Hematology
Abstract
We previously reported that excessive angiotensin-II (AT)->AT receptor-1 (ATR1) signaling results in sickle cell anemia (SCA)-associated nephropathy. Herein, we showed hyperangiotensinemia in SCA results from high erythroid cell-generated reactive oxygen species (ROS), which oxidized angiotensinogen (ATGN) and favored its rapid conversion to AT. Increased AT->ATR1 signaling in SCA erythroid cells generated ROS and created a positive feedback loop of ROS->oxidized ATGN->AT->ATR1-> ROS, perpetuating the hyperangiotensinemia. ATR1-blocker, losartan, reduced erythrocyte ROS, oxidized-AGTN, and AT levels. The ROS->AT->ATR1->ROS loop was driven by sickle erythropoiesis as it was reproduced when WT mice were transplanted with SCA hematopoiesis. Using SCA and WT mice with germline- and erythroid-specific ATR1-deficiency, we found that stress-erythropoiesis, but not steady-state-erythropoiesis, was critically dependent on erythroid AT->ATR1 signaling, which acted in harmony with increased erythropoietin signaling. Further, instead of the canonical AT->ATR1-> NADPH-oxidase->ROS signaling in steady-state erythropoiesis, AT->ATR1 signaling in stress-erythroid cells increased mitochondrial mass and dysfunctional mitochondria, which thereby increased ROS. SCA mice with erythroid-specific ATR1 deficiency had decreased RBC accumulation of dysfunctional mitochondria and decreased ROS, which reduced SCA-associated nephropathy. Overall, we demonstrated that AT->ATR1 signaling was essential for stress-erythropoiesis but led to increased dysfunctional mitochondria retention in mature RBCs, which generated ROS and perpetuated hyperangiotensinemia, resulting in end-organ damage.
Authors
Parul Rai, Swarnava Roy, Paritha Arumugam, Diamantis G. Konstantinidis, Sithara Raju Ponny, Marthe-Sandrine Eiymo Mwa Mpollo, Archana Shrestha, Theodosia A. Kalfa, Punam Malik
Abstract
Vaso-occlusive episodes (VOEs) or acute pain events, involving complex interactions between sickle erythrocytes and other blood cells, are a hallmark of sickle cell disease (SCD). In this study, we analyzed changes in peripheral blood transcriptomes between steady state and VOEs in individuals with SCD. We followed a cohort of 174 individuals with SCD with or without chronic pain and collected peripheral blood at clinic visits (steady state) and during hospitalizations (VOEs). We performed RNA-Seq profiling of CD45+ leukocytes and CD71+ erythroid cells. Pathways linked to complement activation, coagulation, and IL-6/JAK/STAT3 signaling were enriched during VOEs in the CD45+ cells. Contrastingly, the CD71+ cells showed an enrichment of pathways related to the cell cycle, such as mTORC1 signaling and the G2M checkpoint during VOEs. We then analyzed the expression changes of genes in patients with longitudinal data to determine potential biomarkers for VOEs. Expression of 4 genes — FAM20A, IL1B, MS4A4A, and SERPINB2 — was elevated during VOEs compared with steady state in the majority of patients. Furthermore, our results indicate that patients experiencing chronic pain exhibited 44% increased enrichment of significant pathways during VOEs when compared with patients without chronic pain.
Authors
Varsha Bhat, Justin J. Yoo, Srija Ponna, Alka A. Potdar, Ashwin P. Patel, G. Karen Yu, Greg Gibson, Vivien A. Sheehan
Abstract
Fanconi anemia (FA) is the most common bone marrow failure (BMF) syndrome. Beyond a role in DNA repair, FA genes have a role in suppressing DNA-RNA hybrids, termed R-loops, which can be generated via RNA polymerase (RNAP)-mediated transcription. However, how these processes, including a role in fate determination of hematopoietic stem cells (HSCs), are related to BMF is largely unknown. Additionally, single FA gene knockouts in mice do not recapitulate most phenotypes observed in FA patients. Thus, we generated a mouse model for FA by introducing heterozygous Setd2, which restricts RNAP-dependent transcription. Here, we show that FA patient-derived cells and Setd2+/– Fanca–/– HSCs share increased R-loop as well as dsRNA levels, and a ribosomal biogenesis defect. Further, Setd2+/– Fanca–/– HSCs display cell cycle arrest, mitotic errors and BMF phenotypes. Importantly, utilizing our Setd2+/– Fanca–/– mice, we discovered that Juglone, a pan RNAP inhibitor, reduces R-loop and dsRNA and reverses ribosomal biogenesis defects and mitotic errors, thereby rescuing BMF. In conclusion, this study establishes a novel mouse model that underscores a key role for R-loop formation, ribosomal biogenesis defects and mitotic errors in HSCs in driving BMF in Fanconi anemia. We also introduce a potential therapeutic avenue based upon pan-inhibition of RNA polymerases utilizing Juglone.
