Hematopoietic Dysfunction Research Articles

Coexistence of aberrant hematopoietic and stromal elements in myelodysplastic syndromes

Myelodysplastic syndromes (MDS) are a group of clonal hematopoietic disorders related to hematopoietic stem and progenitor cell dysfunction. Several studies have shown the role of the bone marrow microenvironment in regulating hematopoietic stem, and progenitor function and their individual abnormalities have been associated with disease pathogenesis. In this study, we simultaneously evaluated hematopoietic stem cells (HSC), hematopoietic stem progenitor cells (HSPCs) and different stromal elements in a cohort of patients with MDS-refractory cytopenia with multilineage dysplasia (RCMD). Karyotyping of these patients revealed variable chromosomal abnormalities in 73.33% of patients. Long-term HSC and lineage-negative CD34+CD38− cells were reduced while among the HPCs, there was an expansion of common myeloid progenitor and loss of granulocyte-monocyte progenitors. Interestingly, loss of HSCs was accompanied by aberrant frequencies of endothelial (ECs) (CD31+CD45−CD71−) and mesenchymal stem cells (MSCs) (CD31−CD45−71−) and its subsets associated with HSC niche. We further demonstrate down-regulation of HSC maintenance genes such as Cxcl12, VEGF in mesenchymal cells and a parallel upregulation in endothelial cells. Altogether we report for the first time quantitative and qualitative de novo changes in hematopoietic stem and its associated niche in a cohort of MDS-RCMD patients. These findings further reinforce the role of different components of the bone marrow microenvironment in MDS pathogenesis and emphasize the need for comprehensive simultaneous evaluation of all niche elements in such studies.

T cell mediated mechanisms underlying HSC dysfunction and bone marrow failure

Abstract Bone marrow failure occurs when the bone marrow cannot produce sufficient numbers of red blood cells, white blood cells or platelets. Bone marrow failure disorders are rare, yet serious conditions, and are often thought to be the result of immune destruction within the bone marrow. IL-2-deficient mice, which have a deficit in functional regulatory T cells, develop spontaneous bone marrow failure. We show that disease occurs in these mice with a specific accumulation of T cells in the bone marrow, a shift in the CD4/CD8 ratio, IFNγ-mediated hematopoietic stem cell dysfunction, and antibodies directed against bone marrow and bone tissue, recapitulating human disease, and suggesting that this disease may be caused by self-reactive immune responses. Disease onset is rapid at 12 days of age with death by 27 days, and T cell responses are essential for disease progression, providing a novel model for defining the immune cell dysregulation in this autoimmune disease. We use this model to define the mechanisms underlying the deficit in myelopoiesis observed during bone marrow failure, including the contribution of activated lymphocyte subsets to disease induction. Our study describes the influence of IFNγ, CD4 and CD8 T cell subsets, and local bone marrow proliferation and migration on the kinetics and severity of disease.

Role of Aberrant Splicing in Pathogenesis of Myelodysplastic Syndromes-Review

The spectrum of genetic abnormalities in myelodysplastic syndromes(MDS) has been revealed by high-throughput sequencing. However, the functional role of these mutations in occurrence and development of MDS was not delineated. The mutations in splicing factors have been identified as the commonest gene mutations in MDS. Recently, it was reported that the mutated or dysregulated splicing factors, including SF3B1, SRSF2 and U2AF1, attribute to aberrant mRNA splicing, which leads to hematopoietic dysfunction and drives MDS. These findings will be of great help in searching for candidate therapeutic targets in mis-splicing pathway in MDS. In this review the role of aberrant splicing in pathogenesis of MDS is summarized briefly.

多発性骨髄腫に起因すると考えられた下顎骨関節突起部病的骨折の1例

Multiple myeloma (MM) is a malignant tumor of plasma cells and presents with various clinical symptoms, including hematopoietic disorder, osteolytic lesions, organ dysfunction, and susceptibility to infection. Furthermore, skeletal-related events (SREs) such as bone pain, pathological fractures, and hypercalcemia should be managed. We describe a case of pathological fracture of mandibular condyle considered to be a SRE of MM.

Distinct Brca1 Mutations Differentially Reduce Hematopoietic Stem Cell Function

BRCA1 is a well-known DNA repair pathway component and a tissue-specific tumor suppressor. However, its role in hematopoiesis is uncertain. Here, we report that a cohort of patients heterozygous for BRCA1 mutations experienced more hematopoietic toxicity from chemotherapy than those with BRCA2 mutations. To test whether this reflects a requirementfor BRCA1 in hematopoiesis, we generated mice with Brca1 mutations in hematopoietic cells. Mice homozygous for a null Brca1 mutation in theembryonic hematopoietic system (Vav1-iCre;Brca1F22-24/F22-24) developed hematopoietic defects in early adulthood that included reduced hematopoieticstem cells (HSCs). Although mice homozygous for a huBRCA1 knockin allele (Brca1BRCA1/BRCA1) were normal, mice with a mutant huBRCA1/5382insC allele and a null allele (Mx1-Cre;Brca1F22-24/5382insC) had severe hematopoietic defects marked by a complete loss of hematopoietic stem and progenitor cells. Our data show that Brca1 is necessary for HSC maintenance and normal hematopoiesis and that distinct mutations lead to different degrees of hematopoietic dysfunction.

