Thrombosis, or blood clot formation, and its sequelae remain a leading cause of morbidity and mortality, and recurrent thrombosis is common despite current optimal therapy. Protein disulfide isomerase (PDI) is an oxidoreductase that has recently been shown to participate in thrombus formation. While currently available antithrombotic agents inhibit either platelet aggregation or fibrin generation, inhibition of secreted PDI blocks the earliest stages of thrombus formation, suppressing both pathways. Here, we explored extracellular PDI as an alternative target of antithrombotic therapy. A high-throughput screen identified quercetin-3-rutinoside as an inhibitor of PDI reductase activity in vitro. Inhibition of PDI was selective, as quercetin-3-rutinoside failed to inhibit the reductase activity of several other thiol isomerases found in the vasculature. Cellular assays showed that quercetin-3-rutinoside inhibited aggregation of human and mouse platelets and endothelial cell–mediated fibrin generation in human endothelial cells. Using intravital microscopy in mice, we demonstrated that quercetin-3-rutinoside blocks thrombus formation in vivo by inhibiting PDI. Infusion of recombinant PDI reversed the antithrombotic effect of quercetin-3-rutinoside. Thus, PDI is a viable target for small molecule inhibition of thrombus formation, and its inhibition may prove to be a useful adjunct in refractory thrombotic diseases that are not controlled with conventional antithrombotic agents.
Reema Jasuja, Freda H. Passam, Daniel R. Kennedy, Sarah H. Kim, Lotte van Hessem, Lin Lin, Sheryl R. Bowley, Sucharit S. Joshi, James R. Dilks, Bruce Furie, Barbara C. Furie, Robert Flaumenhaft
Hematopoietic stem and progenitor cell (HSPC) functions are governed by intricate signaling networks. The tyrosine kinase JAK2 plays an essential role in cytokine signaling during hematopoiesis. The adaptor protein LNK is a critical determinant of this process through its inhibitory interaction with JAK2, thereby limiting HSPC self-renewal. LNK deficiency promotes myeloproliferative neoplasm (MPN) development in mice, and LNK loss-of-function mutations are found in human MPNs, emphasizing its pivotal role in normal and malignant HSPCs. Here, we report the identification of 14-3-3 proteins as LNK binding partners. 14-3-3 interfered with the LNK-JAK2 interaction, thereby alleviating LNK inhibition of JAK2 signaling and cell proliferation. Binding of 14-3-3 required 2 previously unappreciated serine phosphorylation sites in LNK, and we found that their phosphorylation is mediated by glycogen synthase kinase 3 and PKA kinases. Mutations of these residues abrogated the interaction and augmented the growth inhibitory function of LNK. Conversely, forced 14-3-3 binding constrained LNK function. Furthermore, interaction with 14-3-3 sequestered LNK in the cytoplasm away from the plasma membrane-proximal JAK2. Importantly, bone marrow transplantation studies revealed an essential role for 14-3-3 in HSPC reconstitution that can be partially mitigated by LNK deficiency. We believe that, together, this work implicates 14-3-3 proteins as novel and positive HSPC regulators by impinging on the LNK/JAK2 pathway.
Jing Jiang, Joanna Balcerek, Krasimira Rozenova, Ying Cheng, Alexey Bersenev, Chao Wu, Yiwen Song, Wei Tong
Massive transfusion of blood can lead to clinical complications, including multiorgan dysfunction and even death. Such severe clinical outcomes have been associated with longer red blood cell (rbc) storage times. Collectively referred to as the rbc storage lesion, rbc storage results in multiple biochemical changes that impact intracellular processes as well as membrane and cytoskeletal properties, resulting in cellular injury in vitro. However, how the rbc storage lesion triggers pathophysiology in vivo remains poorly defined. In this study, we developed a guinea pig transfusion model with blood stored under standard blood banking conditions for 2 (new), 21 (intermediate), or 28 days (old blood). Transfusion with old but not new blood led to intravascular hemolysis, acute hypertension, vascular injury, and kidney dysfunction associated with pathophysiology driven by hemoglobin (Hb). These adverse effects were dramatically attenuated when the high-affinity Hb scavenger haptoglobin (Hp) was administered at the time of transfusion with old blood. Pathologies observed after transfusion with old blood, together with the favorable response to Hp supplementation, allowed us to define the in vivo consequences of the rbc storage lesion as storage-related posttransfusion hemolysis producing Hb-driven pathophysiology. Hb sequestration by Hp might therefore be a therapeutic modality for enhancing transfusion safety in severely ill or massively transfused patients.
