Hematopoietic Stem Cell Dysfunction Research Articles

Upfront Alternative Donor Transplant Versus Immunosuppressive Therapy in Patients with Severe Aplastic Anemia Who Lack Fully HLA Matched Related Donor: Systematic Review and Meta-Analysis of Retrospective Studies. on Behalf of the Severe Aplastic Anemia Working Party of European Group for Blood and Marrow Transplantation (SAAWP of EBMT)

Upfront Alternative Donor Transplant Versus Immunosuppressive Therapy in Patients with Severe Aplastic Anemia Who Lack Fully HLA Matched Related Donor: Systematic Review and Meta-Analysis of Retrospective Studies. on Behalf of the Severe Aplastic Anemia Working Party of European Group for Blood and Marrow Transplantation (SAAWP of EBMT)

Can molecular targeting the TNFα-ERK-ETS1-IL27Rα pathway keep us young and healthy by protecting HSCs from aging?

The world's older population grows at an unprecedented rate. Today, there are about 8.5% of people worldwide (617 million) aged 65 and older today, which is projected to continue growing to nearly 17% of the world's population by 2050 (1.6 billion). World population aging has profound implications because aging represents a progressive deterioration of physiological function accompanied by an increase in vulnerability for disease and mortality. Although aging itself is not a disease, it is the greatest risk factor for most chronic human diseases, including cardiovascular and neurodegenerative disease, cancer, diabetes, and osteoarthritis; and a major contributing factor for the increased susceptibility to various infectious diseases such as COVID-19. Age-related changes in the hematopoietic and immune system likely play an important role in the pathogenesis of these diseases in part via induction of immunosenescence and inflammaging.1 These changes have been largely attributed to hematopoietic stem cell (HSC) aging, which leads to a significant decline in HSC self-renewal and long-term hematopoietic reconstitution, a myeloid skewing (ie, increase in myeloid cell but decrease in lymphoid cell production), and clonal hematopoiesis and increased hematological malignancies.2 Both cell-intrinsic and cell-extrinsic alterations have been implicated in driving HSC aging. The cell-intrinsic mechanisms of HSC aging include increased DNA damage,3 loss of polarity and proteostasis,4 impaired autophagy and mitochondrial activities,5 epigenetic reprogramming and senescence.6,7 The cell-extrinsic mechanisms are less studied but may include alterations in bone marrow stromal cell populations, changes in the production of hematopoietic stimulators (such as osteopontin) and inhibitors (such as CCL5) by the stromal cells, and increased production of inflammatory cytokines.8 However, how these cell-extrinsic factors communicate with HSCs to promote HSC aging has not been well established. In a recent Blood publication,9 Dr. Jianwei Wang's group investigated the mechanistic link between age-related inflammation and HSC aging. They found that aging is associated with an upregulation of interleukin 27 receptor α (IL27Rα) expression in HSCs from mouse bone marrow, resulting in a significant expansion of IL27Rα+ HSCs. IL27Rα+ HSCs from both young and old mice exhibited all the phenotypes of aged or senescent HSCs, including inability to self-renew, produce long-term hematopoietic reconstitution, and differentiate into both myeloid and lymphoid cells in a balanced manner after transplantation. In contrast, IL27Rα- HSCs from both old mice and HSCs from old IL27Rα knockout (IL27Rα−/−) mice exhibited significant improvement in HSC functions compared to IL27Rα+ HSCs from the same aged mice. These findings indicate that IL27Rα can be used as a biomarker for aged and senescent HSCs. Next, they investigated the underlying mechanisms contributing to the age-related upregulation of IL27Rα in HSCs. They found that NF-κB and EST1 are important transcription factors that regulate the expression of IL27Rα. However, aging is associated with an increased expression of phosphorylated ERK and ETS1 in HSCs, but with no changes in the NF-κB activation. The activation of the ERK-ETS1 pathway and increased expression of IL27Rα in old HSCs may be attributable to the stimulation by the HSC-extrinsic proinflammatory cytokine TNFα. This is because mouse bone marrow plasma from old mice contained a significantly higher level of TNFα than that from young mice. TNFα stimulated the expression of IL27Rα in c-Kit+ Sca1+ Lineage- (KLS) cells and HSCs in vitro and in vivo, respectively, in an ETS1-dependent manner. The upregulation of IL27Rα in HSCs is likely responsible for mediating TNFα-induced HSC dysfunction because knockout IL27Rα could prevent TNFα-induced HSC dysfunction. Therefore, these findings are highly significant by providing the first link between cell-extrinsic and cell-intrinsic communications that lead to HSC aging via the activation of the TNFα-ERK-ETS1-IL27Rα pathway. It will be of great interest to determine whether molecular targeting this pathway with a small molecule inhibitor or an antibody has the potential to be developed as a novel therapeutic strategy to slowdown HSC aging, which may lead to reduced inflammaging and improvement of hematopoietic and immune functions, particularly under various age-related pathological conditions in which TNFα production is upregulated systemically or locally in the bone marrow microenvironment. However, cautions have to be take into consideration before translating this approach into the clinic because IL27Rα+ HSCs may be primarily responsible for emergency hematopoiesis in the fight against bacterial infection as shown by the authors in this publication.

