HBS1L-MYB intergenic variants modulate fetal hemoglobin via long-range MYB enhancers

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Abstract

Genetic studies have identified common variants within the intergenic region (HBS1L-MYB) between GTP-binding elongation factor HBS1L and myeloblastosis oncogene MYB on chromosome 6q that are associated with elevated fetal hemoglobin (HbF) levels and alterations of other clinically important human erythroid traits. It is unclear how these noncoding sequence variants affect multiple erythrocyte characteristics. Here, we determined that several HBS1L-MYB intergenic variants affect regulatory elements that are occupied by key erythroid transcription factors within this region. These elements interact with MYB, a critical regulator of erythroid development and HbF levels. We found that several HBS1L-MYB intergenic variants reduce transcription factor binding, affecting long-range interactions with MYB and MYB expression levels. These data provide a functional explanation for the genetic association of HBS1L-MYB intergenic polymorphisms with human erythroid traits and HbF levels. Our results further designate MYB as a target for therapeutic induction of HbF to ameliorate sickle cell and β-thalassemia disease severity.

Authors

Ralph Stadhouders, Suleyman Aktuna, Supat Thongjuea, Ali Aghajanirefah, Farzin Pourfarzad, Wilfred van IJcken, Boris Lenhard, Helen Rooks, Steve Best, Stephan Menzel, Frank Grosveld, Swee Lay Thein, Eric Soler

Thrombocytopenia-associated mutations in the ANKRD26 regulatory region induce MAPK hyperactivation

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Abstract

Point mutations in the 5′ UTR of ankyrin repeat domain 26 (ANKRD26) are associated with familial thrombocytopenia 2 (THC2) and a predisposition to leukemia. Here, we identified underlying mechanisms of ANKRD26-associated thrombocytopenia. Using megakaryocytes (MK) isolated from THC2 patients and healthy subjects, we demonstrated that THC2-associated mutations in the 5′ UTR of ANKRD26 resulted in loss of runt-related transcription factor 1 (RUNX1) and friend leukemia integration 1 transcription factor (FLI1) binding. RUNX1 and FLI1 binding at the 5′ UTR from healthy subjects led to ANKRD26 silencing during the late stages of megakaryopoiesis and blood platelet development. We showed that persistent ANKRD26 expression in isolated MKs increased signaling via the thrombopoietin/myeloproliferative leukemia virus oncogene (MPL) pathway and impaired proplatelet formation by MKs. Importantly, we demonstrated that ERK inhibition completely rescued the in vitro proplatelet formation defect. Our data identify a mechanism for development of the familial thrombocytopenia THC2 that is related to abnormal MAPK signaling.

Authors

Dominique Bluteau, Alessandra Balduini, Nathalie Balayn, Manuela Currao, Paquita Nurden, Caroline Deswarte, Guy Leverger, Patrizia Noris, Silverio Perrotta, Eric Solary, William Vainchenker, Najet Debili, Remi Favier, Hana Raslova

Hematopoietic stem cells are acutely sensitive to Acd shelterin gene inactivation

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Abstract

The shelterin complex plays dual functions in telomere homeostasis by recruiting telomerase and preventing the activation of a DNA damage response at telomeric ends. Somatic stem cells require telomerase activity, as evidenced by progressive stem cell loss leading to bone marrow failure in hereditary dyskeratosis congenita. Recent work demonstrates that dyskeratosis congenita can also arise from mutations in specific shelterin genes, although little is known about shelterin functions in somatic stem cells. We found that mouse hematopoietic stem cells (HSCs) are acutely sensitive to inactivation of the shelterin gene Acd, encoding TPP1. Homozygosity for a hypomorphic acd allele preserved the emergence and expansion of fetal HSCs but led to profoundly defective function in transplantation assays. Upon complete Acd inactivation, HSCs expressed p53 target genes, underwent cell cycle arrest, and were severely depleted within days, leading to hematopoietic failure. TPP1 loss induced increased telomeric fusion events in bone marrow progenitors. However, unlike in epidermal stem cells, p53 deficiency did not rescue TPP1-deficient HSCs, indicating that shelterin dysfunction has unique effects in different stem cell populations. Because the consequences of telomere shortening are progressive and unsynchronized, acute loss of shelterin function represents an attractive alternative for studying telomere crisis in hematopoietic progenitors.

