Hematopoietic transitions that accompany fetal development, such as erythroid globin chain switching, play important roles in normal physiology and disease development. In the megakaryocyte lineage, human fetal progenitors do not execute the adult morphogenesis program of enlargement, polyploidization, and proplatelet formation. Although these defects decline with gestational stage, they remain sufficiently severe at birth to predispose newborns to thrombocytopenia. These defects may also contribute to inferior platelet recovery after cord blood stem cell transplantation and may underlie inefficient platelet production by megakaryocytes derived from pluripotent stem cells. In this study, comparison of neonatal versus adult human progenitors has identified a blockade in the specialized positive transcription elongation factor b (P-TEFb) activation mechanism that is known to drive adult megakaryocyte morphogenesis. This blockade resulted from neonatal-specific expression of an oncofetal RNA-binding protein, IGF2BP3, which prevented the destabilization of the nuclear RNA 7SK, a process normally associated with adult megakaryocytic P-TEFb activation. Knockdown of IGF2BP3 sufficed to confer both phenotypic and molecular features of adult-type cells on neonatal megakaryocytes. Pharmacologic inhibition of IGF2BP3 expression via bromodomain and extraterminal domain (BET) inhibition also elicited adult features in neonatal megakaryocytes. These results identify IGF2BP3 as a human ontogenic master switch that restricts megakaryocyte development by modulating a lineage-specific P-TEFb activation mechanism, revealing potential strategies toward enhancing platelet production.
Kamaleldin E. Elagib, Chih-Huan Lu, Goar Mosoyan, Shadi Khalil, Ewelina Zasadzińska, Daniel R. Foltz, Peter Balogh, Alejandro A. Gru, Deborah A. Fuchs, Lisa M. Rimsza, Els Verhoeyen, Miriam Sansó, Robert P. Fisher, Camelia Iancu-Rubin, Adam N. Goldfarb
Quiescent and proliferating leukemia cells accumulate highly lethal DNA double-strand breaks that are repaired by 2 major mechanisms: BRCA-dependent homologous recombination and DNA-dependent protein kinase–mediated (DNA-PK–mediated) nonhomologous end-joining, whereas DNA repair pathways mediated by poly(ADP)ribose polymerase 1 (PARP1) serve as backups. Here we have designed a personalized medicine approach called gene expression and mutation analysis (GEMA) to identify BRCA- and DNA-PK–deficient leukemias either directly, using reverse transcription-quantitative PCR, microarrays, and flow cytometry, or indirectly, by the presence of oncogenes such as BCR-ABL1. DNA-PK–deficient quiescent leukemia cells and BRCA/DNA-PK–deficient proliferating leukemia cells were sensitive to PARP1 inhibitors that were administered alone or in combination with current antileukemic drugs. In conclusion, GEMA-guided targeting of PARP1 resulted in dual cellular synthetic lethality in quiescent and proliferating immature leukemia cells, and is thus a potential approach to eradicate leukemia stem and progenitor cells that are responsible for initiation and manifestation of the disease. Further, an analysis of The Cancer Genome Atlas database indicated that this personalized medicine approach could also be applied to treat numerous solid tumors from individual patients.
