Hematology
Abstract
Individuals with beta-thalassemia or Sickle Cell Disease and hereditary persistence of fetal hemoglobin (HPFH) possessing 30% HbF appear to be symptom-free. Here, we used a non-integrating HDAd5/35++ vector expressing a highly efficient and accurate version of an adenine base editor (ABE8e) to install, in vivo, a -113A>G HPFH mutation in the gamma-globin promoters in “healthy” CD46/β-YAC mice carrying the human β-globin locus. Our in vivo hematopoietic stem cell (HSC) editing/selection strategy involves only subcutaneous and intravenous injections and does not require myeloablation and HSC transplantation. In vivo HSC base editing in CD46/β-YAC mice resulted in >60% -113A>G conversion with 30% γ-globin of human beta globin expressed in 70% of erythrocytes. Importantly, no off-target editing at sites predicted by CIRCLE-Seq or in silico was detected. Furthermore, no critical alterations in the transcriptome of in vivo edited mice were found by RNA-seq. In vitro, in HSCs from beta-thalassemia and Sickle Cell Disease patients, transduction with the base editor vector mediated efficient -113 A>G conversion and reactivation of γ-globin expression with subsequent phenotypic correction of erythroid cells. Because our in vivo base editing strategy is safe and technically simple, it has the potential for clinical application in developing countries where hemoglobinopathies are prevalent.
Authors
Chang Li, Aphrodite Georgakopoulou, Gregory A. Newby, Kelcee A. Everette, Evangelos Nizamis, Kiriaki Paschoudi, Efthymia Vlachaki, Sucheol Gil, Anna K. Anderson, Theodore Koob, Lishan Huang, Hongjie Wang, Hans-Peter Kiem, David R. Liu, Evangelia Yannaki, André Lieber
Abstract
Increased red cell distribution width (RDW), which measures erythrocyte volume (MCV) variability (anisocytosis), has been linked to early mortality in many diseases and in older adults through unknown mechanisms. Hypoxic stress has been proposed as a potential mechanism. However, experimental models to investigate the link between increased RDW and reduced survival are lacking. Here, we show that lifelong hypobaric hypoxia (~10% O2) increases erythrocyte numbers, hemoglobin and RDW, while reducing longevity in male mice. Compound heterozygous knockout (chKO) mutations in succinate dehydrogenase (Sdh; mitochondrial complex II) genes Sdhb, Sdhc and Sdhd reduce Sdh subunit protein levels, RDW, and increase healthy lifespan compared to wild-type (WT) mice in chronic hypoxia. RDW-SD, a direct measure of MCV variability, and the standard deviation of MCV (1SD-RDW) show the most statistically significant reductions in Sdh hKO mice. Tissue metabolomic profiling of 147 common metabolites shows the largest increase in succinate with elevated succinate to fumarate and succinate to oxoglutarate (2-ketoglutarate) ratios in Sdh hKO mice. These results demonstrate that mitochondrial complex II level is an underlying determinant of both RDW and healthy lifespan in hypoxia, and suggest that therapeutic targeting of Sdh might reduce high RDW-associated clinical mortality in hypoxic diseases.
Authors
Bora E. Baysal, Abdulrahman A. Alahmari, Tori C. Rodrick, Debra Tabaczynski, Leslie Curtin, Mukund Seshadri, Drew R. Jones, Sandra Sexton
Abstract
NLRP3 inflammasome and interferon stimulated gene (ISG) induction are key biological drivers of ineffective hematopoiesis and inflammation in Myelodysplastic Syndromes (MDS). Gene mutations involving messenger RNA splicing and epigenetic regulatory pathways induce inflammasome activation and myeloid lineage skewing in MDS through yet undefined mechanisms. Using immortalized murine hematopoietic stem and progenitor cells harboring these somatic gene mutations and primary MDS bone marrow specimens, we show accumulation of unresolved R-loops and micronuclei with concurrent activation of the cytosolic sensor, cGAS. cGAS-STING signaling caused interferon stimulated gene (ISG) induction, NLRP3 inflammasome activation, and maturation of the effector protease, caspase-1. Deregulation of RNA polymerase III drives cytosolic R-loop generation, which upon inhibition, extinguishes ISG and inflammasome response. Mechanistically, caspase-1 degrades the master erythroid transcription factor, GATA1, provoking anemia and myeloid lineage bias that is reversed by cGAS inhibition in vitro and in Tet2-/- hematopoietic stem and progenitor cell transplanted mice. Together, these data identity a novel mechanism by which functionally distinct mutations converge upon the cGAS-STING-NLRP3 axis in MDS directing ISG induction, pyroptosis and myeloid lineage skewing.
