Mechanisms mediating the cardioprotective actions of glucagon-like peptide 1 (GLP-1) were unknown. Here, we show in both ex vivo and in vivo models of ischemic injury that treatment with GLP-1(28–36), a neutral endopeptidase–generated (NEP-generated) metabolite of GLP-1, was as cardioprotective as GLP-1 and was abolished by scrambling its amino acid sequence. GLP-1(28–36) enters human coronary artery endothelial cells (caECs) through macropinocytosis and acts directly on mouse and human coronary artery smooth muscle cells (caSMCs) and caECs, resulting in soluble adenylyl cyclase Adcy10–dependent (sAC-dependent) increases in cAMP, activation of protein kinase A, and cytoprotection from oxidative injury. GLP-1(28–36) modulates sAC by increasing intracellular ATP levels, with accompanying cAMP accumulation lost in sAC–/– cells. We identify mitochondrial trifunctional protein-α (MTPα) as a binding partner of GLP-1(28–36) and demonstrate that the ability of GLP-1(28–36) to shift substrate utilization from oxygen-consuming fatty acid metabolism toward oxygen-sparing glycolysis and glucose oxidation and to increase cAMP levels is dependent on MTPα. NEP inhibition with sacubitril blunted the ability of GLP-1 to increase cAMP levels in coronary vascular cells in vitro. GLP-1(28–36) is a small peptide that targets novel molecular (MTPα and sAC) and cellular (caSMC and caEC) mechanisms in myocardial ischemic injury.
M. Ahsan Siraj, Dhanwantee Mundil, Sanja Beca, Abdul Momen, Eric A. Shikatani, Talat Afroze, Xuetao Sun, Ying Liu, Siavash Ghaffari, Warren Lee, Michael B. Wheeler, Gordon Keller, Peter Backx, Mansoor Husain
Posttranslational modifications (PTMs) are common among proteins that aggregate in neurodegenerative disease, yet how PTMs impact the aggregate conformation and disease progression remains unclear. By engineering knockin mice expressing prion protein (PrP) lacking 2 N-linked glycans (Prnp180Q/196Q), we provide evidence that glycans reduce spongiform degeneration and hinder plaque formation in prion disease. Prnp180Q/196Q mice challenged with 2 subfibrillar, non–plaque-forming prion strains instead developed plaques highly enriched in ADAM10-cleaved PrP and heparan sulfate (HS). Intriguingly, a third strain composed of intact, glycophosphatidylinositol-anchored (GPI-anchored) PrP was relatively unchanged, forming diffuse, HS-deficient deposits in both the Prnp180Q/196Q and WT mice, underscoring the pivotal role of the GPI-anchor in driving the aggregate conformation and disease phenotype. Finally, knockin mice expressing triglycosylated PrP (Prnp187N) challenged with a plaque-forming prion strain showed a phenotype reversal, with a striking disease acceleration and switch from plaques to predominantly diffuse, subfibrillar deposits. Our findings suggest that the dominance of subfibrillar aggregates in prion disease is due to the replication of GPI-anchored prions, with fibrillar plaques forming from poorly glycosylated, GPI-anchorless prions that interact with extracellular HS. These studies provide insight into how PTMs impact PrP interactions with polyanionic cofactors, and highlight PTMs as a major force driving the prion disease phenotype.
Alejandro M. Sevillano, Patricia Aguilar-Calvo, Timothy D. Kurt, Jessica A. Lawrence, Katrin Soldau, Thu H. Nam, Taylor Schumann, Donald P. Pizzo, Sofie Nyström, Biswa Choudhury, Hermann Altmeppen, Jeffrey D. Esko, Markus Glatzel, K. Peter R. Nilsson, Christina J. Sigurdson
Increases in the number of cell therapies in the preclinical and clinical phases have prompted the need for reliable and noninvasive assays to validate transplant function in clinical biomanufacturing. We developed a robust characterization methodology composed of quantitative bright-field absorbance microscopy (QBAM) and deep neural networks (DNNs) to noninvasively predict tissue function and cellular donor identity. The methodology was validated using clinical-grade induced pluripotent stem cell–derived retinal pigment epithelial cells (iPSC-RPE). QBAM images of iPSC-RPE were used to train DNNs that predicted iPSC-RPE monolayer transepithelial resistance, predicted polarized vascular endothelial growth factor (VEGF) secretion, and matched iPSC-RPE monolayers to the stem cell donors. DNN predictions were supplemented with traditional machine-learning algorithms that identified shape and texture features of single cells that were used to predict tissue function and iPSC donor identity. These results demonstrate noninvasive cell therapy characterization can be achieved with QBAM and machine learning.
