Nicholas DeVito

The tumor-intrinsic NOD-, LRR- and pyrin domain-containing protein-3 (NLRP3) inflammasome-heat shock protein 70 (HSP70) signaling axis is triggered by CD8+ T cell cytotoxicity and contributes to the development of adaptive resistance to anti-programmed cell death protein 1 (PD-1) immunotherapy by recruiting granulocytic polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) into the tumor microenvironment. Here, we demonstrate that the tumor NLRP3-HSP70 axis also drives the accumulation of PMN-MDSCs into distant lung tissues in a manner that depends on lung epithelial cell Toll-like receptor 4 (TLR4) signaling, establishing a premetastatic niche that supports disease hyperprogression in response to anti-PD-1 immunotherapy. Lung epithelial HSP70-TLR4 signaling induces the downstream Wnt5a-dependent release of granulocyte colony-stimulating factor (G-CSF) and C-X-C motif chemokine ligand 5 (CXCL5), thus promoting myeloid granulopoiesis and recruitment of PMN-MDSCs into pulmonary tissues. Treatment with anti-PD-1 immunotherapy enhanced the activation of this pathway through immunologic pressure and drove disease progression in the setting of Nlrp3 amplification. Genetic and pharmacologic inhibition of NLRP3 and HSP70 blocked PMN-MDSC accumulation in the lung in response to anti-PD-1 therapy and suppressed metastatic progression in preclinical models of melanoma and breast cancer. Elevated baseline concentrations of plasma HSP70 and evidence of NLRP3 signaling activity in tumor tissue specimens correlated with the development of disease hyperprogression and inferior survival in patients with stage IV melanoma undergoing anti-PD-1 immunotherapy. Together, this work describes a pathogenic mechanism underlying the phenomenon of disease hyperprogression in melanoma and offers candidate targets and markers capable of improving the management of patients with melanoma.

The mechanisms underlying tumor immunosurveillance and their association with the immune-related adverse events (irAEs) associated with checkpoint inhibitor immunotherapies remain poorly understood. We describe a metastatic melanoma patient exhibiting multiple episodes of spontaneous disease regression followed by the development of several irAEs during the course of anti-programmed cell death protein 1 antibody immunotherapy. Whole-exome next-generation sequencing studies revealed this patient to harbor a pyrin inflammasome variant previously described to be associated with an atypical presentation of familial Mediterranean fever. This work highlights a potential role for inflammasomes in the regulation of tumor immunosurveillance and the pathogenesis of irAEs.

AIMS: Use of comprehensive genomic profiling (CGP) in metastatic colorectal cancer (mCRC) is limited. We estimated impacts of expanded 1 L CGP, using the Tempus xT test, on detection of actionable alterations and testing budgets in a modeled US health plan over two-years. MATERIALS AND METHODS: A decision analytic model was developed to estimate the impact of replacing 20% of usual testing (a mix of CGP and non-CGP) with Tempus xT CGP. Actionable alterations for matched treatments or clinical trial included KRAS, NRAS, RAF, BRAF, deficient mismatch repair (dMMR)/microsatellite instability (MSI), NTRK, RET, EGFR, HER2, MET, PIK3CA and POLE1. Costs included initial and repeat testing, physician-associated and administrative costs. RESULTS: In a hypothetical five-million-member plan, 50% Medicare and 50% commercial, 1,112 new cases of mCRC were expected per year. Of these, 566 (51%) would undergo 1 L molecular testing, with 55 re-tested upon progression. Based on current testing rates, there were an expected 521 missed opportunities for genomically informed treatment (47% of new cases), with 442 missed due to lack of testing and 79 due to testing without CGP. Replacing 20% of usual testing with Tempus xT CGP was associated with up to a $0.003 per member per month testing cost increase (net total cost of $202,102 for the five-million-member plan) and 15.5 additional patients with an opportunity for genomically informed care (12.7 patients for treatment and 2.8 for clinical trial). The testing total cost (initial test, repeat test, biopsy and physician services, and administrative cost) to put one additional patient with mCRC on matched therapy or matched clinical trial was estimated to be $13,005. Number needed to test to identify one actionable alteration with Tempus xT CGP versus usual testing was 7.8 patients. LIMITATIONS: Conservative assumptions were made for inputs with limited evidence. Based on high concordance rates with dMMR/MSI status, tumor mutational burden (TMB) status was not calculated separately. CONCLUSIONS: Replacing 20% of usual testing with Tempus xT CGP was associated with a small incremental testing cost and can identify meaningfully more actionable alterations.