Margaret Nieborowska-Skorska, Katherine Sullivan, Yashodhara Dasgupta, Paulina Podszywalow-Bartnicka, Grazyna Hoser, Silvia Maifrede, Esteban Martinez, Daniela Di Marcantonio, Elisabeth Bolton-Gillespie, Kimberly Cramer-Morales, Jaewong Lee, Min Li, Artur Slupianek, Daniel Gritsyuk, Sabine Cerny-Reiterer, Ilona Seferynska, Tomasz Stoklosa, Lars Bullinger, Huaqing Zhao, Vera Gorbunova, Katarzyna Piwocka, Peter Valent, Curt I. Civin, Markus Muschen, John E. Dick, Jean C.Y. Wang, Smita Bhatia, Ravi Bhatia, Kolia Eppert, Mark D. Minden, Stephen M. Sykes, Tomasz Skorski
Mutations of the splicing factor–encoding gene
Bon Ham Yip, Violetta Steeples, Emmanouela Repapi, Richard N. Armstrong, Miriam Llorian, Swagata Roy, Jacqueline Shaw, Hamid Dolatshad, Stephen Taylor, Amit Verma, Matthias Bartenstein, Paresh Vyas, Nicholas C.P. Cross, Luca Malcovati, Mario Cazzola, Eva Hellström-Lindberg, Seishi Ogawa, Christopher W.J. Smith, Andrea Pellagatti, Jacqueline Boultwood
The eleven-nineteen leukemia (ENL) protein family, composed of ENL and AF9, is a common component of 3 transcriptional modulators: AF4–ENL–P-TEFb complex (AEP), DOT1L-AF10-ENL complex (referred to as the DOT1L complex) and polycomb-repressive complex 1 (PRC1). Each complex associates with chromatin via distinct mechanisms, conferring different transcriptional properties including activation, maintenance, and repression. The mixed-lineage leukemia (
Hiroshi Okuda, Boban Stanojevic, Akinori Kanai, Takeshi Kawamura, Satoshi Takahashi, Hirotaka Matsui, Akifumi Takaori-Kondo, Akihiko Yokoyama
Developing erythrocytes take up exceptionally large amounts of iron, which must be transferred to mitochondria for incorporation into heme. This massive iron flux must be precisely controlled to permit the coordinated synthesis of heme and hemoglobin while avoiding the toxic effects of chemically reactive iron. In cultured animal cells, iron chaperones
Moon-Suhn Ryu, Deliang Zhang, Olga Protchenko, Minoo Shakoury-Elizeh, Caroline C. Philpott
The mTOR pathway is a critical determinant of cell persistence and growth wherein mTOR complex 1 (mTORC1) mediates a balance between growth factor stimuli and nutrient availability. Amino acids or glucose facilitates mTORC1 activation by inducing RagA GTPase recruitment of mTORC1 to the lysosomal outer surface, enabling activation of mTOR by the Ras homolog Rheb. Thereby, RagA alters mTORC1-driven growth in times of nutrient abundance or scarcity. Here, we have evaluated differential nutrient-sensing dependence through RagA and mTORC1 in hematopoietic progenitors, which dynamically drive mature cell production, and hematopoietic stem cells (HSC), which provide a quiescent cellular reserve. In nutrient-abundant conditions, RagA-deficient HSC were functionally unimpaired and upregulated mTORC1 via nutrient-insensitive mechanisms. RagA was also dispensable for HSC function under nutritional stress conditions. Similarly, hyperactivation of RagA did not affect HSC function. In contrast, RagA deficiency markedly altered progenitor population function and mature cell output. Therefore, RagA is a molecular mechanism that distinguishes the functional attributes of reactive progenitors from a reserve stem cell pool. The indifference of HSC to nutrient sensing through RagA contributes to their molecular resilience to nutritional stress, a characteristic that is relevant to organismal viability in evolution and in modern HSC transplantation approaches.
Demetrios Kalaitzidis, Dongjun Lee, Alejo Efeyan, Youmna Kfoury, Naema Nayyar, David B. Sykes, Francois E. Mercier, Ani Papazian, Ninib Baryawno, Gabriel D. Victora, Donna Neuberg, David M. Sabatini, David T. Scadden
Alloimmune T cell responses induce graft-versus-host disease (GVHD), a serious complication of allogeneic bone marrow transplantation (allo-BMT). Although Notch signaling mediated by Delta-like 1/4 (DLL1/4) Notch ligands has emerged as a major regulator of GVHD pathogenesis, little is known about the timing of essential Notch signals and the cellular source of Notch ligands after allo-BMT. Here, we have shown that critical DLL1/4-mediated Notch signals are delivered to donor T cells during a short 48-hour window after transplantation in a mouse allo-BMT model. Stromal, but not hematopoietic, cells were the essential source of Notch ligands during in vivo priming of alloreactive T cells. GVHD could be prevented by selective inactivation of