Authors
Michihiro Hashimoto, Xiaomin Feng, Jie Bai, Huimin Zeng, Tian Li, Jue Li, Terumasa Umemoto, Paul R. Andreassen, Gang Huang
Abstract
Mutations in protein tyrosine phosphatase non-receptor type 11 (PTPN11) have been considered late acquired mutations in acute myeloid leukemia (AML) development. Using single-cell DNA sequencing, we found that PTPN11 mutations can occur as initiating events in some patients with AML when accompanied by strong oncogenic drivers, commonly NPM1 mutations. The resulting AML has a diverse set of variably differentiated myeloid cells with few myeloid cells that lack leukemic mutations. The role of Ptpn11 as a codriver was confirmed in a murine model that exhibits an AML phenotype with a comparable immune diversity that is serially engraftable and reconstituted from early precursor cells. Furthermore, lineage-negative bone marrow cells from these mice reconstitute the full diversity of mature myeloid cells, and these cells exhibit an altered cytokine response after physiologic stimulation. Our work highlights how PTPN11-mutated AML is derived from a multitude of codominant and late acquired aberrations that have a previously unrecognized differentiated myeloid clonal expansion potentially contributing to pathogenesis of the disease.
Authors
Sydney Fobare, Chia Sharpe, Kate Quinn, Kinsey Bryant, Linde A. Miles, Robert L. Bowman, Carolyn Cheney, Casie Furby, Marissa Long, Kaytlynn Fyock, Ben Wronowski, James R. Lerma, Krzysztof Mrózek, Deedra Nicolet, Thomas M. Sesterhenn, Megan E. Johnstone, Jianmin Pan, Shesh N. Rai, Chandrashekhar Pasare, Nives Zimmermann, Wen-Mei Yu, Cheng-Kui Qu, Andrew Carroll, Richard Stone, Eunice S. Wang, Jonathan Kolitz, Bayard Powell, John P. Perentesis, Ann-Kathrin Eisfeld, Erin Hertlein, John C. Byrd
Abstract
Systemic Epstein-Barr virus–positive (EBV-positive) T/NK cell lymphoproliferative diseases of childhood (sEBV+T/NK-LPD) are a spectrum of rare diseases that have highly variable biological behavior, from indolent conditions to highly aggressive malignancies. Clinicians currently face substantial challenges in promptly assessing disease severity and predicting patient outcomes, leading to limitations in treatment planning. To address this challenge, we constructed a comprehensive triage system to aid in rapid clinical interventions. The study included 156 patients with newly diagnosed sEBV+T/NK-LPD from 42 institutions. An independent prospective cohort of 35 newly enrolled patients was further included to evaluate the model’s performance. An additional 45 patients from the literature and 18 patients who underwent hematopoietic stem cell transplantation were included to test the score’s generalizability. An integrative machine learning strategy was applied to identify robust and optimal factors and to integrate multiple algorithms to enhance the system’s performance and stability. This system, termed COLLAPSED, identifies critical factors and provides a stable, high-performing ensemble. This model was validated externally and simplified into a risk score to improve interpretability and accessibility. The COLLAPSED system substantially enhances clinicians’ ability to rapidly and precisely identify high-risk patients, thus enabling timely clinical decision-making and expedited initiation of potentially lifesaving treatments.