Authors
Amy F. McLemore, Hsin-An Hou, Benjamin S. Meyer, Nghi B. Lam, Grace A. Ward, Amy L. Aldrich, Matthew A. Rodrigues, Alexis Vedder, Ling Zhang, Eric Padron, Nicole D. Vincelette, David A. Sallman, Omar Abdel-Wahab, Alan F. List, Kathy L. McGraw
Abstract
Rearrangements that drive ectopic MEF2C expression have recurrently been found in human early thymocyte progenitor acute lymphoblastic leukemia (ETP-ALL) patients. Here we show high levels of MEF2C expression in ETP-ALL patients. Using both in vivo and in vitro models of ETP-ALL, we demonstrate that elevated MEF2C expression blocks NOTCH-induced T cell differentiation while promoting a B-lineage program. MEF2C activates a B cell transcriptional program in addition to RUNX1, GATA3 and LMO2, upregulates the IL7R and boosts cell survival by upregulation of BCL2. MEF2C and the Notch pathway therefore demarcate opposite regulators of B- or T-lineage choices, respectively. Enforced MEF2C expression in mouse or human progenitor cells effectively blocks early T cell differentiation and promotes the development of bi-phenotypic lymphoid tumors that co-express CD3 and CD19, resembling human mixed phenotype acute leukemia (MPAL). SIK inhibitors impair MEF2C activity and alleviate the T cell development block. Importantly, this sensitizes cells to prednisolone treatment. Therefore, SIK inhibiting compounds such as dasatinib are potentially a valuable addition to standard chemotherapy for human ETP-ALL.
Authors
Kirsten Canté-Barrett, Mariska T. Meijer, Valentina Cordo', Rico Hagelaar, Wentao Yang, Jiyang Yu, Willem K. Smits, Marloes E. Nulle, Joris P. Jansen, Rob Pieters, Jun J. Yang, Jody J. Haigh, Steven Goossens, Jules P.P. Meijerink
Abstract
Chronic lymphocytic leukemia (CLL) results from expansion of a CD5+ B-cell clone that requires interactions with other cell types, including T cells. Moreover, CLL patients have elevated circulating IL17A+ and IL17F+ CD4+ T cells (Th17s), with higher IL17A+Th17s correlating with better outcomes. We report that CLL Th17s express more miR155, a Th17 differentiation regulator, than control Th17s, despite naïve CD4+ T cell (TN) basal miR155 levels being similar in both. We also found that CLL cells directly regulate miR155 levels in TN, thereby affecting Th17 differentiation by documenting that: co-culturing TN with resting (Brest) or activated (Bact) CLL cells alters the magnitude and direction of T-cell miR155 levels; CLL Bact promote IL17A+ and IL17F+ T cell generation by a miR155-dependent mechanism, confirmed by miR155 inhibition; co-cultures of TN with CLL Bact lead to a linear correlation between the degree and direction of T-cell miR155 expression changes and IL17F production, but not IL17A; Bact-mediated changes in TN miR155 expression correlate with outcome, irrespective of IGHV mutation status, a strong prognostic indicator. Together, the results identify a previously unrecognized CLL Bact-dependent mechanism, upregulation of TN miR155 expression and subsequent enhancement of IL17F+ Th17 generation, that favors better clinical courses.