Authors

Morgan Jones, Gail Osawa, Joshua A. Regal, Daniel N. Weinberg, James Taggart, Hande Kocak, Ann Friedman, David O. Ferguson, Catherine E. Keegan, Ivan Maillard

Platelets mediate lymphovenous hemostasis to maintain blood-lymphatic separation throughout life

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Abstract

Mammals transport blood through a high-pressure, closed vascular network and lymph through a low-pressure, open vascular network. These vascular networks connect at the lymphovenous (LV) junction, where lymph drains into blood and an LV valve (LVV) prevents backflow of blood into lymphatic vessels. Here we describe an essential role for platelets in preventing blood from entering the lymphatic system at the LV junction. Loss of CLEC2, a receptor that activates platelets in response to lymphatic endothelial cells, resulted in backfilling of the lymphatic network with blood from the thoracic duct (TD) in both neonatal and mature mice. Fibrin-containing platelet thrombi were observed at the LVV and in the terminal TD in wild-type mice, but not Clec2-deficient mice. Analysis of mice lacking LVVs or lymphatic valves revealed that platelet-mediated thrombus formation limits LV backflow under conditions of impaired valve function. Examination of mice lacking integrin-mediated platelet aggregation indicated that platelet aggregation stabilizes thrombi that form in the lymphatic vascular environment to prevent retrograde blood flow. Collectively, these studies unveil a newly recognized form of hemostasis that functions with the LVV to safeguard the lymphatic vascular network throughout life.

Authors

Paul R. Hess, David R. Rawnsley, Zoltán Jakus, Yiqing Yang, Daniel T. Sweet, Jianxin Fu, Brett Herzog, MinMin Lu, Bernhard Nieswandt, Guillermo Oliver, Taija Makinen, Lijun Xia, Mark L. Kahn

Induction of myelodysplasia by myeloid-derived suppressor cells

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Abstract

Myelodysplastic syndromes (MDS) are age-dependent stem cell malignancies that share biological features of activated adaptive immune response and ineffective hematopoiesis. Here we report that myeloid-derived suppressor cells (MDSC), which are classically linked to immunosuppression, inflammation, and cancer, were markedly expanded in the bone marrow of MDS patients and played a pathogenetic role in the development of ineffective hematopoiesis. These clonally distinct MDSC overproduce hematopoietic suppressive cytokines and function as potent apoptotic effectors targeting autologous hematopoietic progenitors. Using multiple transfected cell models, we found that MDSC expansion is driven by the interaction of the proinflammatory molecule S100A9 with CD33. These 2 proteins formed a functional ligand/receptor pair that recruited components to CD33’s immunoreceptor tyrosine-based inhibition motif (ITIM), inducing secretion of the suppressive cytokines IL-10 and TGF-β by immature myeloid cells. S100A9 transgenic mice displayed bone marrow accumulation of MDSC accompanied by development of progressive multilineage cytopenias and cytological dysplasia. Importantly, early forced maturation of MDSC by either all-trans-retinoic acid treatment or active immunoreceptor tyrosine-based activation motif–bearing (ITAM-bearing) adapter protein (DAP12) interruption of CD33 signaling rescued the hematologic phenotype. These findings indicate that primary bone marrow expansion of MDSC driven by the S100A9/CD33 pathway perturbs hematopoiesis and contributes to the development of MDS.