1016 – REGULATION OF RIBOSOME BIOGENESIS IN HEMATOPOIETIC STEM CELLS

Dysregulation of ribosome biogenesis is the underlying etiology in a group of bone marrow failure syndromes called ribosomopathies. As prominently illustrated by one type of ribosomopathies, Shwachman-Diamond Syndrome (SDS), germline biallelic mutations in three assembly factors essential for the maturation of the 60S ribosome subunit result in progressive hematopoietic stem and progenitor cell (HSPC) failure, suggesting that HSPCs are sensitive to perturbations in ribosome assembly. However, how ribosome assembly is regulated in HSPCs remains poorly understood, as are approaches to restore in hematopoietic stem cell (HSC) function in the context of defective ribosome biogenesis. Here we uncover a novel role for the E3 ubiquitin ligase, HectD1, in regulating HSC function via ribosome biogenesis. HSCs from hematopoietic-specific <i>Hectd1</i> knockout mice exhibit a striking defect in transplantation ability and self-renewal. <i>Hectd1</i>-deficient HSCs fail to be maintained ex vivo, concomitant with a reduction in global protein synthesis and growth rate. The mechanism underlying HSC dysfunction upon <i>Hectd1</i> deficiency is directly linked to aberrant ribosome assembly. We demonstrate that HectD1 ubiquitinates and degrades an assembly factor for the polypeptide exit tunnel of the 60S ribosome in the cytoplasm. Depletion of <i>HectD1</i> led to an accumulation of the anti-association factor eIF6 on the 60S and decreased 80S to 60S ratio, consistent with a subunit joining defect associated with SDS-like diseases. Importantly, we found that restoration of ribosome assembly in <i>HectD1</i>-deficient cells promoted protein synthesis and enhanced HSC reconstitution capacity. These findings highlight the importance of ribosome assembly in HSC regeneration and represent a rare <i>in vivo</i> example of genetic suppression of HSC defects associated with dysfunctional ribosome biogenesis.

Epidermal growth factor receptor–dependent DNA repair promotes murine and human hematopoietic regeneration

AbstractChemotherapy and irradiation cause DNA damage to hematopoietic stem cells (HSCs), leading to HSC depletion and dysfunction and the risk of malignant transformation over time. Extrinsic regulation of HSC DNA repair is not well understood, and therapies to augment HSC DNA repair following myelosuppression remain undeveloped. We report that epidermal growth factor receptor (EGFR) regulates DNA repair in HSCs following irradiation via activation of the DNA-dependent protein kinase–catalytic subunit (DNA-PKcs) and nonhomologous end joining (NHEJ). We show that hematopoietic regeneration in vivo following total body irradiation is dependent upon EGFR-mediated repair of DNA damage via activation of DNA-PKcs. Conditional deletion of EGFR in hematopoietic stem and progenitor cells (HSPCs) significantly decreased DNA-PKcs activity following irradiation, causing increased HSC DNA damage and depressed HSC recovery over time. Systemic administration of epidermal growth factor (EGF) promoted HSC DNA repair and rapid hematologic recovery in chemotherapy-treated mice and had no effect on acute myeloid leukemia growth in vivo. Further, EGF treatment drove the recovery of human HSCs capable of multilineage in vivo repopulation following radiation injury. Whole-genome sequencing analysis revealed no increase in coding region mutations in HSPCs from EGF-treated mice, but increased intergenic copy number variant mutations were detected. These studies demonstrate that EGF promotes HSC DNA repair and hematopoietic regeneration in vivo via augmentation of NHEJ. EGF has therapeutic potential to promote human hematopoietic regeneration, and further studies are warranted to assess long-term hematopoietic effects.

MYSM1 maintains ribosomal protein gene expression in hematopoietic stem cells to prevent hematopoietic dysfunction.

Ribosomopathies are congenital disorders caused by mutations in the genes encoding ribosomal and other functionally related proteins. They are characterized by anemia, other hematopoietic and developmental abnormalities, and p53 activation. Ribosome assembly requires coordinated expression of many ribosomal protein (RP) genes; however, the regulation of RP gene expression, especially in hematopoietic stem cells (HSCs), remains poorly understood. MYSM1 is a transcriptional regulator essential for HSC function and hematopoiesis. We established that HSC dysfunction in Mysm1 deficiency is driven by p53; however, the mechanisms of p53 activation remained unclear. Here, we describe the transcriptome of Mysm1-deficient mouse HSCs and identify MYSM1 genome-wide DNA binding sites. We establish a direct role for MYSM1 in RP gene expression and show a reduction in protein synthesis in Mysm1-/- HSCs. Loss of p53 in mice fully rescues Mysm1-/- anemia phenotype but not RP gene expression, indicating that RP gene dysregulation is a direct outcome of Mysm1 deficiency and an upstream mediator of Mysm1-/- phenotypes through p53 activation. We characterize a patient with a homozygous nonsense MYSM1 gene variant, and we demonstrate reduced protein synthesis and increased p53 levels in patient hematopoietic cells. Our work provides insights into the specialized mechanisms regulating RP gene expression in HSCs and establishes a common etiology of MYSM1 deficiency and ribosomopathy syndromes.