Authors
Byeongho Jung, Gerardo Ferrer, Pui Yan Chiu, Rukhsana Aslam, Anita Ng, Florencia Palacios, Michael Wysota, Martina Cardillo, Jonathan E. Kolitz, Steven L. Allen, Jacqueline C. Barrientos, Kanti R. Rai, Nicholas Chiorazzi, Barbara Sherry
Abstract
Long-term impairment in T cell mediated adaptive immunity is a major clinical obstacle following treatment of blood disorders with Hematopoietic Stem Cell Transplantation (HSCT). Though T cell development in the thymus has been extensively characterized, there are significant gaps in our understanding of pre-thymic processes which influence early T cell potential. We have uncovered a Notch-IL21 signaling axis in Bone Marrow (BM) Common Lymphoid Progenitor (CLP) cells. IL21r expression is driven by Notch activation in CLPs, and in vivo treatment with IL21 induces Notch-dependent CLP proliferation. Taking advantage of this novel signaling axis, we have generated T cell progenitors ex vivo which better repopulate the thymus and peripheral lymphoid organs of mice in an allogeneic transplant model. Importantly, Notch and IL21 activation is equally effective in the priming and expansion of human Cord Blood (CB) cells toward the T cell fate, confirming the translational potential of the combined treatment.
Authors
Kilian Sottoriva, Na Yoon Paik, Zachary White, Thilinie Bandara, Lijian Shao, Teruyuki Sano, Kostandin Pajcini
Abstract
Platelet homeostasis is dependent on a tight regulation of both platelet production and clearance. The small GTPase Rap1 mediates platelet adhesion and hemostatic plug formation. However, Rap1 signaling is also critical for platelet homeostasis as both Rap1 deficiency and uninhibited Rap1 signaling lead to marked thrombocytopenia in mice. Here we investigated the mechanism by which deficiency in Rasa3, a critical negative regulator of Rap1, causes macrothrombocytopenia in mice. Despite marked morphological and ultrastructural abnormalities, megakaryocytes in hypomorphic Rasa3hlb/hlb or Rasa3-/- mice demonstrated robust proplatelet formation in vivo, suggesting that defective thrombopoiesis is not the main cause of thrombocytopenia. Rather, we observed that Rasa3hlb/hlb platelets become trapped in the spleen marginal zone/red pulp interface, with evidence of platelet phagocytosis by macrophages. Clearance of mutant platelets was also observed in the liver, especially in splenectomized mice. Platelet count and platelet lifespan in Rasa3 mutant mice were restored by genetic or pharmacological approaches to inhibit the Rap1/Talin1/αIIbβ3 integrin axis. A similar pattern of splenic clearance was observed in mice injected with anti-αIIbβ3 but not anti-GPIbα platelet-depleting antibodies. In summary, we describe a novel, integrin-based mechanism of platelet clearance that could be critical for our understanding of select inherited and acquired thrombocytopenias.
Authors
Robert H Lee, Dorsaf Ghalloussi, Gabriel L. Harousseau, Joseph P. Kenny, Patrick A. Kramer, Fabienne Proamer, Bernhard Nieswandt, Matthew J. Flick, Christian Gachet, Caterina Casari, Anita Eckly, Wolfgang Bergmeier
Abstract
BACKGROUND. Gut decontamination (GD) can decrease the incidence and severity of acute graft-versus-host-disease (aGVHD) in murine models of allogeneic hematopoietic cell transplantation (HCT). In this pilot study, we examined the impact of GD on the gut microbiome composition and incidence of aGVHD in HCT patients. METHODS. We randomized 20 pediatric patients undergoing allogeneic HCT to receive (GD) or not receive (no-GD) oral vancomycin-polymyxin B from day -5 through neutrophil engraftment. We evaluated shotgun metagenomic sequencing of serial stool samples to compare the composition and diversity of the gut microbiome between study arms. We assessed clinical outcomes in the 2 arms and performed strain-specific analyses of pathogens that caused bloodstream infections (BSI). RESULTS. The two arms did not differ in the predefined primary outcome of Shannon diversity of the gut microbiome at two weeks post-HCT (Genus, p=0.8; Species, p=0.44) or aGVHD incidence (p=0.58). Immune reconstitution of T-cell and B-cell subsets was similar between groups. Five patients in the no-GD arm had eight BSI episodes vs one episode in the GD arm (p=0.09). The BSI-causing pathogens were traceable to the gut in seven of eight BSI episodes in the no-GD arm, including Staphylococcus species. CONCLUSIONS. While GD did not differentially impact Shannon diversity or clinical outcomes, our findings suggest that GD may protect against gut-derived BSI in HCT patients by decreasing the prevalence or abundance of gut pathogens. TRIAL REGISTRATION. ClinicalTrials.gov NCT02641236 FUNDING. NIH, Damon Runyon Cancer Research Foundation, V Foundation, Sloan Foundation, Emerson Collective, Stanford MCHRI.
