JAK2-V617F-positive chronic myeloproliferative neoplasia (CMN) is marked by dysfunction of integrins and adhesion molecules expressed on platelets, erythrocytes and leukocytes. However, the mechanism by which the two major leukocyte integrin chains, β1 and β2, mediate CMN pathophysiology remained unclear. β1 (α4β1; VLA-4) and β2 (αLβ2; LFA-1) integrins are essential regulators for attachment of leukocytes to endothelial cells. We here show enhanced adhesion of granulocytes from JAK2+/VF knock-in mice to vascular cell adhesion molecule 1 (VCAM1) and intercellular adhesion molecule 1 (ICAM1) coated surfaces. Soluble VCAM1 and ICAM1 ligand binding assays revealed increased affinity of β1 and β2 integrins for their respective ligands. For β1 integrins, this correlated with a structural change from the low to the high affinity conformation induced by JAK2-V617F. JAK2-V617F triggers constitutive activation of the integrin inside-out signaling molecule Rap1 resulting in translocation towards the cell membrane. Employing a venous thrombosis model, we demonstrate that neutralizing anti-VLA4 and anti-β2 integrin antibodies suppress pathologic thrombosis as observed in JAK2+/VF mice. In addition, aberrant homing of JAK2+/VF leukocytes to the spleen is inhibited by neutralizing anti-β2 antibodies and by pharmacologic inhibition of Rap1. Thus, our findings identify a cross talk between JAK2-V617F and integrin activation promoting pathologic thrombosis and abnormal trafficking of leukocytes to the spleen. .
Bärbel Edelmann, Nibedita Gupta, Tina M. Schnöder, Anja M. Oelschlegel, Khurrum Shahzad, Jürgen Goldschmidt, Lars Philipsen, Sönke Weinert, Aniket Ghosh, Felix C. Saalfeld, Subbaiah Chary Nimmagadda, Peter Müller, Rüdiger C. Braun-Dullaeus, Juliane Mohr, Denise Wolleschak, Stefanie Kliche, Holger Amthauer, Florian H. Heidel, Burkhart Schraven, Berend Isermann, Andreas Müller, Thomas Fischer
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. Herein, 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 decline of glomerular filtration rate. 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 diabetic kidney disease.
John T. Liles, Britton K. Corkey, Gregory T. Notte, Grant Budas, Eric B. Lansdon, Ford Hinojosa-Kirschenbaum, Shawn S. Badal, Michael Lee, Brian E. Schultz, Sarah Wise, Swetha Pendem, Michael Graupe, Laurie Castonguay, Keith A. Koch, Melanie H. Wong, Giuseppe A. Papalia, Dorothy M. French, Theodore Sullivan, Erik G. Huntzicker, Frank Y. Ma, David J. Nikolic-Paterson, Tareq Altuhaifi, Haichun Yang, Agnes B. Fogo, David G. Breckenridge
The adjuvanted varicella-zoster virus glycoprotein E (VZV gE) subunit herpes zoster vaccine (HZ/su) confers higher protection against HZ than the live attenuated zoster vaccine (ZV). To understand the immunologic basis for the different efficacies of the vaccines, we compared immune responses to the vaccines in adults 50- to 85-year-old. gE-specific T cells were very low/undetectable before vaccination when analyzed by FluoroSpot and flow cytometry. Both ZV and HZ/su increased gE-specific responses, but at peak memory response (PMR) after vaccination (30 days after ZV or after the second dose of HZ/su) gE-specific CD4+ and CD8+ T-cell responses were ≥ 10-fold higher in HZ/su compared with ZV recipients. Comparing the vaccines, T cell memory responses, including gE- and VZV-IL2+ spot-forming cells (SFC), were higher in HZ/su recipients and cytotoxic and effector responses were lower. At 1 year after vaccination, all gE-Th1 and VZV-IL2+ SFC remained higher in HZ/su compared to ZV recipients. Mediation analyses showed that IL2+ PMR were necessary for the persistence of Th1 responses to either vaccine and VZV-IL2+ PMR explained 73% of the total effect of HZ/su on persistence. This emphasizes the biological importance of the memory responses, which were clearly superior in HZ/su compared with ZV participants.
