A family of pH-responsive diblock polymers composed of poly[(ethylene glycol)–b-[(2-(dimethylamino)ethyl methacrylate)-co-(butyl methacrylate)] PEG-(DMAEMA-co-BMA) was reversible addition fragmentation chain transfer (RAFT) synthesized with 0-75 mole% BMA in the second polymer block. The relative mole% of DMAEMA and BMA was varied in order to identify a polymer that can be used to formulate PEGylated, siRNA-loaded polyplex nanoparticles (NPs) with an optimized balance of cationic and hydrophobic content in the NP core based on siRNA packaging, cytocompatibility, blood circulation half-life, endosomal escape, and in vivo bioactivity. The polymer with 50:50 mole% of DMAEMA:BMA (polymer “50B”) in the RAFT-polymerized block efficiently condensed siRNA into 100-nm NPs that displayed pH-dependent membrane disruptive behavior finely tuned for endosomal escape. In vitro delivery of siRNA with polymer 50B produced up to 94% protein-level knockdown of the model gene luciferase. The PEG corona of the NPs blocked nonspecific interactions with constituents of human whole blood, and the relative hydrophobicity of polymer 50B increased NP stability in the presence of human serum or the polyanion heparin. When injected intravenously, 50B NPs enhanced blood circulation half-life 3-fold relative to more standard PEG-DMAEMA (0B) NPs (p<0.05), due to improved stability and a reduced rate of renal clearance. The 50B NPs enhanced siRNA biodistribution to the liver and other organs and significantly increased gene silencing in the liver, kidneys, and spleen relative to the benchmark polymer 0B (p<0.05). These collective findings validate the functional significance of tuning the balance of cationic and hydrophobic content of polyplex NPs utilized for systemic siRNA delivery in vivo.

Immune-related adverse events, particularly severe toxicities such as myocarditis, are major challenges to immune checkpoint inhibitor (ICI) utility in anti-cancer therapy1. The pathogenesis of ICI-myocarditis is poorly understood. Pdcd1-/-Ctla4+/-mice recapitulate clinicopathologic features of ICI-myocarditis, including myocardial T cell in ltration2. Single cell RNA/T cell receptor (TCR) sequencing on the cardiac immune in ltrate of Pdcd1-/-Ctla4+/-mice identi ed activated, clonal CD8+ T cells as the dominant cell population. Treatment with anti-CD8, but not anti-CD4, depleting antibodies rescued survival of Pdcd1-/-Ctla4+/-mice. Adoptive transfer of immune cells from mice with myocarditis induced fatal myocarditis in recipients which required CD8+ T cells. Alpha-myosin, a cardiac speci c protein absent from the thymus3,4, was identi ed as the cognate antigen source for three MHC-I restricted TCRs derived from mice with fulminant myocarditis. Peripheral blood T cells from two patients with ICI-myocarditis were expanded by alpha-myosin peptides, and these alpha-myosin expanded T cells shared TCR clonotypes with diseased heart and skeletal muscles, indicating that alpha-myosin may be a clinically important autoantigen in ICI-myocarditis. These studies underscore the critical role for cytotoxic CD8+ T cells, are the rst to identify a candidate autoantigen in ICI-myocarditis and yield new insights into ICI toxicity pathogenesis.Grant 5P30 CA68485-19 and the Shared Instrumentation Grant S10 OD023475-01A1 for the Leica Bond RX. The Vanderbilt VANTAGE Core, including A. Jones and L. Raju, provided technical assistance for this work. VANTAGE is supported in part by a CTSA Grant (5UL1 RR024975-03), the Vanderbilt Ingram Cancer Center (P30 CA68485), the Vanderbilt Vision Center (P30 EY08126) and the NIH/NCRR (G20 RR030956). Figures 1a and 4b were created with BioRender.com.Con ict Interest Disclosure M.L. Axelrod is listed as a coinventor on a provisional patent application for methods to predict therapeutic outcomes using blood-based gene expression patterns, that is owned by Vanderbilt University Medical Center, and is currently unlicensed. S.C. Wei is an employee of Spotlight Therapeutics, a consultant for BioEntre, and an inventor on a patent for a genetic mouse model of autoimmune adverse events and immune checkpoint blockade therapy (PCT/US2019/050551) pending to Board of Regents, The University of Texas System. J.C. Rathmell is a founder, scienti c advisory board member, and stockholder of Sitryx Therapeutics, a scienti c advisory board member and stockholder of Caribou Biosciences, a member of the scienti c advisory board of Nirogy Therapeutics, has consulted for Merck, P zer, and Mitobridge within the past three years, and has received research support from Incyte Corp., Calithera Biosciences, and Tempest Therapeutics. P.B. Ferrell receives research support from Incyte Corporation. D.B.Johnson has served on advisory boards or as a consultant for BMS, Catalyst