Altered microRNA expression links IL6 and TNF-induced inflammaging with myeloid malignancy in humans and mice

AbstractAging is associated with significant changes in the hematopoietic system, including increased inflammation, impaired hematopoietic stem cell (HSC) function, and increased incidence of myeloid malignancy. Inflammation of aging (“inflammaging”) has been proposed as a driver of age-related changes in HSC function and myeloid malignancy, but mechanisms linking these phenomena remain poorly defined. We identified loss of miR-146a as driving aging-associated inflammation in AML patients. miR-146a expression declined in old wild-type mice, and loss of miR-146a promoted premature HSC aging and inflammation in young miR-146a–null mice, preceding development of aging-associated myeloid malignancy. Using single-cell assays of HSC quiescence, stemness, differentiation potential, and epigenetic state to probe HSC function and population structure, we found that loss of miR-146a depleted a subpopulation of primitive, quiescent HSCs. DNA methylation and transcriptome profiling implicated NF-κB, IL6, and TNF as potential drivers of HSC dysfunction, activating an inflammatory signaling relay promoting IL6 and TNF secretion from mature miR-146a−/− myeloid and lymphoid cells. Reducing inflammation by targeting Il6 or Tnf was sufficient to restore single-cell measures of miR-146a−/− HSC function and subpopulation structure and reduced the incidence of hematological malignancy in miR-146a−/− mice. miR-146a−/− HSCs exhibited enhanced sensitivity to IL6 stimulation, indicating that loss of miR-146a affects HSC function via both cell-extrinsic inflammatory signals and increased cell-intrinsic sensitivity to inflammation. Thus, loss of miR-146a regulates cell-extrinsic and -intrinsic mechanisms linking HSC inflammaging to the development of myeloid malignancy.

Chronic activation of endothelial MAPK disrupts hematopoiesis via NFKB dependent inflammatory stress reversible by SCGF

Inflammatory signals arising from the microenvironment have emerged as critical regulators of hematopoietic stem cell (HSC) function during diverse processes including embryonic development, infectious diseases, and myelosuppressive injuries caused by irradiation and chemotherapy. However, the contributions of cellular subsets within the microenvironment that elicit niche-driven inflammation remain poorly understood. Here, we identify endothelial cells as a crucial component in driving bone marrow (BM) inflammation and HSC dysfunction observed following myelosuppression. We demonstrate that sustained activation of endothelial MAPK causes NF-κB-dependent inflammatory stress response within the BM, leading to significant HSC dysfunction including loss of engraftment ability and a myeloid-biased output. These phenotypes are resolved upon inhibition of endothelial NF-κB signaling. We identify SCGF as a niche-derived factor that suppresses BM inflammation and enhances hematopoietic recovery following myelosuppression. Our findings demonstrate that chronic endothelial inflammation adversely impacts niche activity and HSC function which is reversible upon suppression of inflammation.

Abstract 1172: JAK2V617F-mutant megakaryocytes contribute to stem cell aging in myeloproliferative neoplasms

Abstract The myeloproliferative neoplasms (MPNs) are stem cell disorders characterized by hematopoietic stem cell (HSC) expansion and an increased propensity to develop hematologic malignancies. The acquired kinase mutation JAK2V617F plays a central role in MPNs. In this work we tested the hypothesis that the JAK2V617F-bearing megakaryocytes (MKs), an important component of the hematopoietic niche, promotes HSC dysfunction in MPNs. Methods JAK2V617F Flip-Flop (FF1) mice and Pf4-Cre mice (Radek Skoda, Switzerland) were crossed to generate MK lineage-specific human JAK2V617F knock-in mouse line (Pf4/FF1). Animal experiments were performed in accordance with the Institutional Animal Care and Use Committee guideline. Results We previously reported that, at 28 wks of age, the Pf4/FF1 mice developed a murine myeloproliferative syndrome with moderate thrombocytosis, splenomegaly, increased marrow MKs, and a 3-fold increase of CD45+CD201+CD150+CD48- HSCs with increased donor engraftment in a competitive repopulation assay compared to Pf4-cre control mice. (Zhan Leukemia 2016; Zhang Stem Cells 2018) Rigorous, extremely sensitive RT-PCR assays eliminated the possibility that HSCs in Pf4/FF1 mice express the mutant kinase. To study the effect of JAK2V617F-bearing MKs on HSC function during aging, we followed the Pf4/FF1 mice up to 2 years of age. At 2 yr, there were significant neutrophilia in Pf4/FF1 mice but no change was observed in hemoglobin, platelet count, or marrow HSC cell numbers compared to age-matched control mice. Indeed, while HSC numbers significantly increased in old (2-yr) control mice compared to young (6-mo) control mice (3.3-fold, p=0.0002, n=4-8), there was no significant increase of HSCs between old and young Pf4/FF1 mice (1.6-fold, p=0.158, n=6-8). A competitive marrow transplantation experiment was performed in which CD45.2 donor marrow cells from 2-yr old Pf4/FF1 or control mice were injected intravenously together with competitor CD45.1 wild-type marrow cells intro lethally irradiated wild-type recipient (CD45.1) (n=8-9 in each group). During a 4-month follow up, old HSCs from the Pf4/FF1 mice displayed hallmarks of murine hematopoietic aging including a lower peripheral blood donor chimerism, increased myeloid blood cell production, and decreased lymphoid cells compared to old HSCs from control mice. To begin to understand how JAK2V617F-bearing MKs promote the aging phenotype of HSCs in old Pf4/FF1 mice, qRT-PCR of purified HSCs identified up-regulation of p21 (2-fold, p = 0.019) in Pf4/FF1 mice compared to controls. Conclusion The Pf4/FF1 mice, in which the JAK2V617F expression is restricted to MKs, developed a myeloproliferative phenotype at young age and accelerated stem cell aging at old age. The precise mechanism by which JAK2V617F-bearing MKs affect the MPN marrow niche and stem cell dysfunction during disease progression deserve further investigation. Citation Format: Helen Wong, Melissa Castiglione, Molly Quan, Huichun Zhan. JAK2V617F-mutant megakaryocytes contribute to stem cell aging in myeloproliferative neoplasms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1172.