Myron J. Levin, Miranda E. Kroehl, Michael J. Johnson, Andrew Hammes, Dominik Reinhold, Nancy Lang, Adriana Weinberg
The MALT1 paracaspase plays an essential role in Activated B-cell like Diffuse Large B cell Lymphoma (ABC DLBCL) downstream of B cell and Toll-like receptor pathway genes mutated in these tumors. Although MALT1 is considered to be a compelling therapeutic target, development of tractable and specific MALT1 protease inhibitors has thus far been elusive. Herein, we developed a target engagement assay that provides a quantitative readout for specific MALT1 inhibitory effects in living cells. This enabled a structure-guided medicinal chemistry effort culminating in the discovery of pharmacologically tractable irreversible substrate-mimetic compounds that bind the MALT1 active site. We confirmed MALT1 targeting with compound #3 is effective at suppressing ABC DLBCL cells in vitro and in vivo. We show that reduction in serum IL10 levels exquisitely correlates with drug PK and degree of MALT1 inhibition in vitro and in vivo and could constitute a useful pharmacodynamic biomarker to evaluate these compounds in clinical trials. Compound #3 revealed insights into the biology of MALT1 in ABC DLBCL, such as driving JAK-STAT signaling and suppressing type I interferon (IFN) response and MHC class II expression, suggesting that MALT1 inhibition could prime lymphomas for immune recognition by cytotoxic immune cells.
Lorena Fontán, Qi Qiao, John M. Hatcher, Gabriella Casalena, Ilkay Us, Matt Teater, Matthew Durant, Guangyan Du, Min Xia, Natalia Bilchuk, Spandan Chennamadhavuni, Giuseppe Palladino, Giorgio Inghirami, Ulrike Philippar, Hao Wu, David A. Scott, Nathanael S. Gray, Ari Melnick
Nucleophosmin (NPM1) is amongst the most frequently mutated genes in acute myeloid leukemia (AML). It is not known, however, how the resulting oncoprotein mutant-NPM1 is leukemogenic. To reveal the cellular machinery in which NPM1 participates in myeloid cells, we analyzed the endogenous NPM1 protein-interactome by mass-spectrometry, and discovered abundant amounts of the master transcription factor driver of monocyte lineage-differentiation PU.1 (SPI1). Mutant-NPM1, which aberrantly accumulates in cytoplasm, dislocated PU.1 into cytoplasm with it. CEBPA and RUNX1, the master transcription factors that collaborate with PU.1 to activate granulo-monocytic lineage-fates, remained nuclear, but without PU.1, their coregulator interactions were toggled from coactivators to corepressors, repressing instead of activating greater than 500 granulocyte and monocyte terminal-differentiation genes. An inhibitor of nuclear export, selinexor, by locking mutant-NPM1/PU.1 in the nucleus, activated terminal monocytic fates. Direct depletion of the corepressor DNA methyltransferase 1 (DNMT1) from the CEBPA/RUNX1 protein interactome using the clinical drug decitabine activated terminal granulocytic fates. Together, these non-cytotoxic treatments extended survival by greater than 160 days versus vehicle in a patient-derived xenotransplant model of NPM1/FLT3-mutated AML. In sum, mutant-NPM1 represses monocyte and granulocyte terminal-differentiation by disrupting PU.1/CEBPA/RUNX1 collaboration, a transforming action that can be reversed by pharmacodynamically-directed dosing of clinical small molecules.
Xiaorong Gu, Quteba Ebrahem, Reda Z. Mahfouz, Metis Hasipek, Francis Enane, Tomas Radivoyevitch, Nicolas Rapin, Bartlomiej Przychodzen, Zhenbo Hu, Ramesh Balusu, Claudiu V. Cotta, David Wald, Christian Argueta, Yosef Landesman, Maria Paola Martelli, Brunangelo Falini, Hetty Carraway, Bo T. Porse, Jaroslaw P. Maciejewski, Babal K. Jha, Yogen Saunthararajah
Genome-wide association studies have repeatedly mapped susceptibility loci for emphysema to genes that modify hedgehog signaling, but the functional relevance of hedgehog signaling to this morbid disease remains unclear. In the current study, we identified a broad population of mesenchymal cells in the adult murine lung receptive to hedgehog signaling, characterized by higher activation of hedgehog surrounding the proximal airway relative to the distal alveoli. Single cell RNA-sequencing showed that the hedgehog-receptive mesenchyme is composed of mostly fibroblasts with distinct proximal and distal subsets with discrete identities. Ectopic hedgehog activation in the distal fibroblasts promoted expression of proximal fibroblast markers, and promoted loss of distal alveoli and airspace enlargement of over twenty percent compared to controls. We found that hedgehog suppressed mesenchymal-derived mitogens enriched in distal fibroblasts that regulate alveolar stem cell regeneration and airspace size. Finally, single cell analysis of the human lung mesenchyme showed that segregated proximal-distal identity with preferential hedgehog activation in the proximal fibroblasts is conserved between mice and humans. In conclusion, we showed that differential hedgehog activation segregates mesenchymal identities of distinct fibroblast subsets, and disruption of fibroblast identity can alter the alveolar stem cell niche leading to emphysematous changes in the murine lung.
