and CXCL5 expression and inhibited the recruitment of PMN-MDSCs to the lung in response to anti-PD-1. Tumor-specific inhibition of NLRP3/HSP70 and lung-specific ablation of TLR4 suppressed metastatic progression following anti-PD-1 in the BRAFV600E melanoma model. Combination anti-PD-1 and NLRP3 inhibition suppressed primary melanoma progression and distant melanoma metastases versus anti-PD-1 monotherapy. Elevated plasma levels of HSP70 were associated with disease hyperprogression in metastatic melanoma patients undergoing anti-PD-1Abstract 319 Figure 1Tumor-intrinsic NLRP3 inflammasome and metastasisConclusionsTogether, these results describe a novel cross-talk mechanism between the primary tumor and the lung that mediates distant metastatic progression that is accentuated following anti-PD-1 (figure 1). Future clinical studies are needed to evaluate the pharmacologic inhibition of this tumor-lung NLRP3/HSP70/TLR4/Wnt5a/CXCL5 axis on melanoma metastasis and disease hyperprogression during checkpoint inhibitor immunotherapy

The vast majority of cancer-related deaths are due to metastasis, a process that requires evasion of the host immune system. In addition, a significant percentage of cancer patients do not benefit from our current immunotherapy arsenal due to either primary or secondary immunotherapy resistance. Importantly, select subsets of dendritic cells (DCs) have been shown to be indispensable for generating responses to checkpoint inhibitor immunotherapy. These observations are consistent with the critical role of DCs in antigen cross-presentation and the generation of effective anti-tumor immunity. Therefore, the evolution of efficient tumor-extrinsic mechanisms to modulate DCs is expected to be a potent strategy to escape immunosurveillance and various immunotherapy strategies. Despite this critical role, little is known regarding the methods by which cancers subvert DC function. Herein, we focus on those select mechanisms utilized by developing cancers to co-opt and tolerize local DC populations. We discuss the reported mechanisms utilized by cancers to induce DC tolerization in the tumor microenvironment, describing various parallels between the evolution of these mechanisms and the process of mesenchymal transformation involved in tumorigenesis and metastasis, and we highlight strategies to reverse these mechanisms in order to enhance the efficacy of the currently available checkpoint inhibitor immunotherapies.

An in-depth understanding of immune escape mechanisms in cancer is likely to lead to innovative advances in immunotherapeutic strategies. However, much remains unknown regarding these mechanisms and how they impact immunotherapy resistance. Using several preclinical tumor models as well as clinical specimens, we identified a mechanism whereby CD8(+) T cell activation in response to programmed cell death 1 (PD-1) blockade induced a programmed death ligand 1/NOD-, LRR-, and pyrin domain–containing protein 3 (PD-L1/NLRP3) inflammasome signaling cascade that ultimately led to the recruitment of granulocytic myeloid-derived suppressor cells (PMN-MDSCs) into tumor tissues, thereby dampening the resulting antitumor immune response. The genetic and pharmacologic inhibition of NLRP3 suppressed PMN-MDSC tumor infiltration and significantly augmented the efficacy of anti–PD-1 antibody immunotherapy. This pathway therefore represents a tumor-intrinsic mechanism of adaptive resistance to anti–PD-1 checkpoint inhibitor immunotherapy and is a promising target for future translational research.