Nicholas J. Schaub, Nathan A. Hotaling, Petre Manescu, Sarala Padi, Qin Wan, Ruchi Sharma, Aman George, Joe Chalfoun, Mylene Simon, Mohamed Ouladi, Carl G. Simon Jr., Peter Bajcsy, Kapil Bharti
Hereditary hemorrhagic telangiectasia (HHT), a genetic bleeding disorder leading to systemic arteriovenous malformations (AVMs), is caused by loss-of-function mutations in the ALK1/ENG/Smad1/5/8 pathway. Evidence suggests that HHT pathogenesis strongly relies on overactivated PI3K/Akt/mTOR and VEGFR2 pathways in endothelial cells (ECs). In the BMP9/10-immunoblocked (BMP9/10ib) neonatal mouse model of HHT, we report here that the mTOR inhibitor, sirolimus, and the receptor tyrosine kinase inhibitor, nintedanib, could synergistically fully block, but also reversed, retinal AVMs to avert retinal bleeding and anemia. Sirolimus plus nintedanib prevented vascular pathology in the oral mucosa, lungs, and liver of the BMP9/10ib mice, as well as significantly reduced gastrointestinal bleeding and anemia in inducible ALK1-deficient adult mice. Mechanistically, in vivo in BMP9/10ib mouse ECs, sirolimus and nintedanib blocked the overactivation of mTOR and VEGFR2, respectively. Furthermore, we found that sirolimus activated ALK2-mediated Smad1/5/8 signaling in primary ECs — including in HHT patient blood outgrowth ECs — and partially rescued Smad1/5/8 activity in vivo in BMP9/10ib mouse ECs. These data demonstrate that the combined correction of endothelial Smad1/5/8, mTOR, and VEGFR2 pathways opposes HHT pathogenesis. Repurposing of sirolimus plus nintedanib might provide therapeutic benefit in patients with HHT.
Santiago Ruiz, Haitian Zhao, Pallavi Chandakkar, Julien Papoin, Hyunwoo Choi, Aya Nomura-Kitabayashi, Radhika Patel, Matthew Gillen, Li Diao, Prodyot K. Chatterjee, Mingzhu He, Yousef Al-Abed, Ping Wang, Christine N. Metz, S. Paul Oh, Lionel Blanc, Fabien Campagne, Philippe Marambaud
The protein-protein interaction between menin and mixed lineage leukemia 1 (MLL1) plays a critical role in acute leukemias with translocations of the MLL1 gene or with mutations in the nucleophosmin 1 (NPM1) gene. As a step toward clinical translation of menin-MLL1 inhibitors, we report development of MI-3454, a highly potent and orally bioavailable inhibitor of the menin-MLL1 interaction. MI-3454 profoundly inhibited proliferation and induced differentiation in acute leukemia cells and primary patient samples with MLL1 translocations or NPM1 mutations. When applied as a single agent, MI-3454 induced complete remission or regression of leukemia in mouse models of MLL1-rearranged or NPM1-mutated leukemia, including patient-derived xenograft models, through downregulation of key genes involved in leukemogenesis. We also identified MEIS1 as a potential pharmacodynamic biomarker of treatment response with MI-3454 in leukemia, and demonstrated that this compound is well tolerated and did not impair normal hematopoiesis in mice. Overall, this study demonstrates, for the first time to our knowledge, profound activity of the menin-MLL1 inhibitor as a single agent in clinically relevant PDX models of leukemia. These data provide a strong rationale for clinical translation of MI-3454 or its analogs for leukemia patients with MLL1 rearrangements or NPM1 mutations.