Jin Hyen Baek, Felice D’Agnillo, Florence Vallelian, Claudia P. Pereira, Matthew C. Williams, Yiping Jia, Dominik J. Schaer, Paul W. Buehler
Individuals with Down syndrome (DS; also known as trisomy 21) have a markedly increased risk of leukemia in childhood but a decreased risk of solid tumors in adulthood. Acquired mutations in the transcription factor–encoding GATA1 gene are observed in nearly all individuals with DS who are born with transient myeloproliferative disorder (TMD), a clonal preleukemia, and/or who develop acute megakaryoblastic leukemia (AMKL). Individuals who do not have DS but bear germline GATA1 mutations analogous to those detected in individuals with TMD and DS-AMKL are not predisposed to leukemia. To better understand the functional contribution of trisomy 21 to leukemogenesis, we used mouse and human cell models of DS to reproduce the multistep pathogenesis of DS-AMKL and to identify chromosome 21 genes that promote megakaryoblastic leukemia in children with DS. Our results revealed that trisomy for only 33 orthologs of human chromosome 21 (Hsa21) genes was sufficient to cooperate with GATA1 mutations to initiate megakaryoblastic leukemia in vivo. Furthermore, through a functional screening of the trisomic genes, we demonstrated that DYRK1A, which encodes dual-specificity tyrosine-(Y)-phosphorylation–regulated kinase 1A, was a potent megakaryoblastic tumor–promoting gene that contributed to leukemogenesis through dysregulation of nuclear factor of activated T cells (NFAT) activation. Given that calcineurin/NFAT pathway inhibition has been implicated in the decreased tumor incidence in adults with DS, our results show that the same pathway can be both proleukemic in children and antitumorigenic in adults.
Sébastien Malinge, Meghan Bliss-Moreau, Gina Kirsammer, Lauren Diebold, Timothy Chlon, Sandeep Gurbuxani, John D. Crispino
Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults. Long-term survival of patients with AML has changed little over the past decade, necessitating the identification and validation of new AML targets. Integration of genomic approaches with small-molecule and genetically based high-throughput screening holds the promise of improved discovery of candidate targets for cancer therapy. Here, we identified a role for glycogen synthase kinase 3α (GSK-3α) in AML by performing 2 independent small-molecule library screens and an shRNA screen for perturbations that induced a differentiation expression signature in AML cells. GSK-3 is a serine-threonine kinase involved in diverse cellular processes, including differentiation, signal transduction, cell cycle regulation, and proliferation. We demonstrated that specific loss of GSK-3α induced differentiation in AML by multiple measurements, including induction of gene expression signatures, morphological changes, and cell surface markers consistent with myeloid maturation. GSK-3α–specific suppression also led to impaired growth and proliferation in vitro, induction of apoptosis, loss of colony formation in methylcellulose, and anti-AML activity in vivo. Although the role of GSK-3β has been well studied in cancer development, these studies support a role for GSK-3α in AML.