Epidermal Growth Factor Receptor - Mediated DNA Repair Regulates Hematopoietic Stem Cell Regeneration

Hematopoietic myelosuppression is the principal dose-limiting toxicity of curative chemotherapy and radiotherapy in patients with cancer. Both chemotherapy and radiotherapy deplete hematopoietic stem cells (HSCs) via induction of DNA damage. DNA damage contributes to both HSC dysfunction and risk for malignant transformation over time. Extrinsic signals capable of promoting DNA repair in HSCs following injury can potentially improve HSC function and may decrease risk for dysplasia and leukemia over time. Here, we show that treatment with epidermal growth factor (EGF) decreases DNA damage in murine HSCs following irradiation via activation of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and nonhomologous end joining (NHEJ) repair. Specifically, we observed that in vitro treatment of irradiated bone marrow (BM) ckit+sca-1+lin- (KSL) hematopoietic stem/progenitor cells (HSPCs) with 100 ng/ml EGF decreased HSPC DNA damage at 1 hour as measured by gamma-H2AX foci and Comet assay (p<0.0001, p<0.0001). Further, EGF treatment increased phosphorylation and nuclear localization of DNA-PKcs, which is integral to NHEJ repair, and increased phosphorylation of Akt in irradiated HSPCs. Inhibition of Akt or DNA-PKcs completely abrogated EGF - mediated DNA repair and EGF - mediated recovery of HSPCs following irradiation in vitro. In irradiated (500 cGy) C57BL/6 mice, subcutaneous administration of 10 μg/day of EGF significantly increased recovery of peripheral blood white blood cells and lymphocytes (p=0.03, p=0.04), as well as BM SLAM+KSL cells (HSCs) at day +14 (p=0.0004). Treatment with the DNA-PKcs inhibitor, NU7441, abrogated EGF - mediated recovery of peripheral blood WBCs in irradiated mice (p=0.007), suggesting that EGF - mediated mitigation of radiation injury in vivo was dependent on DNA-PKcs - mediated NHEJ repair. Importantly, EGF treatment of irradiated mice also substantially increased the survival of irradiated mice compared to irradiated control mice (p=0.009) and caused the recovery of long-term HSCs capable of 20 week competitive repopulation in congenic recipient mice (p=0.01, 20 weeks).Conversely, doxycycline inducible, cell - specific suppression of EGFR in hematopoietic cells using SCL-tTA;EGFR-DN mice caused a significant depressed regeneration of BM SLAM+KSL cells and KSL cells at day +10 after irradiation (p=0.005, p=0.02, respectively). SCL-tTA;EGFR-DN mice also displayed significantly decreased survival at day +30 compared to EGFR wild type mice (23.1% survival vs. 58.3% survival, p=0.04). Consistent with our hypothesis, BM HSPCs from EGFR-DN mice displayed increased DNA damage in response to total body irradiation, as measured by gamma H2AX foci and the Comet assay (p<0.0001, p=0.04). Coordinately, BM HSPCs from EGFR-DN mice displayed decreased phosphorylation of Akt and DNA-PKcs in response to irradiation in vitro (p<0.0001, p=0.004).These studies suggest that EGFR - mediated DNA repair is necessary for HSC and progenitor cell recovery following irradiation and that EGF treatment accelerates hematopoietic regeneration via augmentation of NHEJ repair in HSCs. EGF treatment following high dose chemotherapy and/or radiotherapy may increase HSC function and lessen near - term hematologic toxicities and morbidities. DisclosuresNo relevant conflicts of interest to declare.

Rheumatoid Arthritis Causes Hematopoietic Stem Cell Reprogramming to Maintain Functionality

