Albert G. Zimmermann

NLRX1 is a regulator of mitochondrial antiviral immunity

The RIG-like helicase (RLH) family of intracellular receptors detect viral nucleic acid and signal through the mitochondrial antiviral signalling adaptor MAVS (also known as Cardif, VISA and IPS-1) during a viral infection. MAVS activation leads to the rapid production of antiviral cytokines, including type 1 interferons. Although MAVS is vital to antiviral immunity, its regulation from within the mitochondria remains unknown. Here we describe human NLRX1, a highly conserved nucleotide-binding domain (NBD)- and leucine-rich-repeat (LRR)-containing family member (known as NLR) that localizes to the mitochondrial outer membrane and interacts with MAVS. Expression of NLRX1 results in the potent inhibition of RLH- and MAVS-mediated interferon-β promoter activity and in the disruption of virus-induced RLH–MAVS interactions. Depletion of NLRX1 with small interference RNA promotes virus-induced type I interferon production and decreases viral replication. This work identifies NLRX1 as a check against mitochondrial antiviral responses and represents an intersection of three ancient cellular processes: NLR signalling, intracellular virus detection and the use of mitochondria as a platform for anti-pathogen signalling. This represents a conceptual advance, in that NLRX1 is a modulator of pathogen-associated molecular pattern receptors rather than a receptor, and identifies a key therapeutic target for enhancing antiviral responses.

Cryopyrin/NALP3 binds ATP/dATP, is an ATPase, and requires ATP binding to mediate inflammatory signaling

The CATERPILLER (CLR/NLR) gene family encodes a family of putative nucleotide-binding proteins important for host defense. Although nucleotide binding is thought to be central to this family, this aspect is largely unstudied. The CATERPILLER protein cryopyrin/NALP3 regulates IL-1β processing by assembling the multimeric inflammasome complex. Mutations within the exon encoding the nucleotide-binding domain are associated with hereditary periodic fevers characterized by constitutive IL-1β production. We demonstrate that purified cryopyrin binds ATP, dATP, and ATP-agarose, but not CTP, GTP, or UTP, and exhibits ATPase activity. Mutation of the nucleotide-binding domain reduces ATP binding, caspase-1 activation, IL-1β production, cell death, macromolecular complex formation, self-association, and association with the inflammasome component ASC. Disruption of nucleotide binding abolishes the constitutive activation of disease-associated mutants, identifying nucleotide binding by cryopyrin as a potential target for antiinflammatory pharmacologic intervention.

Tissue-specific Regulation of the Ecto-5′-nucleotidase Promoter ROLE OF THE cAMP RESPONSE ELEMENT SITE IN MEDIATING REPRESSION BY THE UPSTREAM REGULATORY REGION

We have isolated the 5′ region of the ecto-5′-nucleotidase (low K m 5′-NT) gene and established that a 969-base pair (bp) fragment confers cell-specific expression of a CAT reporter gene that correlates with the expression of endogenous ecto-5′-NT mRNA and enzymatic activity. A 768-bp upstream negative regulatory region has been identified that conferred lymphocyte-specific negative regulation in a heterologous system with a 244-bp deoxycytidine kinase core promoter. DNase I footprinting identified several protected areas including Sp1, Sp1/AP-2, and cAMP response element (CRE) binding sites within the 201-bp core promoter region and Sp1, NRE-2a, TCF-1/LEF-1, and Sp1/NF-AT binding sites in the upstream regulatory region. Whereas the CRE site was essential in mediating the negative activity of the upstream regulatory region in Jurkat but not in HeLa cells, mutation of the Sp1/AP-2 site decreased promoter activity in both cell lines. Electrophoretic mobility shift assay analysis of proteins binding to the CRE site identified both ATF-1 and ATF-2 in Jurkat cells. Finally, phorbol 12-myristate 13-acetate increased the activity of both the core and the 969-bp promoter fragments, and this increase was abrogated by mutations at the CRE site. In summary, we have identified a tissue-specific regulatory region 5′ of the ecto-5′-NT core promoter that requires the presence of a functional CRE site within the basal promoter for its suppressive activity.

Regulation of Inosine-5′-monophosphate Dehydrogenase Type II Gene Expression in Human T Cells ROLE FOR A NOVEL 5′ PALINDROMIC OCTAMER SEQUENCE

Expression of the gene encoding human inosine- 5′-monophosphate dehydrogenase (IMPDH) type II, an enzyme catalyzing the rate-limiting step in the generation of guanine nucleotides, is increased more than 10-fold in activated peripheral blood T lymphocytes and is required for T cell activation. We have examined the 5′-regulatory sequences that are important for the transcriptional regulation of this gene in T cells. DNase I mapping of genomic DNA identified a hypersensitive element near the transcription initiation site. Fine mapping by in vivo footprinting demonstrated five transcription factor binding sites that are occupied in both resting and activated peripheral blood T lymphocytes; these are tandem CRE motifs, a Sp1 site, an overlapping Egr-1/Sp1 site, and a novel palindromic octamer sequence (POS). The tandem CRE and POS sites are of major functional importance as judged by mutational and electrophoretic mobility shift analyses. These data provide evidence that expression of the human IMPDH type II gene is predominantly regulated by the nuclear factors ATF-2 and an as yet unidentified POS-binding protein. Additional major protein-DNA interactions do not occur within the promoter region after T lymphocyte activation, indicating a requirement for additional protein-protein interactions and/or post-translational modifications of pre-bound transcription factors to account for the observed increase in IMPDH type II gene expression.