Versha Banerji, Stacey M. Frumm, Kenneth N. Ross, Loretta S. Li, Anna C. Schinzel, Cynthia K. Hahn, Rose M. Kakoza, Kwan T. Chow, Linda Ross, Gabriela Alexe, Nicola Tolliday, Haig Inguilizian, Ilene Galinsky, Richard M. Stone, Daniel J. DeAngelo, Giovanni Roti, Jon C. Aster, William C. Hahn, Andrew L. Kung, Kimberly Stegmaier
Chronic myelogenous leukemia (CML) results from a chromosomal translocation in hematopoietic stem or early progenitor cells that gives rise to the oncogenic BCR/ABL fusion protein. Clinically, CML has a chronic phase that eventually evolves into an accelerated stage and blast crisis. A CML-specific immune response is thought to contribute to the control of disease. Whether the immune system can also promote disease progression is not known. In the present study, we investigated the possibility that the TNF receptor family member CD27 is present on leukemia stem cells (LSCs) and mediates effects of the immune system on CML. In a mouse model of CML, BCR/ABL+ LSCs and leukemia progenitor cells were found to express CD27. Binding of CD27 by its ligand, CD70, increased expression of Wnt target genes in LSCs by enhancing nuclear localization of active β-catenin and TRAF2- and NCK-interacting kinase (TNIK). This resulted in increased proliferation and differentiation of LSCs. Blocking CD27 signaling in LSCs delayed disease progression and prolonged survival. Furthermore, CD27 was expressed on CML stem/progenitor cells in the bone marrow of CML patients, and CD27 signaling promoted growth of BCR/ABL+ human leukemia cells by activating the Wnt pathway. Since expression of CD70 is limited to activated lymphocytes and dendritic cells, our results reveal a mechanism by which adaptive immunity contributes to leukemia progression. In addition, targeting CD27 on LSCs may represent an attractive therapeutic approach to blocking the Wnt/β-catenin pathway in CML.
Christian Schürch, Carsten Riether, Matthias S. Matter, Alexandar Tzankov, Adrian F. Ochsenbein
In hematologic diseases, such as sickle cell disease (SCD) and hemolytic uremic syndrome (HUS), pathological biophysical interactions among blood cells, endothelial cells, and soluble factors lead to microvascular occlusion and thrombosis. Here, we report an in vitro “endothelialized” microfluidic microvasculature model that recapitulates and integrates this ensemble of pathophysiological processes. Under controlled flow conditions, the model enabled quantitative investigation of how biophysical alterations in hematologic disease collectively lead to microvascular occlusion and thrombosis. Using blood samples from patients with SCD, we investigated how the drug hydroxyurea quantitatively affects microvascular obstruction in SCD, an unresolved issue pivotal to understanding its clinical efficacy in such patients. In addition, we demonstrated that our microsystem can function as an in vitro model of HUS and showed that shear stress influences microvascular thrombosis/obstruction and the efficacy of the drug eptifibatide, which decreases platelet aggregation, in the context of HUS. These experiments establish the versatility and clinical relevance of our microvasculature-on-a-chip model as a biophysical assay of hematologic pathophysiology as well as a drug discovery platform.
Michelle Tsai, Ashley Kita, Joseph Leach, Ross Rounsevell, James N. Huang, Joel Moake, Russell E. Ware, Daniel A. Fletcher, Wilbur A. Lam
DNA methyltransferase 3B (Dnmt3b) belongs to a family of enzymes responsible for methylation of cytosine residues in mammals. DNA methylation contributes to the epigenetic control of gene transcription and is deregulated in virtually all human tumors. To better understand the generation of cancer-specific methylation patterns, we genetically inactivated Dnmt3b in a mouse model of MYC-induced lymphomagenesis. Ablation of Dnmt3b function using a conditional knockout in T cells accelerated lymphomagenesis by increasing cellular proliferation, which suggests that Dnmt3b functions as a tumor suppressor. Global methylation profiling revealed numerous gene promoters as potential targets of Dnmt3b activity, the majority of which were demethylated in Dnmt3b–/– lymphomas, but not in Dnmt3b–/– pretumor thymocytes, implicating Dnmt3b in maintenance of cytosine methylation in cancer. Functional analysis identified the gene Gm128 (which we termed herein methylated in normal thymocytes [Ment]) as a target of Dnmt3b activity. We found that Ment was gradually demethylated and overexpressed during tumor progression in Dnmt3b–/– lymphomas. Similarly, MENT was overexpressed in 67% of human lymphomas, and its transcription inversely correlated with methylation and levels of DNMT3B. Importantly, knockdown of Ment inhibited growth of mouse and human cells, whereas overexpression of Ment provided Dnmt3b+/+ cells with a proliferative advantage. Our findings identify Ment as an enhancer of lymphomagenesis that contributes to the tumor suppressor function of Dnmt3b and suggest it could be a potential target for anticancer therapies.