Rheumatoid arthritis (RA) is a debilitating autoimmune disease resulting from autoantibodies that cause damage to synovial joints. Joint damage causes increased systemic inflammatory cytokines which may lead to aberrant hematopoiesis. Indeed, RA is accompanied by many hematological complications including anemia, cytopenias, and suppressed bone marrow function. Hematopoietic stem cells (HSC) at root of the blood system can respond to inflammatory signals by activating the cell cycle and preferentially generating myeloid cells. However, chronic inflammation can also lead to HSC dysfunction. Previous studies using genetic mouse models of RA have identified myeloid overproduction in this context; however, HSC long-term reconstitution activity was maintained.To better understand hematopoietic alterations in RA, our group used the collagen induced arthritis (CIA) mouse model, which is inducible in adult mice and recapitulates many immunological features of the human disease, including elevated inflammatory cytokine levels in the bone marrow (BM) and peripheral blood (PB). Confirming prior reports, we found increased numbers of myeloid lineage cells in the PB and BM of CIA mice. We also found reduced erythroid and lymphoid lineage progenitor cell numbers in the BM, consistent with anemia and immunosenescence phenotypes in RA patients. Interestingly, these features were accompanied by a significant increase in the number of myeloid-biased multipotent progenitor-3 (MPP3) cells, suggesting increased activation of myeloid differentiation pathways. However, we found no changes to the number of activated (MPP1), short-term HSC (ST-HSC), or long-term HSC (LT-HSC) in CIA mice. Likewise, and in line with previous reports, long-term HSC potential was not reduced in CIA mice, as assessed by transplantation of either purified HSC or with unfractionated BM into irradiated recipient mice. While HSC from control and CIA donor mice displayed similar blood chimerism and lineage distribution over a 16-week period, we did observe increased proportions of donor-derived MPP3 in CIA recipient animals, further supporting an activation of myeloid HSC differentiation pathways. Overall, these results reveal underlying changes in the BM driving aberrant hematopoiesis, with the HSC pool remaining intact despite activation of a myeloid differentiation pathway.To better understand how HSC are impacted by arthritic inflammation, we assessed the molecular state of control and CIA HSC using RNA-seq. We found 292 genes significantly upregulated and 237 genes significantly downregulated in CIA HSC. Analysis of these genes using Ingenuity, GSEA, and DAVID tools revealed broad downregulation of inflammatory and proliferation signaling pathways including IL-1β, NFκB, MYC, and ERK. Genes for G1/S cell cycle transition, transcription, protein translation, and proliferation pathways were also significantly downregulated in CIA HSC. On the other hand, genes corresponding to cell cycle arrest and negative regulation of transcription were significantly upregulated. Notably, we find that IL-1β, which is produced in the BM of CIA mice, is sufficient to induce this molecular program. We find that HSC in CIA mice have a global downregulation of transcripts required for activation and proliferation, and a global upregulation of transcripts that would promote quiescence. Hence, HSC are forced back into a quiescent state by broad downregulation of cell growth and proliferation genes even during chronic inflammation caused by RA.Altogether, our data show that a mouse model of rheumatoid arthritis causes hematopoietic lineage skewing towards the myeloid lineage with simultaneous loss of lymphoid and erythroid lineage potential. Interestingly, in this chronic inflammatory setting HSC downregulate pathways involved in cytokine signaling, cell cycle activation, and translation. This mechanism can be triggered by chronic exposure to pro-inflammatory cytokines, and may serve to limit HSC proliferation and potential for damage in disease settings. These results may explain the relative rarity of outright bone marrow failure in autoimmune disease patients, while providing insight into mechanisms driving aberrant hematopoiesis in these individuals. Lastly, they provide functional evidence for cytokine blockade to normalize HSC function in the setting of RA and other chronic inflammatory diseases. DisclosuresNo relevant conflicts of interest to declare.

RARE CASE OF SERRATIA PNEUMONIA CAUSING TRANSIENT APLASTIC ANEMIA

SESSION TITLE: Critical Care 1 SESSION TYPE: Med Student/Res Case Report PRESENTED ON: 10/09/2018 03:45 PM - 04:45 PM INTRODUCTION: Aplastic anemia (AA) is defined as two or three cell line cytopenias due to hematopoietic stem cell (HSC) dysfunction. Incidence is 2 cases/million people. Symptoms include fatigue, dyspnea, cardiac symptoms, bruising, mucosal bleeding and increased infection risk. The most common causes include PNH, drugs, toxins and viruses (parvovirus B19, EBV, CMV, hepatitis, HSV6 and dengue). Overall prognosis for AA is poor. CASE PRESENTATION: 71 year-old female with past medical history of hypertension and hypothyroidism presented with weakness and word slurring. Five days prior, she awoke with dizziness and difficulty swallowing, then developed vomiting and diarrhea. Travel in the past 3 months included Morocco, France and Chile. Patient denied sick contacts, chills, night sweats or weight loss; endorsed hematuria one month ago, which resolved after nitrofurantoin course. Initial vitals: BP 84/50, HR 127, RR 32, T 101.8F, SpO2 89% on 3LPM O2. She appeared toxic with bronchial breath sounds and abdominal tenderness. Initial labs: WBC 0.2K/uL, Hb 9.9g/dL, plt 4K/uL, reticulocytes 0.6%, ESR 124, INR 1.5, D-dimer 2.35 mg/L, lactic acid 1.5 mmol/L, troponin undetectable, and BNP 287 pg/mL. CXR showed right lower lobe hazy opacity with a small right pleural effusion. CT abdomen/pelvis showed ascites and hepatomegaly. CTA chest was negative for PE. The patient was intubated and given IV fluids, broad spectrum antibiotics and 2U platelets. Respiratory cultures grew Serratia (not speciated) and antibiotic treatment was narrowed to meropenem and vancomycin. Thrombocyto- and leukocytopenia persisted, requiring multiple transfusions. Serum rheumatology, infectious, vasculitis, SIFE/SPEP, G6PD deficiency, hepatitis and labs PNH studies were negative. Positive findings included high IgE, parvovirus/EBV IgG and ferritin 5544 ng/mL. Bone marrow (BM) biopsy showed hypocellular BM (0-15%) with rare megakaryocytes, scanty myeloid and erythroid precursors, and slight plasmacytosis. The patient was started on anti-thymocyte globulin and cyclosporine, and then switched to a thrombopoietin nonpeptide agonist, for severe AA. After 14 days of antibiotics and 10 days of AA treatment, the patient’s WBC and platelets increased then stabilized. DISCUSSION: The above case of Serratia as the cause of transient AA suggests that the infectious mechanism leading to BM failure was likely T-cell mediated. Most viruses, like parvovirus B19, directly destroy HSCs while other pathogens, like bacteria, cause AA secondary to excessive immune activation via T-cells. Thus, the transient nature of bacterial-induced AA likely occurs after treating the inciting pathogen, like in this case. CONCLUSIONS: Bacterial causes of transient AA are rare and the specific mechanisms of bacterial-induced, T-cell-mediated HSC injury are largely unknown. This case highlights Serratia as a likely cause of HSC dysfunction causing transient AA. Reference #1: Espinoza, J. L., Kotecha, R., & Nakao, S. (2017). Microbe-induced inflammatory signals triggering acquired bone marrow failure syndromes. Frontiers in Immunology, 8, 186. 10.3389/fimmu.2017.00186 [doi] Reference #2: Keisu, M., Heit, W., Lambertenghi-Deliliers, G., Parcells-Kelly, J., Polliack, A., & Heimpel, H. (1990). Transient pancytopenia. A report from the international agranulocytosis and aplastic study. Blut, 61(4), 240-244. Reference #3: Young, N. S. (2002). Acquired aplastic anemia. Annals of Internal Medicine, 136(7), 534-546. 200204020-00011 [pii] DISCLOSURES: No relevant relationships by Eric Bondarsky, source=Web Response No relevant relationships by Lindsay Hammons, source=Web Response