Bone Marrow (BM) Hematopoietic Dysfunction in Chronic Lymphocytic Leukemia (CLL) - Association with Leukemic Burden and Reversibility with Therapeutic Responses.

Introduction: CLL is the most prevalent leukemia in the United States (≥120,000 cases) with approximately 5000 deaths per year. The dominant cause of mortality in CLL patients is serious infection. Indeed, about 80% of CLL patients will sustain infectious complications at some point during their disease course and infection risk increases with length of the disease. Steady-state hematopoiesis in BM is required to sustain a peripheral repertoire of blood cells and maintain immune competence. There have been few studies evaluating the hematopoietic capacity of CLL BM and how it may relate to immune competence. Thus, the goal of this study was to determine the status of BM hematopoiesis in untreated and treated CLL patients.Methods and findings: The hematopoietic stem (HSC) and progenitor cell pool (HSPC) in human BM is defined by expression of CD34 and lack of surface markers (Lin-) expressed on their mature progeny. Frequencies of Lin- CD34+ progenitors in BM between age matched healthy subjects (HS) (n=22) and untreated CLL patients (n=10) were compared. We note that the Lin+ cocktail used included an antibody to CD19, eliminating dilution of CD34+ HSPC frequency due to variation in leukemic B cell infiltration. CD34+ HSPCs were significantly reduced in untreated CLL patients (6.33±3.01% vs. 2.04±2.33% in HS vs. CLL, p=0.0004). Next we evaluated total CFU responding cells within the residual CD34+ cells to determine if they were functionally compromised. Numbers of total CFU-responsive CD34+ progenitors (CFU-GM, CFU-E, and CFU-GEMM) were reduced when assayed from purified CLL patient CD34+ cells compared to HS. Thus, while HSPC are present in CLL patient BM, they are functionally compromised even after removal from the tumor microenvironment. To determine if reductions in CFUs were accompanied by decreased myeloid and erythroid lineage differentiated progeny, we evaluated frequencies and absolute numbers of CD14+ monocytes and CD71+ erythroid progenitors by flow cytometry and found both were severely reduced in CLL patient BM. To determine if the reductions in myeloid/erythroid lineage cells was due to deficiencies in their precursor HSPCs, we evaluated frequencies and absolute numbers of HSC, multipotent progenitors (MPP), and common myeloid progenitors (CMP). Proportions, but not absolute numbers, of HSC, MPP, and CMP were significantly reduced in CLL patient BM. Frequencies of CD14+ monocytes and CD71+ erythroid precursors were also significantly reduced in patients with low BM tumor burden (designated as 20-40% BM involvement), suggesting that the hematopoietic dysfunction is not solely due to tumor burden. Comparative analysis of frequencies of CD14+ monocytes in BM to frequencies of monocytes in peripheral blood revealed a strong association, suggesting that for some immune cell types the blood immune repertoire may be reflective of BM dysfunction. In addition to BM evaluation of untreated CLL patients, we also studied a cohort of post-therapy patients (n=23). Not surprisingly, we found considerable heterogeneity in the extent of BM hematopoietic reconstitution. Nevertheless, we observed robust BM hematopoiesis post therapy in a subset of treated patients (n=9 of 23).Summary: Our findings reveal BM hematopoietic dysfunction (HD) in CLL not solely due to the degree of tumor burden. Importantly, the BM HD is reversible by effective therapies. The HD is manifested by reduced numbers and function of HSPCs and production of their mature progeny. Of significance is the severe reduction in monocytic and granulocytic cells found in CLL BM. This is an important finding as these two innate cell types provide critical first responder protection against invading pathogens. Our study also uncovered that levels of blood monocytes can mirror their diminished production in BM. Future studies will focus on determining the mechanism(s) that reduce the ability of CLL BM to sustain immune cell production in addition to determining if restoration of hematopoiesis is a necessary outcome for more effective therapies. DisclosuresDing:Merck: Research Funding. Kay:Pharmacyclics: Research Funding; Tolero Pharmaceuticals: Research Funding; Acerta: Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Morpho-Sys: Membership on an entity's Board of Directors or advisory committees; Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

The Effects of Galactic Cosmic Radiation Exposure on Hematopoietic Stem Cell Dysfunction and Oncogenesis