Ryan A. Hlady, Slavomira Novakova, Jana Opavska, David Klinkebiel, Staci L. Peters, Juraj Bies, Jay Hannah, Javeed Iqbal, Kristi M. Anderson, Hollie M. Siebler, Lynette M. Smith, Timothy C. Greiner, Dhundy Bastola, Shantaram Joshi, Oksana Lockridge, Melanie A. Simpson, Dean W. Felsher, Kay-Uwe Wagner, Wing C. Chan, Judith K. Christman, Rene Opavsky
Cutaneous T cell lymphomas (CTCLs) represent a heterogeneous group of non-Hodgkin lymphomas that affect the skin. The pathogenesis of these conditions is poorly understood. For example, the signaling mechanisms contributing to the dysregulated growth of the neoplastic T cells are not well defined. Here, we demonstrate that loss of nuclear localization of pro–IL-16 facilitates CTCL cell proliferation by causing a decrease in expression of the cyclin dependent–kinase inhibitor p27Kip1. The decrease in p27Kip1 expression was directly attributable to an increase in expression of S-phase kinase-associated protein 2 (Skp2). Regulation of Skp2 is in part attributed to the nuclear presence of the scaffold protein pro–IL-16. T cells isolated from 11 patients with advanced CTCL, but not those from healthy controls or patients with T cell acute lymphocytic leukemia (T-ALL), demonstrated reduction in nuclear pro–IL-16 levels. Sequence analysis identified the presence of mutations in the 5ι end of the PDZ1 region of pro–IL-16, a domain required for association of pro–IL-16 with the nuclear chaperone HSC70 (also known as HSPA8). HSC70 knockdown led to loss of nuclear translocation by pro–IL-16 and subsequent increases in Skp2 levels and decreases in p27Kip1 levels, which ultimately enhanced T cell proliferation. Thus, our data indicate that advanced CTCL cell growth is facilitated, at least in part, by mutations in the scaffold protein pro–IL-16, which directly regulates Skp2 synthesis.
Clara Curiel-Lewandrowski, Hisato Yamasaki, Chuan Ping Si, Xiaoyi Jin, Yujun Zhang, Jillian Richmond, Marina Tuzova, Kevin Wilson, Beth Sullivan, David Jones, Nataliya Ryzhenko, Frederick Little, Thomas S. Kupper, David M. Center, William W. Cruikshank
Erythropoiesis, the production of red blood cells, must be tightly controlled to ensure adequate oxygen delivery to tissues without causing thrombosis or stroke. Control of physiologic and pathologic erythropoiesis is dependent predominantly on erythropoietin (EPO), the expression of which is regulated by hypoxia-inducible factor (HIF) activity in response to low oxygen tension. Accumulating evidence indicates that oxygen-independent mediators, including inflammatory stimuli, cytokines, and growth factors, also upregulate HIF activity, but it is unclear whether these signals also result in EPO production and erythropoiesis in vivo. Here, we found that signaling through herpesvirus entry mediator (HVEM), a molecule of the TNF receptor superfamily, promoted HIF-1α activity in the kidney and subsequently facilitated renal Epo production and erythropoiesis in vivo under normoxic conditions. This Epo upregulation was mediated by increased production of NO by renal macrophages. Hvem-deficient mice displayed impaired Epo expression and aggravated anemia in response to erythropoietic stress. These data reveal that HVEM signaling functions to promote HIF-1α activity and Epo production, and thus to regulate erythropoiesis. Furthermore, our findings suggest that this molecular mechanism could represent a therapeutic target for Epo-responsive diseases, including anemia.
Yukimi Sakoda, Sudarshan Anand, Yuming Zhao, Jang-June Park, Yingjia Liu, Atsuo Kuramasu, Nico van Rooijen, Ling Chen, Scott E. Strome, Wayne W. Hancock, Lieping Chen, Koji Tamada