Authors

Xianghong Chen, Erika A. Eksioglu, Junmin Zhou, Ling Zhang, Julie Djeu, Nicole Fortenbery, Pearlie Epling-Burnette, Sandra Van Bijnen, Harry Dolstra, John Cannon, Je-in Youn, Sarah S. Donatelli, Dahui Qin, Theo De Witte, Jianguo Tao, Huaquan Wang, Pingyan Cheng, Dmitry I. Gabrilovich, Alan List, Sheng Wei

Myelodysplastic syndromes are induced by histone methylation–altering ASXL1 mutations

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Abstract

Recurrent mutations in the gene encoding additional sex combs-like 1 (ASXL1) are found in various hematologic malignancies and associated with poor prognosis. In particular, ASXL1 mutations are common in patients with hematologic malignancies associated with myelodysplasia, including myelodysplastic syndromes (MDSs), and chronic myelomonocytic leukemia. Although loss-of-function ASXL1 mutations promote myeloid transformation, a large subset of ASXL1 mutations is thought to result in stable truncation of ASXL1. Here we demonstrate that C-terminal–truncating Asxl1 mutations (ASXL1-MTs) inhibited myeloid differentiation and induced MDS-like disease in mice. ASXL1-MT mice displayed features of human-associated MDS, including multi-lineage myelodysplasia, pancytopenia, and occasional progression to overt leukemia. ASXL1-MT resulted in derepression of homeobox A9 (Hoxa9) and microRNA-125a (miR-125a) expression through inhibition of polycomb repressive complex 2–mediated (PRC2-mediated) methylation of histone H3K27. miR-125a reduced expression of C-type lectin domain family 5, member a (Clec5a), which is involved in myeloid differentiation. In addition, HOXA9 expression was high in MDS patients with ASXL1-MT, while CLEC5A expression was generally low. Thus, ASXL1-MT–induced MDS-like disease in mice is associated with derepression of Hoxa9 and miR-125a and with Clec5a dysregulation. Our data provide evidence for an axis of MDS pathogenesis that implicates both ASXL1 mutations and miR-125a as therapeutic targets in MDS.

Authors

Daichi Inoue, Jiro Kitaura, Katsuhiro Togami, Koutarou Nishimura, Yutaka Enomoto, Tomoyuki Uchida, Yuki Kagiyama, Kimihito Cojin Kawabata, Fumio Nakahara, Kumi Izawa, Toshihiko Oki, Akie Maehara, Masamichi Isobe, Akiho Tsuchiya, Yuka Harada, Hironori Harada, Takahiro Ochiya, Hiroyuki Aburatani, Hiroshi Kimura, Felicitas Thol, Michael Heuser, Ross L. Levine, Omar Abdel-Wahab, Toshio Kitamura

Extracellular hemin crisis triggers acute chest syndrome in sickle mice

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Abstract

The prevention and treatment of acute chest syndrome (ACS) is a major clinical concern in sickle cell disease (SCD). However, the mechanism underlying the pathogenesis of ACS remains elusive. We tested the hypothesis that the hemolysis byproduct hemin elicits events that induce ACS. Infusion of a low dose of hemin caused acute intravascular hemolysis and autoamplification of extracellular hemin in transgenic sickle mice, but not in sickle-trait littermates. The sickle mice developed multiple symptoms typical of ACS and succumbed rapidly. Pharmacologic inhibition of TLR4 and hemopexin replacement therapy prior to hemin infusion protected sickle mice from developing ACS. Replication of the ACS-like phenotype in nonsickle mice revealed that the mechanism of lung injury due to extracellular hemin is independent of SCD. Using genetic and bone marrow chimeric tools, we confirmed that TLR4 expressed in nonhematopoietic vascular tissues mediated this lethal type of acute lung injury. Respiratory failure was averted after the onset of ACS-like symptoms in sickle mice by treating them with recombinant hemopexin. Our results reveal a mechanism that helps to explain the pathogenesis of ACS, and we provide proof of principle for therapeutic strategies to prevent and treat this condition in mice.