Jooho Chung, Christen L. Ebens, Eric Perkey, Vedran Radojcic, Ute Koch, Leonardo Scarpellino, Alexander Tong, Frederick Allen, Sherri Wood, Jiane Feng, Ann Friedman, David Granadier, Ivy T. Tran, Qian Chai, Lucas Onder, Minhong Yan, Pavan Reddy, Bruce R. Blazar, Alex Y. Huang, Todd V. Brennan, D. Keith Bishop, Burkhard Ludewig, Christian W. Siebel, Freddy Radtke, Sanjiv A. Luther, Ivan Maillard
Primary myelofibrosis (PMF) is a clonal hematologic malignancy characterized by BM fibrosis, extramedullary hematopoiesis, circulating CD34+ cells, splenomegaly, and a propensity to evolve to acute myeloid leukemia. Moreover, the spleen and BM of patients harbor atypical, clustered megakaryocytes, which contribute to the disease by secreting profibrotic cytokines. Here, we have revealed that megakaryocytes in PMF show impaired maturation that is associated with reduced GATA1 protein. In investigating the cause of GATA1 downregulation, our gene-expression study revealed the presence of the
Laure Gilles, Ahmet Dirim Arslan, Christian Marinaccio, Qiang Jeremy Wen, Priyanka Arya, Maureen McNulty, Qiong Yang, Jonathan C. Zhao, Katerina Konstantinoff, Terra Lasho, Animesh Pardanani, Brady Stein, Isabelle Plo, Sriram Sundaravel, Amittha Wickrema, Annarita Migliaccio, Sandeep Gurbuxani, William Vainchenker, Leonidas C. Platanias, Ayalew Tefferi, John D. Crispino
Heparin-induced thrombocytopenia (HIT) is a prothrombotic disorder initiated by antibodies against complexes between human platelet factor 4 (hPF4) and heparin. A better understanding of the events that initiate the prothrombotic state may improve approaches to antithrombotic management. Here, we visualized thrombus formation in an in vivo murine model and an endothelialized microfluidic system that simulate the pathogenesis of HIT. hPF4 released from platelets predominantly bound to peri-injury endothelium and formed HIT antigenic complexes that were dissociated by heparin. In mice expressing both hPF4+ and human platelet IgG Fc receptor IIA (FcγRIIA), infusion of the HIT-like monoclonal antibody KKO increased fibrin and platelet deposition at sites of injury, followed immediately by antigen formation on proximate endothelial cells. After a few minutes, HIT antigen was detected within the thrombus itself at the interface between the platelet core and the surrounding shell. We observed similar results in the humanized, endothelialized microfluidic system. hPF4 and KKO selectively bound to photochemically injured endothelium at sites where surface glycocalyx was reduced. These studies support the concept that the perithrombus endothelium is the predominant site of HIT antigen assembly. This suggests that disrupting antigen formation along the endothelium or protecting the endothelium may provide a therapeutic opportunity to prevent thrombotic complications of HIT, while sparing systemic hemostatic pathways.
Vincent Hayes, Ian Johnston, Gowthami M. Arepally, Steven E. McKenzie, Douglas B. Cines, Lubica Rauova, Mortimer Poncz
Platelets are anuclear cells that are essential for blood clotting. They are produced by large polyploid precursor cells called megakaryocytes. Previous genome-wide association studies in nearly 70,000 individuals indicated that single nucleotide variants (SNVs) in the gene encoding the actin cytoskeletal regulator tropomyosin 4 (TPM4) exert an effect on the count and volume of platelets. Platelet number and volume are independent risk factors for heart attack and stroke. Here, we have identified 2 unrelated families in the BRIDGE Bleeding and Platelet Disorders (BPD) collection who carry a
Irina Pleines, Joanne Woods, Stephane Chappaz, Verity Kew, Nicola Foad, José Ballester-Beltrán, Katja Aurbach, Chiara Lincetto, Rachael M. Lane, Galina Schevzov, Warren S. Alexander, Douglas J. Hilton, William J. Astle, Kate Downes, Paquita Nurden, Sarah K. Westbury, Andrew D. Mumford, Samya G. Obaji, Peter W. Collins, NIHR BioResource, Fabien Delerue, Lars M. Ittner, Nicole S. Bryce, Mira Holliday, Christine A. Lucas, Edna C. Hardeman, Willem H. Ouwehand, Peter W. Gunning, Ernest Turro, Marloes R. Tijssen, Benjamin T. Kile