Chaoqun Wang, Nabora S. Reyes de Mochel, Stephanie A. Christenson, Monica Cassandras, Rebecca Moon, Alexis N. Brumwell, Lauren E. Byrnes, Alfred Li, Yasuyuki Yokosaki, Peiying Shan, Julie B. Sneddon, David Jablons, Patty J. Lee, Michael A. Matthay, Harold A. Chapman, Tien Peng
Induction of TLR2 activation depends on its association with adapter protein MyD88. We have found that levels of TLR2 and MyD88 are elevated in the hippocampus and cortex of Alzheimer’s disease (AD) patients and 5XFAD mouse model of AD. Since there is no specific inhibitor of TLR2, to target induced TLR2 from therapeutic angle, we engineered a peptide corresponding to the TLR2-interacting domain of MyD88 (TIDM) that binds to the BB loop of only TLR2, but not other TLRs. Interestingly, wild type (wt) TIDM peptide inhibited microglial activation induced by fibrillar Aβ1-42 and lipoteichoic acid, but not 1-methyl-4-phenylpyridinium, double-stranded RNA, bacterial lipopolysaccharide, flagellin, and CpG DNA. After intranasal administration, wtTIDM peptide reached the hippocampus, reduced hippocampal glial activation, lowered Aβ burden, attenuated neuronal apoptosis, and improved memory and learning in 5XFAD mice. However, wtTIDM peptide was not effective in 5XFAD mice lacking TLR2. In addition to 5XFAD mice, wtTIDM peptide also suppressed the disease process in mice with experimental allergic encephalomyelitis and collagen-induced arthritis. Therefore, selective targeting of activated status of one component of the innate immune system by wtTIDM peptide may be beneficial in AD as well as other disorders in which TLR2-MyD88 signaling plays a role in disease pathogenesis.
Suresh B. Rangasamy, Malabendu Jana, Avik Roy, Grant T. Corbett, Madhuchhanda Kundu, Sujyoti Chandra, Susanta Mondal, Sridevi Dasarathi, Elliott J. Mufson, Rama K. Mishra, Chi-Hao Luan, David A. Bennett, Kalipada Pahan
Anaplastic thyroid carcinomas (ATC) have a high prevalence of BRAF and TP53 mutations. A trial of vemurafenib in non-melanoma BRAFV600E-mutant cancers showed significant, although short-lived, responses in ATCs, indicating that these virulent tumors remain addicted to BRAF despite their high mutation burden. To explore the mechanisms mediating acquired resistance to BRAF blockade we generated mice with thyroid-specific deletion of p53 and dox-dependent expression of BRAFV600E, 50% of which developed ATCs after dox treatment. Upon dox withdrawal there was complete regression in all mice, although recurrences were later detected in 85% of animals. The relapsed tumors had elevated MAPK transcriptional output, and retained responses to the MEK/RAF inhibitor CH5126766 in vivo and in vitro. Whole exome sequencing identified recurrent focal amplifications of chromosome 6, with a minimal region of overlap that included Met. Met-amplified recurrences overexpressed the receptor as well as its ligand Hgf. Growth, signaling and viability of Met-amplified tumor cells were suppressed in vitro and in vivo by the Met kinase inhibitors PF-04217903 and crizotinib, whereas primary ATCs and Met-diploid relapses were resistant. Hence, recurrences are the rule after BRAF suppression in murine ATCs, most commonly due to activation of HGF/MET signaling, which generates exquisite dependency to MET kinase inhibitors.
Jeffrey A. Knauf, Kathleen A. Luckett, Kuen-Yuan Chen, Francesca Voza, Nicholas D. Socci, Ronald Ghossein, James A. Fagin
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