Cancer poses a serious health problem in society and is increasingly surpassing cardiovascular disease as the leading cause of mortality in the United States. Current therapeutic strategies for cancer are extreme and harsh to patients and often have limited success; the danger of cancer is intensified as it metastasizes to secondary locations such as lung, bone, and liver, posing a dire threat to patient treatment and survival. Hedgehog signaling is an important pathway for normal development. Initially identified in Drosophila, the vertebrate and mammalian equivalent of the pathway has been studied extensively for its role in cancer development and progression. As this pathway regulates key target genes involved in development, its action also allows for the modulation of the microenvironment to prepare a tumor-suitable niche by manipulating tumor cell growth, differentiation, and immune regulation, thus creating an enabling environment for progression and metastasis. In this review, we will summarize recent scientific discoveries reporting the impact of the Hedgehog signaling pathway on the tumor initiation process and metastatic cascade, shedding light on the ability of the tumor to take over a mechanism crucially intended for development and normal function.

Host responses to tumor cells include tumor suppressing or promoting mechanisms. We sought to detail the effect of Hedgehog (Hh) pathway inhibition on the composition of the mammary tumor immune portfolio. We hypothesized that Hh signaling mediates a crosstalk between breast cancer cells and macrophages that dictates alternative polarization of macrophages and consequently supports a tumor-promoting microenvironment. We used an immunocompetent, syngeneic mouse mammary cancer model to inhibit Hh signaling with the pharmacological inhibitor, Vismodegib. Using molecular and functional assays, we identified that Hedgehog (Hh) signaling mediates a molecular crosstalk between mammary cancer cells and macrophages that culminates in alternative polarization of macrophages. We carried out an unbiased kinomics and genomics assessment to unravel changes in global kinomic and gene signatures impacted by Hh signaling. Our investigations reveal that in an immunocompetent mammary cancer model, the administration of Vismodegib led to changes in the portfolio of tumor-infiltrating immune cells. This was characterized by a marked reduction in immune-suppressive innate and adaptive cells concomitant with an enrichment of cytotoxic immune cells. Breast cancer cells induce M2 polarization of macrophages via a crosstalk mediated by Hh ligands that alters critical kinomic and genomic signatures. Macrophage depletion improved the benefit of Hedgehog inhibition on eliciting an immunogenic, pro-inflammatory profile. We define a novel role for Hh signaling in disabling anti-tumor immunity. Inhibition of Hh signaling presents with dual advantages of tumor cell-targeting as well as re-educating a dysfunctional tumor microenvironment.

Elevated infiltration of immunosuppressive alternatively polarized (M2) macrophages is associated with poor prognosis in patients with cancer. The tumor microenvironment remarkably orchestrates molecular mechanisms that program these macrophages. Here we identify a novel role for oncogenic Hedgehog (Hh) signaling in programming signature metabolic circuitries that regulate alternative polarization of tumor-associated macrophages. Two immunocompetent orthotopic mouse models of mammary tumors were used to test the effect of inhibiting Hh signaling on tumor-associated macrophages. Treatment with the pharmacologic Hh inhibitor vismodegib induced a significant shift in the profile of tumor-infiltrating macrophages. Mass spectrometry-based metabolomic analysis showed Hh inhibition induced significant alterations in metabolic processes, including metabolic sensing, mitochondrial adaptations, and lipid metabolism. In particular, inhibition of Hh in M2 macrophages reduced flux through the UDP-GlcNAc biosynthesis pathway. Consequently, O-GlcNAc-modification of STAT6 decreased, mitigating the immune-suppressive program of M2 macrophages, and the metabolically demanding M2 macrophages shifted their metabolism and bioenergetics from fatty acid oxidation to glycolysis. M2 macrophages enriched from vismodegib-treated mammary tumors showed characteristically decreased O-GlcNAcylation and altered mitochondrial dynamics. These Hh-inhibited macrophages are reminiscent of inflammatory (M1) macrophages, phenotypically characterized by fragmented mitochondria. This is the first report highlighting the relevance of Hh signaling in controlling a complex metabolic network in immune cells. These data describe a novel immunometabolic function of Hh signaling that can be clinically exploited. Significance: These findings illustrate that Hh activity regulates a metabolic and bioenergetic regulatory program in tumor-associated macrophages that promotes their immune-suppressive polarization.