Authors

Samit Ghosh, Olufolake Adetoro Adisa, Prasanthi Chappa, Fang Tan, Kesmic Ann Jackson, David Robert Archer, Solomon Fiifi Ofori-Acquah

Fanconi anemia signaling network regulates the spindle assembly checkpoint

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Abstract

Fanconi anemia (FA) is a heterogenous genetic disease with a high risk of cancer. The FA proteins are essential for interphase DNA damage repair; however, it is incompletely understood why FA-deficient cells also develop gross aneuploidy, leading to cancer. Here, we systematically evaluated the role of the FA proteins in chromosome segregation through functional RNAi screens and analysis of primary cells from patients with FA. We found that FA signaling is essential for the spindle assembly checkpoint and is therefore required for high-fidelity chromosome segregation and prevention of aneuploidy. Furthermore, we discovered that FA proteins differentially localize to key structures of the mitotic apparatus in a cell cycle–dependent manner. The essential role of the FA pathway in mitosis offers a mechanistic explanation for the aneuploidy and malignant transformation known to occur after disruption of FA signaling. Collectively, our findings provide insight into the genetically unstable cancers resulting from inactivation of the FA/BRCA pathway.

Authors

Grzegorz Nalepa, Rikki Enzor, Zejin Sun, Christophe Marchal, Su-Jung Park, Yanzhu Yang, Laura Tedeschi, Stephanie Kelich, Helmut Hanenberg, D. Wade Clapp

Differentiation and functional regulation of human fetal NK cells

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Abstract

The human fetal immune system is naturally exposed to maternal allogeneic cells, maternal antibodies, and pathogens. As such, it is faced with a considerable challenge with respect to the balance between immune reactivity and tolerance. Here, we show that fetal natural killer (NK) cells differentiate early in utero and are highly responsive to cytokines and antibody-mediated stimulation but respond poorly to HLA class I–negative target cells. Strikingly, expression of killer-cell immunoglobulin-like receptors (KIRs) did not educate fetal NK cells but rendered them hyporesponsive to target cells lacking HLA class I. In addition, fetal NK cells were highly susceptible to TGF-β–mediated suppression, and blocking of TGF-β signaling enhanced fetal NK cell responses to target cells. Our data demonstrate that KIR-mediated hyporesponsiveness and TGF-β–mediated suppression are major factors determining human fetal NK cell hyporesponsiveness to HLA class I–negative target cells and provide a potential mechanism for fetal-maternal tolerance in utero. Finally, our results provide a basis for understanding the role of fetal NK cells in pregnancy complications in which NK cells could be involved, for example, during in utero infections and anti-RhD–induced fetal anemia.

Authors

Martin A. Ivarsson, Liyen Loh, Nicole Marquardt, Eliisa Kekäläinen, Lena Berglin, Niklas K. Björkström, Magnus Westgren, Douglas F. Nixon, Jakob Michaëlsson

Increased Fanconi C expression contributes to the emergency granulopoiesis response

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Abstract

Emergency granulopoiesis is a component of the innate immune response that is induced in response to infectious or inflammatory challenge. It is characterized by the rapid expansion and differentiation of granulocyte/monocyte progenitor (GMP) populations, which is due in part to a shortened S-phase of the cell cycle. We found that IRF8 (also known as ICSBP), an interferon regulatory transcription factor that activates phagocyte effector genes during the innate immune response, activates the gene encoding Fanconi C (Fancc) in murine myeloid progenitor cells. Moreover, IRF8-induced Fancc transcription was augmented by treatment with IL-1β, an essential cytokine for emergency granulopoiesis. The Fanconi pathway participates in repair of stalled or collapsed replication forks during DNA replication, leading us to hypothesize that the Fanconi pathway contributes to genomic stability during emergency granulopoiesis. In support of this hypothesis, Fancc^–/– mice developed anemia and neutropenia during repeated, failed episodes of emergency granulopoiesis. Failed emergency granulopoiesis in Fancc^–/– mice was associated with excess apoptosis of HSCs and progenitor cells in the bone marrow and impaired HSC function. These studies have implications for understanding the pathogenesis of bone marrow failure in Fanconi anemia and suggest possible therapeutic approaches.

Authors

Liping Hu, Weiqi Huang, Elizabeth Hjort, Elizabeth A. Eklund