Szymon Klossowski, Hongzhi Miao, Katarzyna Kempinska, Tao Wu, Trupta Purohit, EunGi Kim, Brian M. Linhares, Dong Chen, Gloria Jih, Eric Perkey, Huang Huang, Miao He, Bo Wen, Yi Wang, Ke Yu, Stanley Chun-Wei Lee, Gwenn Danet-Desnoyers, Winifred Trotman, Malathi Kandarpa, Anitria Cotton, Omar Abdel-Wahab, Hongwei Lei, Yali Dou, Monica Guzman, Luke Peterson, Tanja Gruber, Sarah Choi, Duxin Sun, Pingda Ren, Lian-Sheng Li, Yi Liu, Francis Burrows, Ivan Maillard, Tomasz Cierpicki, Jolanta Grembecka
Leptomeningeal anastomoses or pial collateral vessels play a critical role in cerebral blood flow (CBF) restoration following ischemic stroke. The magnitude of this adaptive response is postulated to be controlled by the endothelium, although the underlying molecular mechanisms remain under investigation. Here we demonstrated that endothelial genetic deletion, using EphA4fl/fl/Tie2-Cre and EphA4fl/fl/VeCahderin-CreERT2 mice and vessel painting strategies, implicated EphA4 receptor tyrosine kinase as a major suppressor of pial collateral remodeling, CBF, and functional recovery following permanent middle cerebral artery occlusion. Pial collateral remodeling is limited by the crosstalk between EphA4-Tie2 signaling in vascular endothelial cells, which is mediated through p-Akt regulation. Furthermore, peptide inhibition of EphA4 resulted in acceleration of the pial arteriogenic response. Our findings demonstrate that EphA4 is a negative regulator of Tie2 receptor signaling, which limits pial collateral arteriogenesis following cerebrovascular occlusion. Therapeutic targeting of EphA4 and/or Tie2 represents an attractive new strategy for improving collateral function, neural tissue health, and functional recovery following ischemic stroke.
Benjamin Okyere, William A. Mills III, Xia Wang, Michael Chen, Jiang Chen, Amanda Hazy, Yun Qian, John B. Matson, Michelle H. Theus
Increased rates of locoregional recurrence are observed in patients with basal-like breast cancer (BC) despite the use of radiation therapy (RT); therefore, approaches that result in radiosensitization of basal-like BC are critically needed. Using patients’ tumor gene expression data from 4 independent data sets, we correlated gene expression with recurrence to find genes significantly correlated with early recurrence after RT. The highest-ranked gene, TTK, was most highly expressed in basal-like BC across multiple data sets. Inhibition of TTK by both genetic and pharmacologic methods enhanced radiosensitivity in multiple basal-like cell lines. Radiosensitivity was mediated, at least in part, through persistent DNA damage after treatment with TTK inhibition and RT. Inhibition of TTK impaired homologous recombination (HR) and repair efficiency, but not nonhomologous end-joining, and decreased the formation of Rad51 foci. Reintroduction of wild-type TTK rescued both radioresistance and HR repair efficiency after TTK knockdown, however, reintroduction of kinase-dead TTK did not. In vivo, TTK inhibition combined with RT led to a significant decrease in tumor growth in both heterotopic and orthotopic, including patient-derived xenograft, BC models. These data support the rationale for clinical development of TTK inhibition as a radiosensitizing strategy for patients with basal-like BC, and efforts toward this end are currently underway.
Benjamin C. Chandler, Leah Moubadder, Cassandra L. Ritter, Meilan Liu, Meleah Cameron, Kari Wilder-Romans, Amanda Zhang, Andrea M. Pesch, Anna R. Michmerhuizen, Nicole Hirsh, Marlie Androsiglio, Tanner Ward, Eric Olsen, Yashar S. Niknafs, Sofia Merajver, Dafydd G. Thomas, Powel H. Brown, Theodore S. Lawrence, Shyam Nyati, Lori J. Pierce, Arul Chinnaiyan, Corey Speers
Antigen receptor–dependent (AgR-dependent) stimulation of the NF-κB transcription factor in lymphocytes is a required event during adaptive immune response, but dysregulated activation of this signaling pathway can lead to lymphoma. AgR stimulation promotes assembly of the CARMA1-BCL10-MALT1 complex, wherein MALT1 acts as (a) a scaffold to recruit components of the canonical NF-κB machinery, and (b) a protease to cleave and inactivate specific substrates, including negative regulators of NF-κB. In multiple lymphoma subtypes, malignant B cells hijack AgR signaling pathways to promote their own growth and survival, and inhibiting MALT1 reduces the viability and growth of these tumors. As such, MALT1 has emerged as a potential pharmaceutical target. Here, we identified G protein–coupled receptor kinase 2 (GRK2) as a new MALT1-interacting protein. We demonstrated that GRK2 binds the death domain of MALT1 and inhibits MALT1 scaffolding and proteolytic activities. We found that lower GRK2 levels in activated B cell–type diffuse large B cell lymphoma (ABC-DLBCL) are associated with reduced survival, and that GRK2 knockdown enhances ABC-DLBCL tumor growth in vitro and in vivo. Together, our findings suggest that GRK2 can function as a tumor suppressor by inhibiting MALT1 and provide a roadmap for developing new strategies to inhibit MALT1-dependent lymphomagenesis.