Authors
Christopher J. Severyn, Benjamin A. Siranosian, Sandra Tian-Jiao Kong, Angel Moreno, Michelle M. Li, Nan Chen, Christine N. Duncan, Steven P. Margossian, Leslie E. Lehmann, Shan Sun, Tessa M. Andermann, Olga Birbrayer, Sophie Silverstein, Soomin Kim, Niaz Banaei, Jerome Ritz, Anthony A. Fodor, Wendy B. London, Ami S. Bhatt, Jennifer S. Whangbo
Abstract
Mosaic loss of chromosome Y (mLOY) in blood cells is one of the most frequent chromosome alterations in adult males. It is strongly associated with clonal hematopoiesis, hematopoietic malignancies, and other hematopoietic and nonhematopoietic diseases. However, whether there is a causal relationship between mLOY and human diseases is unknown. Here, we generated mLOY in murine hematopoietic stem and progenitor cells (HSPCs) with CRISPR/Cas9 genome editing. We found that mLOY led to dramatically increased DNA damage in HSPCs. Interestingly, HSPCs with mLOY displayed significantly enhanced reconstitution capacity and gave rise to clonal hematopoiesis in vivo. mLOY, which is associated with AML1-ETO translocation and p53 defects in patients with acute myeloid leukemia (AML), promoted AML in mice. Mechanistically, loss of KDM5D, a chromosome Y–specific histone 3 lysine 4 demethylase in both humans and mice, partially recapitulated mLOY in DNA damage and leukemogenesis. Thus, our study validates mLOY as a functional driver for clonal hematopoiesis and leukemogenesis.
Authors
Qi Zhang, Lei Zhao, Yi Yang, Shujun Li, Yu Liu, Chong Chen
Abstract
BACKGROUND. Red blood cell (RBC) transfusion effectiveness varies due to donor, component, and recipient factors. Prior studies identified characteristics associated with variation in hemoglobin increments following transfusion. We extended these observations, examining donor genetic and non-genetic factors affecting transfusion effectiveness. METHODS. This is a multicenter retrospective study of 46,705 patients, and 102,043 evaluable RBC transfusions from 2013-2016 across 12 hospitals. Transfusion effectiveness was defined as hemoglobin, bilirubin, or creatinine increments following single RBC unit transfusion. Models incorporated a subset of donors with data on single nucleotide polymorphisms associated with osmotic and oxidative hemolysis in vitro. Mixed modelling accounting for repeated transfusion episodes identified predictors of transfusion effectiveness. RESULTS. Blood donor (sex, Rh status, fingerstick hemoglobin, smoking), component (storage duration, gamma irradiation, leukoreduction, apheresis collection, storage solution), and recipient (sex, body mass index, race, age) characteristics were associated with hemoglobin and bilirubin but not creatinine increments following RBC transfusions. Increased storage duration was associated with increased bilirubin and decreased hemoglobin increments, suggestive of in vivo hemolysis following transfusion. Donor G6PD-deficiency and polymorphisms in SEC14L4, HBA2, and MYO9B genes were associated with decreased hemoglobin increments. Donor G6PD-deficiency and polymorphisms in SEC14L4 were associated with increased transfusion requirements in the subsequent 48 hours. CONCLUSIONS. Donor genetic and other factors, such as RBC storage duration, affect transfusion effectiveness as defined by decreased hemoglobin or increased bilirubin increments. Addressing these factors will provide a precision medicine approach to improve patient outcomes, particularly for chronically-transfused RBC recipients, who would most benefit from more effective transfusion products.
Authors
Nareg H. Roubinian, Sarah E. Reese, Hannah Qiao, Colleen Plimier, Fang Fang, Grier P. Page, Ritchard G. Cable, Brian Custer, Mark T. Gladwin, Ruchika Goel, Bob Harris, Jeanne E. Hendrickson, Tamir Kanias, Steve Kleinman, Alan E. Mast, Steven R. Sloan, Bryan R. Spencer, Steven L. Spitalnik, Michael P. Busch, Eldad A. Hod
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