Erik G. Huntzicker

Differential effects of targeting Notch receptors in a mouse model of liver cancer.

Primary liver cancer encompasses both hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA). The Notch signaling pathway, known to be important for the proper development of liver architecture, is also a potential driver of primary liver cancer. However, with four known Notch receptors and several Notch ligands, it is not clear which Notch pathway members play the predominant role in liver cancer. To address this question, we utilized antibodies to specifically target Notch1, Notch2, Notch3, or jagged1 (Jag1) in a mouse model of primary liver cancer driven by v‐akt murine thymoma viral oncogene homolog and neuroblastoma RAS viral oncogene homolog (NRas). We show that inhibition of Notch2 reduces tumor burden by eliminating highly malignant HCC‐ and CCA‐like tumors. Inhibition of the Notch ligand, Jag1, had a similar effect, consistent with Jag1 acting in cooperation with Notch2. This effect was specific to Notch2, because Notch3 inhibition did not decrease tumor burden. Unexpectedly, Notch1 inhibition altered the relative proportion of tumor types, reducing HCC‐like tumors but dramatically increasing CC‐like tumors. Finally, we show that Notch2 and Jag1 are expressed in, and Notch2 signaling is activated in, a subset of human HCC samples. Conclusions: These findings underscore the distinct roles of different Notch receptors in the liver and suggest that inhibition of Notch2 signaling represents a novel therapeutic option in the treatment of liver cancer. (Hepatology 2015;61:942–952)

Controlling Hair Follicle Signaling Pathways through Polyubiquitination

Hair follicle development and maintenance require precise reciprocal signaling interactions between the epithelium and underlying dermis. Three major developmental signaling pathways, Wnt, Sonic hedgehog, and NF-kappaB/Edar, are indispensable for this process and, when aberrantly activated, can lead to skin and appendage neoplasms. Recent data point to protein polyubiquitination as playing a central role in regulating the timing, duration, and location of signaling. Here we review how polyubiquitination regulates the stability and interaction of key signaling components that control hair follicle development and regeneration.

Anti-HBV response to toll-like receptor 7 agonist GS-9620 is associated with intrahepatic aggregates of T cells and B cells

BACKGROUND & AIMS GS-9620, an oral agonist of toll-like receptor 7, is in clinical development for the treatment of chronic hepatitis B (CHB). GS-9620 was previously shown to induce prolonged suppression of serum viral DNA and antigens in the chimpanzee and woodchuck models of CHB. Herein, we investigated the immunomodulatory mechanisms underlying these antiviral effects. METHODS Archived liver biopsies and paired peripheral blood mononuclear cell samples from a previous chimpanzee study were analyzed by RNA sequencing, quantitative reverse transcription PCR, immunohistochemistry (IHC) and in situ hybridization (ISH). RESULTS GS-9620 treatment of CHB chimpanzees induced an intrahepatic transcriptional profile significantly enriched with genes associated with hepatitis B virus (HBV) clearance in acutely infected chimpanzees. Type I and II interferon, CD8+ T cell and B cell transcriptional signatures were associated with treatment response, together with evidence of hepatocyte death and liver regeneration. IHC and ISH confirmed an increase in intrahepatic CD8+ T cell and B cell numbers during treatment, and revealed that GS-9620 transiently induced aggregates predominantly comprised of CD8+ T cells and B cells in portal regions. There were no follicular dendritic cells or IgG-positive cells in these lymphoid aggregates and very few CD11b+ myeloid cells. There was no change in intrahepatic natural killer cell number during GS-9620 treatment. CONCLUSION The antiviral response to GS-9620 treatment in CHB chimpanzees was associated with an intrahepatic interferon response and formation of lymphoid aggregates in the liver. Our data indicate these intrahepatic structures are not fully differentiated follicles containing germinal center reactions. However, the temporal correlation between development of these T and B cell aggregates and the antiviral response to treatment suggests they play a role in promoting an effective immune response against HBV. LAY SUMMARY New therapies to treat chronic hepatitis B (CHB) are urgently needed. In this study we performed a retrospective analysis of liver and blood samples from a chimpanzee model of CHB to help understand how GS-9620, a drug in clinical trials, suppressed hepatitis B virus (HBV). We found that the antiviral response to GS-9620 was associated with accumulation of immune cells in the liver that can either kill cells infected with HBV or can produce antibodies that may prevent HBV from infecting new liver cells. These findings have important implications for how GS-9620 may be used in patients and may also help guide the development of new therapies to treat chronic HBV infection.

ASK1 contributes to fibrosis and dysfunction in models of kidney disease.