Mlh1 deficiency increases the risk of hematopoietic malignancy after simulated space radiation exposure.

Cancer-causing genome instability is a major concern during space travel due to exposure of astronauts to potent sources of high-linear energy transfer (LET) ionizing radiation. Hematopoietic stem cells (HSCs) are particularly susceptible to genotoxic stress, and accumulation of damage can lead to HSC dysfunction and oncogenesis. Our group recently demonstrated that aging human HSCs accumulate microsatellite instability coincident with loss of MLH1, a DNA Mismatch Repair (MMR) protein, which could reasonably predispose to radiation-induced HSC malignancies. Therefore, in an effort to reduce risk uncertainty for cancer development during deep space travel, we employed an Mlh1+/− mouse model to study the effects high-LET 56Fe ion space-like radiation. Irradiated Mlh1+/− mice showed a significantly higher incidence of lymphomagenesis with 56Fe ions compared to γ-rays and unirradiated mice, and malignancy correlated with increased MSI in the tumors. In addition, whole exome sequencing analysis revealed high SNVs and INDELs in lymphomas being driven by loss of Mlh1 and frequently mutated genes had a strong correlation with human leukemias. Therefore, the data suggest that age-related MMR deficiencies could lead to HSC malignancies after space radiation, and that countermeasure strategies will be required to adequately protect the astronaut population on the journey to Mars.

Regulation of microRNA-22 on glycometabolism of hematopoietic stem cell TF-1 and its molecular mechanism

Objective To study the regulation of microRNA-22(miR-22) on glycometabolism of hematopoietic stem cell TF-1 and its molecular mechanism. Methods TF-1 cells were cultured for 2 h under hypoxic conditions.The expression levels of Glut4 and miR-22 was detected by quantitative real-time PCR(qRT-PCR). The sgRNA vector of the miR-22 gene was constructed and miR-22 gene of TK-1 cells was knocked out by the CRISPR/Cas9 technique.Overexpression vectors were constructed, and miR-22 knocked-out cells were introduced to overexpress miR-22, the expression of miR-22 was detected by qRT-PCR and the expression levels of Glut4 and PPAR-γ were detected by qRT-PCR and Western blot. Results Compared with the control group, the expression of miR-22 in TF-1 cells decreased (0.015±0.000 vs. 0.056±0.001) and the expression of Glut4 (0.351±0.038 vs. 0.152±0.005) and PPAR-γ (0.421±0.017 vs. 0.248±0.008) increased, when TF-1 cells were cultured for 2 h under hypoxic conditions, and the differences were statistically significant (all P<0.05). Compared with the control group, the expression levels of Glut4 (0.019±0.00 vs. 0.008±0.000) and PPAR-γ (0.038±0.001 vs. 0.019±0.000) were significantly increased after miR-22 gene silencing, and they were significantly decreased (Glut4: 0.005±0.000 vs. 0.008±0.000; PPAR-γ: 0.137±0.000 vs. 0.019±0.000) after overexpression of miR-22, and the differences were statistically significant (all P<0.05). Conclusions It suggests that miR-22 exerts a negative regulation on glycometabolism of hematopoietic stem cell TF-1 by downregulating the expression of PPAR-γ.A new regulatory factor of TF-1 glycometabolism and the mechanisms are identified, which has provided new ideas for the targeted medication of diseases induced by hematopoietic stem cell dysfunction. Key words: Hematopoietic stem cells; Glycometabolism; MicroRNA; Peroxisome proliferators-activated receptor-γ

Abstract 5053: Mlh1 deficiency increases the risk of hematopoietic malignancies post low- and high-LET radiation exposure