<jats:sec><jats:title>Background</jats:title><jats:p>Conventional dendritic cells (DCs) are essential mediators of anti-tumor immunity and the efficacy of anti-PD-1 checkpoint immunotherapies.<jats:sup>1</jats:sup> Recent studies suggest that tumor-mediated development of a sub-population of tolerogenic DCs plays an important role in immune evasion.<jats:sup>2 3</jats:sup> Metabolic reprogramming regulates tolerogenic DCs in the tumor microenvironment (TME).<jats:sup>4 5</jats:sup> Activation of DCs leads to rewiring of cDC metabolism towards glycolysis to support T cell activation while tolerogenic DCs display enhanced fatty acid oxidation.<jats:sup>6</jats:sup> 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.<jats:sup>7</jats:sup> 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.</jats:p></jats:sec><jats:sec><jats:title>Methods</jats:title><jats:p>We 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.</jats:p><jats:p>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.</jats:p></jats:sec><jats:sec><jats:title>Conclusions</jats:title><jats:p>Lipid 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.</jats:p></jats:sec><jats:sec><jats:title>References</jats:title><jats:list list-type="order"><jats:list-item><jats:p>Gardner A, Ruffell B. Dendritic cells and cancer immunity. <jats:italic>Trends Immunol</jats:italic> 2016;<jats:bold>37</jats:bold>:855–865. doi:10.1016/j.it.2016.09.006</jats:p></jats:list-item><jats:list-item><jats:p>DeVito NC, Plebanek MP, Theivanthiran B, Hanks BA. Role of tumor-mediated dendritic cell tolerization in immune evasion. <jats:italic>Front Immunol</jats:italic> 2019;<jats:bold>10</jats:bold>:2876. doi:10.3389/fimmu.2019.02876</jats:p></jats:list-item><jats:list-item><jats:p>Gerhard GM, Bill R, Messemaker M, Klein AM, Pittet MJ. Tumor-infiltrating dendritic cell states are conserved across solid human cancers. <jats:italic>J Exp Med</jats:italic> 2021;218. doi:10.1084/jem.20200264</jats:p></jats:list-item><jats:list-item><jats:p>Plebanek MP, Sturdivant M, DeVito NC, Hanks BA. Role of dendritic cell metabolic reprogramming in tumor immune evasion. <jats:italic>Int Immunol</jats:italic> 2020;<jats:bold>32</jats:bold>:485–491. doi:10.1093/intimm/dxaa036</jats:p></jats:list-item><jats:list-item><jats:p>Wculek SK, Khouili SC, Priego E, Heras-Murillo I, Sancho D. Metabolic control of dendritic cell functions: digesting information. <jats:italic>Front Immunol</jats:italic> 2019;<jats:bold>10</jats:bold>:775. doi:10.3389/fimmu.2019.00775</jats:p></jats:list-item><jats:list-item><jats:p>Zhao F. et al. Paracrine Wnt5a-beta-Catenin signaling triggers a metabolic program that drives dendritic cell tolerization. <jats:italic>Immunity</jats:italic> 2018;<jats:bold>48</jats:bold>:147-+, doi:10.1016/j.immuni.2017.12.004</jats:p></jats:list-item><jats:list-item><jats:p>Xue L. et al. Targeting SREBP-2-Regulated mevalonate metabolism for cancer therapy. <jats:italic>Front Oncol</jats:italic> 2020;<jats:bold>10</jats:bold>:1510, doi:10.3389/fonc.2020.01510</jats:p></jats:list-item></jats:list></jats:sec><jats:sec><jats:title>Ethics Approval</jats:title><jats:p>Collection 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</jats:p></jats:sec>