Jing Cheng, Linda R. Klei, Nathaniel E. Hubel, Ming Zhang, Rebekka Schairer, Lisa M. Maurer, Hanna B. Klei, Heejae Kang, Vincent J. Concel, Phillip C. Delekta, Eric V. Dang, Michelle A. Mintz, Mathijs Baens, Jason G. Cyster, Narayanan Parameswaran, Margot Thome, Peter C. Lucas, Linda M. McAllister-Lucas
The corneocyte lipid envelope, composed of covalently bound ceramides and fatty acids, is important to the integrity of the permeability barrier in the stratum corneum, and its absence is a prime structural defect in various skin diseases associated with defective skin barrier function. SDR9C7 encodes a short-chain dehydrogenase/reductase family 9C member 7 (SDR9C7) recently found mutated in ichthyosis. In a patient with SDR9C7 mutation and a mouse Sdr9c7 knockout model, we show loss of covalent binding of epidermal ceramides to protein, a structural fault in the barrier. For reasons unresolved, protein binding requires lipoxygenase-catalyzed transformations of linoleic acid (18:2) esterified in ω-O-acylceramides. In Sdr9c7–/– epidermis, quantitative liquid chromatography–mass spectometry (LC-MS) assays revealed almost complete loss of a species of ω-O-acylceramide esterified with linoleate-9,10-trans-epoxy-11E-13-ketone; other acylceramides related to the lipoxygenase pathway were in higher abundance. Recombinant SDR9C7 catalyzed NAD+-dependent dehydrogenation of linoleate 9,10-trans-epoxy-11E-13-alcohol to the corresponding 13-ketone, while ichthyosis mutants were inactive. We propose, therefore, that the critical requirement for lipoxygenases and SDR9C7 is in producing acylceramide containing the 9,10-epoxy-11E-13-ketone, a reactive moiety known for its nonenzymatic coupling to protein. This suggests a mechanism for coupling of ceramide to protein and provides important insights into skin barrier formation and pathogenesis.
Takuya Takeichi, Tetsuya Hirabayashi, Yuki Miyasaka, Akane Kawamoto, Yusuke Okuno, Shijima Taguchi, Kana Tanahashi, Chiaki Murase, Hiroyuki Takama, Kosei Tanaka, William E. Boeglin, M. Wade Calcutt, Daisuke Watanabe, Michihiro Kono, Yoshinao Muro, Junko Ishikawa, Tamio Ohno, Alan R. Brash, Masashi Akiyama
Pattern recognition receptors (PRRs) are crucial for responses to infections and tissue damage; however, their role in autoimmunity is less clear. Herein we demonstrate that 2 C-type lectin receptors (CLRs), Mcl and Mincle, play an important role in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Congenic rats expressing lower levels of Mcl and Mincle on myeloid cells exhibited a drastic reduction in EAE incidence. In vivo silencing of Mcl and Mincle or blockade of their endogenous ligand SAP130 revealed that these receptors’ expression in the central nervous system is crucial for T cell recruitment and reactivation into a pathogenic Th17/GM-CSF phenotype. Consistent with this, we uncovered MCL- and MINCLE-expressing cells in brain lesions of MS patients and we further found an upregulation of the MCL/MINCLE signaling pathway and an increased response following MCL/MINCLE stimulation in peripheral blood mononuclear cells from MS patients. Together, these data support a role for CLRs in autoimmunity and implicate the MCL/MINCLE pathway as a potential therapeutic target in MS.
Marie N’diaye, Susanna Brauner, Sevasti Flytzani, Lara Kular, Andreas Warnecke, Milena Z. Adzemovic, Eliane Piket, Jin-Hong Min, Will Edwards, Filia Mela, Hoi Ying Choi, Vera Magg, Tojo James, Magdalena Linden, Holger M. Reichardt, Michael R. Daws, Jack van Horssen, Ingrid Kockum, Robert A. Harris, Tomas Olsson, Andre O. Guerreiro-Cacais, Maja Jagodic
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