Authors
Pujun Guan, Zihang Chen, Hanze Dong, Xia Guo, Juan Huang, Tian Dong, Mi Wang, Xiaoxi Lu, Fei Huang, Wenbin Li, Yuan Tang, Li Zhang, Ling Pan, Ju Gao, Shikun Wang, Rongbo Liu, Wenyan Zhang, Sha Zhao, Weiping Liu
Abstract
The contribution of 9p deletion to B-cell acute lymphoblastic leukemia (B-ALL) has remained elusive since its discovery more than 40 years ago. Here we show that loss of CD72 is recurrent in B-ALL cases containing PAX5 deletions, and that Cd72 haploinsufficiency drives B-ALL development in Pax5+/− mice. Mechanistically, Cd72+/-;Pax5+/- precursor B cells exhibit an inflammatory transcriptional profile characterized by a decrease in Myd88 expression, a finding that aligns with our previous studies of B-ALL development in Pax5+/- mice following exposure to immune stressors. These combined genomic analyses and functional models provide compelling evidence that co-deletion of two contiguous genes, Pax5 and Cd72, drives B-cell leukemogenesis.
Authors
Belén Ruiz-Corzo, Ana Casado-García, Ninad Oak, Paula Somoza-Cotillas, Andrea López-Álvarez de Neyra, Jorge Martínez-Cano, Alba Pérez-Pons, Elena G. Sánchez, Oscar Blanco, Diego Alonso-López, Javier De Las Rivas, Susana Riesco, Pablo Prieto-Matos, Francisco Javier Garcia-Criado, Maria Begoña Garcia-Cenador, Alberto Orfao, Manuel Ramírez-Orellana, César Cobaleda, Carolina Vicente-Dueñas, Kim E. Nichols, Isidro Sánchez-García
Abstract
We hypothesized that performing bone marrow transplant (BMT) using marrow extracted from the vertebral bodies (VB) of an unrelated deceased lung transplant (LTX) donor would be able to establish persistent hematopoiesis, generate immunity, and tolerance. A teenager with severe combined immunodeficiency with lung failure due to recurrent pneumonias underwent LTX in 2016 from a 1/8 HLA allele-matched unrelated donor, followed by BMT 4 months later using T-cell/B-cell-depleted, cryopreserved VB marrow. Rapid engraftment was followed by accelerating immune competence at 6 months, with independence from immunosuppression by 16 months. Donor T-cell (>95%) and myeloid chimerism (7-10%) have persisted for over nine years. At two years post-BMT, circulating T cells were hyporesponsive to host dendritic cells in vitro. T-cell receptor clonotyping revealed the disappearance of host-reactive clones, and T-cell RNA-sequencing exhibited downmodulated signaling pathways for cytotoxicity/rejection, paired with upregulated immunomodulatory pathways, suggesting active suppression. In parallel, host monocytes upregulated certain signaling pathways, indicating active interactions between post-thymic donor T cells and host monocytes. In summary, durable hematopoietic engraftment, immunity, and tolerance were demonstrable for the first time in a recipient of BMT obtained from VB graft.
Authors
Paul Szabolcs, Xiaohua Chen, Marian G. Michaels, Memphis Hill, Evelyn Garchar, Zarreen Amin, Heather M. Stanczak, Shawna McIntyre, Aleksandra Petrovic, Dhivyaa Rajasundaram, Ansuman Chattopadhyay, Jonathan E. Spahr, Peter D. Wearden, Geoffrey Kurland
Abstract
Growing evidence indicates that PKLR, the gene for pyruvate kinase (PK), is a genetic modifier of the sickle cell phenotype. Co-inheritance of specific PKLR variants is associated with increased pain-related hospitalization and can trigger sickle cell disease (SCD) phenotypes in asymptomatic carriers. PK deficiency disrupts RBC glycolysis, leading to ATP deficits and accumulation of 2,3-diphosphoglycerate, which exacerbates sickling in SCD. Using CRISPR-Cas9, we generated null mutations in Pklr [Pklr(13ntdel/13ntdel) or Pklr(246ntdel/246ntdel)] specific for the RBC isoform (PKR) in Townes mice that were homozygous (SS) or heterozygous (AS) for the human sickle globin gene, or homozygous for human hemoglobin A (AA, controls), to investigate the effect of PKR deficiency on the sickle phenotype in mice. PKR-deficient AA and AS mice developed severe anemia, reticulocytosis, and substantial spleen and liver iron deposits. Unlike what is observed in humans, PKR-deficiency in AS and SS mice surprisingly decreased sickling, but it was also associated with increased extramedullary hematopoiesis and mitochondrial retention in mature RBCs. These results demonstrate the differential effect of Pklr mutations on the phenotype of both AS and SS mouse models, offering new insights into the complex role of PKR deficiency in SCD pathology.