Oxidative stress is an underlying component of acute and chronic kidney disease. Apoptosis signal–regulating kinase 1 (ASK1) is a widely expressed redox-sensitive serine threonine kinase that activates p38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase kinases, and induces apoptotic, inflammatory, and fibrotic signaling in settings of oxidative stress. We describe the discovery and characterization of a potent and selective small-molecule inhibitor of ASK1, GS-444217, and demonstrate the therapeutic potential of ASK1 inhibition to reduce kidney injury and fibrosis. Activation of the ASK1 pathway in glomerular and tubular compartments was confirmed in renal biopsies from patients with diabetic kidney disease (DKD) and was decreased by GS-444217 in several rodent models of kidney injury and fibrosis that collectively represented the hallmarks of DKD pathology. Treatment with GS-444217 reduced progressive inflammation and fibrosis in the kidney and halted glomerular filtration rate decline. Combination of GS-444217 with enalapril, an angiotensin-converting enzyme inhibitor, led to a greater reduction in proteinuria and regression of glomerulosclerosis. These results identify ASK1 as an important target for renal disease and support the clinical development of an ASK1 inhibitor for the treatment of DKD.

Partial proteasome inhibitors induce hair follicle growth by stabilizing β-catenin.

The activation of tissue stem cells from their quiescent state represents the initial step in the complex process of organ regeneration and tissue repair. While the identity and location of tissue stem cells are becoming known, how key regulators control the balance of activation and quiescence remains mysterious. The vertebrate hair is an ideal model system where hair cycling between growth and resting phases is precisely regulated by morphogen signaling pathways, but how these events are coordinated to promote orderly signaling in a spatial and temporal manner remains unclear. Here, we show that hair cycle timing depends on regulated stability of signaling substrates by the ubiquitin‐proteasome system. Topical application of partial proteasomal inhibitors (PaPIs) inhibits epidermal and dermal proteasome activity throughout the hair cycle. PaPIs prevent the destruction of the key anagen signal β‐catenin, resulting in more rapid hair growth and dramatically shortened telogen. We show that PaPIs induce excess β‐catenin, act similarly to the GSK3β antagonist LiCl, and antagonize Dickopf‐related protein‐mediated inhibition of anagen. PaPIs thus represent a novel class of hair growth agents that act through transiently modifying the balance of stem cell activation and quiescence pathways. Stem Cells 2014;32:85–92

Abstract 653: Inhibition of MMP9 improves anti-tumor immunity by changing the tumor microenvironment to promote T cell trafficking and activation

Background: Matrix metalloproteinase 9 (MMP9) acts via diverse mechanisms to promote tumor growth, invasion, and metastasis. It activates growth factors and signaling pathways, promotes angiogenesis, and impedes anti-tumor immune responses. We developed a monoclonal antibody that inhibits mouse MMP9 (AB0046) and assessed its mechanism of action in immunocompetent mouse tumor models and in vitro assays. Methods: We examined MMP9 expression in a variety of human tumor tissues via immunohistochemistry. Human monocytes were differentiated in vitro and protein expression was assessed via enzyme-linked immunosorbent assay. Primary tumor growth in orthotopic, syngeneic tumor models was examined following anti-MMP9 antibody treatment. RNA sequencing, immunohistochemical and flow cytometry analyses were performed on tumor tissues to assess gene expression, stromal remodeling and macrophage polarization in response to AB0046 treatment. Results: MMP9 levels are elevated in human tumors compared to healthy tissues. The protein is expressed predominantly in stromal cells, including macrophages and neutrophils, with more occasional heterogeneous expression in tumor epithelia. Differentiation of human monocyte-derived macrophages in vitro revealed that M2 polarization is associated with increased expression of MMP9 and Th2 markers CCL18 and TGFβ. Anti-MMP9 treatment in three independent mouse tumor models (HC11-NeuT, CT26, Lewis lung carcinoma (LLC)) resulted in decreased primary tumor growth (p=0.001 and p=0.018 for HC11-NeuT and CT26 respectively) and increased animal survival (p=0.024 for LLC). Gene expression profiling of tumors from the various models demonstrated that inhibition of MMP9 resulted in elevated expression of genes associated with immune cell activation pathways (Hallmark Interferon Gamma Response, p Conclusions: These analyses show that MMP9 is expressed in a variety of human tumors. Our data suggest that inhibition of MMP9 promotes anti-tumor immunity and enhances a Th1 immune response. GS-5745, a humanized anti-MMP9 inhibitory antibody, is being evaluated in gastric cancer in phase 3 and 2 studies with chemotherapy and nivolumab, respectively (NCT02545504, NCT02864381). Citation Format: Vladi Juric, Amanda Mikels-Vigdal, Chris O9Sullivan, Andrew Greenstein, Erin Stefanutti, Vivian Barry-Hamilton, Igor Mikaelian, Ted Sullivan, Erik Huntzicker, Jeremiah Degenhardt, Peng Yue, Victoria Smith. Inhibition of MMP9 improves anti-tumor immunity by changing the tumor microenvironment to promote T cell trafficking and activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 653. doi:10.1158/1538-7445.AM2017-653