Abstract Introduction/Aim: Natural sources of radiation in space include galactic cosmic rays (GCR), solar energetic particles and trapped energetic particles in a planetary magnetic field. These sources are difficult to shield, thus posing significant health risks to astronauts on long-term inter-planetary missions. During space travel, genomic instability is a major concern where astronauts are exposed to potent sources of ionizing radiation, namely GCR consisting of high energy and charged atomic nuclei. In particular, hematopoietic stem cells (HSCs) are susceptible to internal and external stresses that threaten the integrity of the cell, and accumulation of damage can lead to HSCs dysfunction and oncogenesis. Recent data has demonstrated that humans accumulate microsatellite instability with acquired loss of MLH1 protein in HSCs as a function of age. Therefore, we hypothesized that high-LET (linear energy transfer) radiation characteristic of the GCR that will confront astronauts on space missions will damage HSCs and contribute to induction and progression of hematopoietic malignancies. Methods: To study this hypothesis, we employed a DNA mismatch repair deficient mouse model (Mlh1+/-) to study the effects of low-LET γ-ray vs high-LET 56Fe ion radiation on HSCs of potential astronaut population. In vitro colony forming unit assays and in vivo complete blood count (CBC) plus competitive repopulation assays were carried out to understand harmful impact of radiation and Mlh1 deficiency on HSCs functions. In addition, mice were followed up to 18 months post irradiation to observe HSC malignancies in Mlh1+/+ and Mlh1+/- mice. Results: HSC short- and long-term functional assays showed defects in HSCs/HPCs function caused by irradiation, but not depending on Mlh1 status. CBC 5 and 9 months post irradiation demonstrated no impact of irradiation or Mlh1 status on HSC differentiation. However, 56Fe-ion irradiated Mlh1+/- mice showed a significant higher incidence of lymphomagenesis compared to γ-rays irradiated and sham-irradiated Mlh1+/- mice. In addition, immunohistochemistry analysis of lymphomas displayed significant higher incidence of T-cell rich B-cell lymphomas. Conclusion: Thus, the data show that MMR defects in HSCs leads to sensitization to radiation induced hematopoietic malignancy, and that radiation quality effects exacerbate the sensitivity. The findings could have profound effects on astronaut screening and designing better mitigators for space missions. Citation Format: Rutulkumar Patel, Stanton L. Gerson, Scott M. Welford. Mlh1 deficiency increases the risk of hematopoietic malignancies post low- and high-LET radiation exposure [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5053.

The transcription factor Zfp90 regulates the self-renewal and differentiation of hematopoietic stem cells

Hematopoietic stem cells (HSCs) can give rise to all blood cells that are essential to defend against pathogen invasion. The defective capability of HSC self-renewal is linked to many serious diseases, such as anemia. However, the potential mechanism regulating HSC self-renewal has not been thoroughly elucidated to date. In this study, we showed that Zfp90 was highly expressed in HSCs. Zfp90 deficiency in the hematopoietic system caused impaired HSPC pools and led to HSC dysfunction. We showed that Zfp90 deletion inhibited HSC proliferation, while HSC apoptosis was not affected. Regarding the mechanism of this effect on HSC proliferation, we found that Zfp90 interacted with Snf2l, a subunit of the NURF complex, to regulate Hoxa9 expression. Ectopic expression of Hoxa9 rescued the HSC repopulation capacity in Zfp90-deficient mice, which indicates that Hoxa9 is the downstream effector of Zfp90. In summary, our findings identify Zfp90 as a key transcription factor in determining the fate of HSCs.

The Critical Role of Inflammation in the Pathogenesis and Progression of Myeloid Malignancies.

Hematopoietic stem cells (HSCs) maintain an organism’s immune system for a lifetime, and derangements in HSC proliferation and differentiation result in hematologic malignancies. Chronic inflammation plays a contributory if not causal role in HSC dysfunction. Inflammation induces HSC exhaustion, which promotes the emergence of mutant clones that may be resistant to an inflammatory microenvironment; this likely promotes the onset of a myeloid hematologic malignancy. Inflammatory cytokines are characteristically high in patients with myeloid malignancies and are linked to disease initiation, symptom burden, disease progression, and worsened prognostic survival. This review will cover our current understanding of the role of inflammation in the initiation, progression, and complications of myeloid hematologic malignancies, drawing from clinical studies as well as murine models. We will also highlight inflammation as a therapeutic target in hematologic malignancies.

Human AK2 links intracellular bioenergetic redistribution to the fate of hematopoietic progenitors

AK2 is an adenylate phosphotransferase that localizes at the intermembrane spaces of the mitochondria, and its mutations cause a severe combined immunodeficiency with neutrophil maturation arrest named reticular dysgenesis (RD). Although the dysfunction of hematopoietic stem cells (HSCs) has been implicated, earlier developmental events that affect the fate of HSCs and/or hematopoietic progenitors have not been reported. Here, we used RD-patient-derived induced pluripotent stem cells (iPSCs) as a model of AK2-deficient human cells. Hematopoietic differentiation from RD-iPSCs was profoundly impaired. RD-iPSC-derived hemoangiogenic progenitor cells (HAPCs) showed decreased ATP distribution in the nucleus and altered global transcriptional profiles. Thus, AK2 has a stage-specific role in maintaining the ATP supply to the nucleus during hematopoietic differentiation, which affects the transcriptional profiles necessary for controlling the fate of multipotential HAPCs. Our data suggest that maintaining the appropriate energy level of each organelle by the intracellular redistribution of ATP is important for controlling the fate of progenitor cells.