Authors
Xunde Wang, Meghann Smith, Sayuri Kamimura, Quan Li, Niharika Shah, Martha Quezado, Luis E.F. Almeida, Sebastian Vogel, Mickias B. Tegegn, Kevin Y. Sun, Rafael Villasmil, Chengyu Liu, William A. Eaton, Swee Lay Thein, Zenaide M.N. Quezado
Abstract
Infection leads to durable cell-autonomous changes in hematopoietic stem and progenitor cells (HSPCs), resulting in production of innate immune cells with heightened immunity. The mechanisms underlying this phenomenon, termed central trained immunity, remain poorly understood. We hypothesized that infection induces histone modifications leading to changes in chromatin accessibility that are conserved during differentiation from HSPCs to myeloid progenitors and monocytes. We conducted genome-wide surveillance of histone marks H3K27ac and H3K4me3 and chromatin accessibility in hematopoietic stem cells, multipotent progenitor 3, granulocyte-monocyte progenitors, monocytes and macrophages of naïve and Mycobacterium avium infected mice. Interferon signaling pathways and related transcription factor binding motifs including IRFs, NF-κB, and CEBP showed increased activating histone marks and chromatin accessibility across cell types. However, histone marks and increased chromatin accessibility were conserved at only a few loci, notably Irf1 and Gbp6. Knock out of IRF1 disrupted enhanced mitochondrial respiration and bacterial killing in human monocyte cell lines, while GBP6 KO monocyte cell lines showed dysregulated mitochondrial respiration. In summary, this study identifies IRF1 and GBP6 as two key loci at which infection-induced systemic inflammation leads to epigenetic changes that are conserved from HSPCs to downstream monocytes, providing a mechanistic avenue for central trained immunity.
Authors
Brandon T. Tran, Pamela N. Luna, Ruoqiong Cao, Duy T. Le, Apoorva Thatavarty, Laure Maneix, Bailee N. Kain, Scott Koh, Andre Catic, Katherine Y. King
Abstract
Vaso-occlusive episodes (VOEs) in the setting of hyperhemolysis can rapidly evolve into multiorgan failure in sickle cell disease (SCD). Although the mechanisms for rapid progression to multiorgan failure are unclear, a systemic vasculopathy with thrombotic microangiopathy-type features has been described. Reduced thrombomodulin (TM) function is implicated in some thrombotic microangiopathy syndromes. We observed a greater decline in platelet count and hemoglobin concentration and increase in vascular injury biomarkers within 24-hours of admission for a VOE in 12 SCD patients with versus 12 without multiorgan failure. We observed decreased TM expression on the lung and kidney vasculature of three additional SCD patients with multiorgan failure and an autopsy performed compared to a non-SCD control. Transgenic SCD mice challenged with cell-free hemoglobin had reduced TM function, increased vascular injury biomarkers, and reduced renal cortical blood flow. Infusion of recombinant TM 2- or 24-hours after the challenge restored cortical blood flow, mitigated increases in vascular injury, complement activation, and tubular injury biomarkers, and protected against acute kidney and lung injury. We demonstrated that impaired TM function may be involved in the systemic vasculopathy of SCD-related multiorgan failure and infusion of recombinant TM may restore vascular function and protect against acute organ damage.
Authors
Guohui Ren, Dustin R. Fraidenburg, Suman Setty, Jiwang Chen, Janae Gonzales, Maria Armila Ruiz, Zalaya Ivy, Najmeh Eskandari, Richard D. Minshall, James P. Lash, Victor R. Gordeuk, Santosh L. Saraf
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