Plexin-D1 Is a Novel Regulator of Germinal Centers and Humoral Immune Responses

Long-lived humoral immune responses depend upon the generation of memory B cells and long-lived plasma cells during the germinal center (GC) reaction. These memory compartments, characterized by class-switched IgG and high-affinity Abs, are the basis for successful vaccination. We report that a new member of the plexin family of molecules, plexin-D1, controls the GC reaction and is required for secondary humoral immune responses. Plexin-D1 was not required for B cell maturation, marginal zone precursor development, dark and light zone formation, Igλ+ and Igκ+ B cell skewing, B1/B2 development, and the initial extrafollicular response. Plexin-D1 expression was increased following B cell activation, and PlxnD1−/− mice exhibited defective GC reactions during T-dependent immune activation. PlxnD1−/− B cells showed a defect in migration toward the GC chemokines, CXCL12, CXCL13, and CCL19. Accordingly, PlxnD1−/− mice exhibited defective production of IgG1 and IgG2b, but not IgG3 serum Ab, accompanied by reductions in long-lived bone marrow plasmacytes and recall humoral memory responses. These data show a new role for immune plexins in the GC reaction and generation of immunologic memory.

Abstract 947: A small molecule Mer tyrosine kinase inhibitor (UNC MerTKi) effectively inhibits growth of murine melanoma

The Mer Receptor Tyrosine Kinase (RTK) is overexpressed in hematologic and epithelial malignancies. Mer is not a proliferative driver but rather produces a cancer cell-intrinsic survival signal. In addition, tumor-associated macrophages (TAMs) express Mer, which upon binding ligand-associated with apoptotic material, triggers engulfment (effercytosis). The Mer signal also polarizes macrophages towards an M2-like, wound healing, anti-inflammatory state, calming the innate immune response when ingesting “self”. We have previously shown that syngeneic polyoma middle T (PyVmT) breast cancer cells demonstrate markedly attenuated growth when orthotopically implanted into Mer knock-out mice, with Mer -/- TAMs of PyVmT tumors producing M1-like inflammatory cytokines (JCI 123:3231, 2013). This result suggests that Mer inhibition could enhance innate anti-tumor responses, and toward that end, we examine the activity of first-in-class, orally bioavailable MerTK inhibitor on tumor growth in autochthonous murine tumor models. UNC MerTKi is 5-fold selective for Mer vs. Axl/Tyro3 and has favorable pharmacokinetics. Once daily, oral dosing inhibits the growth of Mer-expressing leukemia and NSCLC xenografts. UNC MerTKi was assessed in immune-competent, genetically engineered murine models (GEMMs) in the UNC Lineberger Mouse Phase 1 Unit (MP1U). After dose-finding studies in wild-type mice established an MTD, the inhibitor was given at 120 mpk/d in mouse chow, a dose which did not cause weight loss and produced a measurable effect (i.e. inhibition of second phase platelet aggregation, a known Mer pharmacodynamics marker). This dose did not exhibit single agent activity in a murine model of breast cancer (C3TAg), but exhibited pronounced single agent activity in RAS-driven, INK4a/Arf null melanoma GEMM (TRIA). The MP1U has previously reported the efficacy of 15 chemotherapeutic and/or targeted regimens in a large (>220) cohort of TRIA mice (CCR 18:5290, 2012). The overall response was 10% (partial responses and stable disease). There were no complete responses. A combination of MEK (AZD 6244) and PI3K/mTOR (BEZ235) inhibitors were the most active previous regimen (responses seen in 9/18 mice = 50%, with 0 CRs) with moderate toxicity. UNC MerTKi exhibited greater activity (6/8 mice = 75%, with 3 CRs) with mild, well-tolerated toxicity in the TRIA model. TRIA cell lines do not express Mer, suggesting that UNC MerTKi as a monotherapy may induce responses via Mer inhibition in TAMs and the tumor microenvironment, or via inhibition of Axl, Tyro or an unknown target. In summary, a potent and selective Mer inhibitor exhibited greater pre-clinical efficacy in a highly faithful model of RAS-mutant melanoma than any other drug tested to date, including several compounds that are FDA approved for use in metastatic melanoma. Citation Format: H Shelton Earp, David Darr, Albert Zimmermann, Kelly Clark, Norman E. Sharpless, Wolfgang Bergmeier, Weihe Zhang, Xiaodong Wang, Deborah DeRyckere, Stephen Frye, Douglas Graham. A small molecule Mer tyrosine kinase inhibitor (UNC MerTKi) effectively inhibits growth of murine melanoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 947. doi:10.1158/1538-7445.AM2014-947

Conventional Murine Gene Targeting

Murine gene knockout models engineered over the last two decades have continued to demonstrate their potential as invaluable tools in understanding the role of gene function in the context of normal human development and disease. The more recent elucidation of the human and mouse genomes through sequencing has opened up the capability to elucidate the function of every human gene. State-of-the-art mouse model generation allows, through a multitude of experimental steps requiring careful standardization, gene function to be reliably and predictably ablated in a live model system. The application of these standardized methodologies to directly target gene function through murine gene knockout has to date provided comprehensive and verifiable genetic models that have contributed tremendously to our understanding of the cellular and molecular pathways underlying normal and disease states in humans. The ensuing chapter provides an overview of the latest steps and procedures required to ablate gene function in a murine model.