Regulation of hematopoietic stem cell homeostasis by Spred1

Various types of stresses account for the dysregulation of the self-renewal activity of stem cells, resulting in the functional failure of tissues or tumorigenesis promotion. Although diets also affect our health, the effect of harmful dietary stresses on the tissue or stem cell homeostasis remains unclear. Recent research has revealed that Spred1, which negatively regulates RAS-MAPK signaling, protects hematopoietic stem cell (HSC) homeostasis against high-fat diet (HFD) -induced systemic stress. In steady-state conditions, Spred1 negatively regulates HSC self-renewal in a manner supported by the Rho kinase (ROCK) activity. In addition, Spred1 deficiency in mice mitigates HSC dysfunction induced by aging or lipopolysaccharide treatment, enhances the HSC self-renewal capacity, and prolongs HSC lifespan, but does not induce leukemia because of the compensatory upregulation of Spred2-the other Spred family member. Conversely, HFD triggers ERK hyperactivation and aberrant self-renewal in Spred1-deficient HSCs, resulting in HSC dysfunction, severe anemia, and the development of lethal myeloproliferative neoplasm-like disease. The depletion of the gut microbiota by antibiotics restored myeloproliferation, anemia, and HSC reconstitution ability in HFD-fed Spred1-deficient mice, suggesting that HFD-induced hematopoietic abnormalities were partially because of alterations in the gut microbiota composition. Thus, HFD-induced systemic stress affects the regulation of HSC self-renewal, and Spred1 safeguards HSC homeostasis against the diet-induced systemic stress.

Histone demethylase LSD1 regulates hematopoietic stem cells homeostasis and protects from death by endotoxic shock

Hematopoietic stem cells (HSCs) maintain a quiescent state during homeostasis, but with acute infection, they exit the quiescent state to increase the output of immune cells, the so-called "emergency hematopoiesis." However, HSCs' response to severe infection during septic shock and the pathological impact remain poorly elucidated. Here, we report that the histone demethylase KDM1A/LSD1, serving as a critical regulator of mammalian hematopoiesis, is a negative regulator of the response to inflammation in HSCs during endotoxic shock typically observed during acute bacterial or viral infection. Inflammation-induced LSD1 deficiency results in an acute expansion of a pathological population of hyperproliferative and hyperinflammatory myeloid progenitors, resulting in a septic shock phenotype and acute death. Unexpectedly, in vivo administration of bacterial lipopolysaccharide (LPS) to wild-type mice results in acute suppression of LSD1 in HSCs with a septic shock phenotype that resembles that observed following induced deletion of LSD1 The suppression of LSD1 in HSCs is caused, at least in large part, by a cohort of inflammation-induced microRNAs. Significantly, reconstitution of mice with bone marrow progenitor cells expressing inhibitors of these inflammation-induced microRNAs blocked the suppression of LSD1 in vivo following acute LPS administration and prevented mortality from endotoxic shock. Our results indicate that LSD1 activators or miRNA antagonists could serve as a therapeutic approach for life-threatening septic shock characterized by dysfunction of HSCs.

Single Cell-Resolution Analysis of HSC Dysfunction in Mir-146a knockout Mice

MicroRNA miR-146a is frequently depleted in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Loss of miR-146a may be an initiating event in tumorigenesis, as miR-146a loss in mouse models is sufficient to cause features of MDS and eventual progression to AML. To define how miR-146a loss initiates tumorigenesis, we analyzed hematopoietic stem cell (HSC) function from miR-146a knockout (KO) mice prior to onset of an overt malignant phenotype. Tracking cell division kinetics, proliferation, and differentiation of single long-term HSC (LT-HSC; EPCR+CD45+CD48-CD150+) in culture, we found evidence that miR-146a KOreduces HSC quiescence and promotes differentiating cell divisions. Our data show that miR-146a KO HSC dysfunction may stem from loss of a CD150-bright EPCR-bright sub-population, which has previously been associated with robust HSC activity. In line with this, single cell DNA methylation profiling revealed a reduction in a primitive sub-population of LT-HSCs in miR-146a KO animals. In addition, single cell LT-HSC transplants revealed a myeloid repopulation bias. As reduced HSC cell cycle quiescence has been linked to impaired HSC self-renewal upon hematopoietic stress, such as serial transplantation, we assessed the frequency of serially transplantable HSCs by performing secondary transplants with limiting dilution. Serially transplantable HSC frequency was reduced in miR-146a KO compared to wild type, suggesting impaired HSC self-renewal. Transcriptome profiling of miR-146a KO hematopoietic stem and progenitor cells identified tumor necrosis factor (TNF) signaling activation as a potential driver of HSC dysfunction. LT-HSC cell cycle quiescence and the CD150-bright EPCR-bright LT-HSC sub-population were restored in miR-146a/TNF double KO mice, suggesting that aberrant TNF signaling activation drives HSC dysfunction upon loss of miR-146a. Gene expression levels in the TNF signaling network are inversely correlated with miR-146a levels in human AML, implying that TNF signaling may similarly disrupt HSC function in miR-146a- depleted myeloid malignancies. Overall, our findings suggest that miR-146a promotes HSC cell cycle quiescence and inhibits differentiation by antagonizing TNF signaling, in order to maintain a primitive sub-population of long-term self-renewing HSCs. DisclosuresEaves:Experimental Hematology: Other: Editor of journal; StemCell Technologies Inc: Other: Wife of owner.