Natural sources of radiation in space include galactic cosmic rays (GCR), solar energetic particles (SPE) and trapped energetic particles in a planetary magnetic field. These different sources of space radiation consist of protons of various energies, particle nuclei of high energy and charge (HZE) and neutrons of different energies. These sources are difficult to shield because of their high energies and dense ionization patterns, thus posing significant health risks to astronauts on long term inter-planetary missions. Efforts to protect astronauts from harmful cosmic radiation require a deeper understanding of the effects of GCR on human health. In particular, very little is known about the effects of GCR exposure on the hematopoietic stem cell (HSC) population and whether disruptions in genetic stability in HSCs could result in the development of hematopoietic malignancies in astronauts on deep space missions.The average age of shuttle crew has risen above 46 years, and our work and others have shown that HSCs display diminished function with age. Recent data from our group has demonstrated that middle-aged individuals show frequent defects in DNA mismatch repair (MMR) in HSCs. MMR corrects DNA mismatches generated by DNA polymerase during replication which prevents mutations from becoming permanent in dividing cells. Thus, MMR plays a crucial role in the DNA damage response pathway to prevent short-term mutagenesis and long-term tumorigenesis. Several human MMR proteins have been identified as MutS and MutL homologues consisting of MSH2 and MLH1 heterodimers that functions in DNA mismatch/damage recognition, endonuclease activity and termination of mismatch-provoked excision. Our group has shown that humans accumulate microsatellite instability (MSI) with acquired loss of MLH1 protein in hematopoietic stem and progenitor cells as a function of age. Therefore, we employed a DNA mismatch repair deficient mouse model (MLH1+/- and MLH1-/-) to study the effects of different radiation sources including 56Fe, 28Si, 4He, 1H and γ-rays on HSCs to examine HSCs of potential astronaut population under GCR conditions. The complete blood count (CBC) data after 5 months and 9 months of whole body irradiation with different ions showed a slight dose-dependent decrease in all blood counts but absence of any significant difference in CBC of MLH1+/+ vs MLH1+/- mice. In addition, CFU and competitive repopulation data demonstrated a radiation quality effects on HSC function, but not an MLH1 effect. These results demonstrate that hematopoietic stem cell function is normal and that a MLH1 defect does not differentiate progenitor and mature effector cells following HZE radiation.To study long term effects of different ions on the potential for disease progression in a MLH1 dependent manner, we performed whole body irradiation with 56Fe, 28Si, 1H and γ-rays on MLH1+/+ and MLH1+/- mice and followed them up to 18 months post exposure. We observed that MLH1+/- mice show dramatic increases in lymphomagenesis 10-12 months after 56Fe irradiation compared to wild type mice, with greater than 60 % of MLH1+/- mice developed lymphomas at doses 10 cGy and 100 cGy compared to less than 10 % of wild type. For comparison, roughly 10 % of MLH1+/- mice developed lymphomas when mice were treated with whole body sparsely ionizing γ-rays at 100 cGy compared to none of the control. Thus the date show that MMR defects in HSCs lead to sensitization to radiation induced hematopoietic malignancy and that radiation quality effects exacerbate the sensitivity. The findings could have profound effects on astronaut screening, as well as lead to important questions regarding safety of ion therapy and development of second malignancies for cancer patients who remain on Earth. DisclosuresNo relevant conflicts of interest to declare.

Neuropeptide Y Is Critical for Bone Marrow Stromal Cells and Hematopoietic Recovery Following Injury

Bone marrow suppression is the most common limiting side-effect of conventional cancer chemo/radio therapy and is the primary cause of morbidity/mortality after accidental exposure to a high dose of ionizing radiation. The mechanisms mediating radiation-induced hematopoietic stem and stromal cell dysfunction however are not well understood. Radiation therapy causes substantial sensory neuropathy in patients. Recent studies reveal that bone marrow cells are highly innervated by sympathetic nerve fibers and that chemotherapy induced nerve-damage can impair hematopoietic regeneration, suggesting a contribution of nerve fibers in the regulation of hematopoietic stem cell and stromal cell activities. Whether irradiation- mediated nerve injury is a crucial lesion that causes deficits in hematopoietic recovery is not known. We recently discovered that differential signaling from the neuropeptide Y (NPY) receptors on bone marrow endothelial cells regulates vascular permeability and stem cell egress. NPY is an important neurotransmitter of the sympathetic nervous system and the principal adreno-medullary hormone. In this study, we found that NPY is important for reconstitution of the bone marrow niche and hematopoietic regeneration following sublethal irradiation (650 cGy). The levels of NPY were significantly reduced in bone marrow of irradiated mice suggesting damage to nerve fibers. Treatment of wild-type mice with full length NPY (1µg/mouse/day) for 3 consecutive days after irradiation markedly reduced the loss of mesenchymal stem cells (CD45-Ter119-CD31-Nestin+PDGFR+CD51+), endothelial cells (CD45-Ter119-CD31+VE-cadherin+) and hematopoietic stem and progenitor cells (SLAM LSK and LSK) in the bone marrow and promoted faster hematopoietic recovery. In addition, pharmacological NPY treatment prevented irradiation mediated nerve fiber damage. In contrast, in NPY knockout mice, regeneration of CD45neg stromal cells, SLAM LSK and LSK cells after irradiation was significantly reduced compared to wild-type controls. This reduced hematopoietic recovery in NPY deficient mice following irradiation was associated with increased apoptosis/necrosis of stromal cells and hematopoietic stem and progenitor cells. We also examined whether NPY played an intrinsic or extrinsic role in stem cell homing. Wild-type or NPY deficient BM cells were transplanted into wild-type or NPY knockout recipients. Strikingly, the homing of wild-type donor cells into NPY deficient recipients or NPY knockout donor cells into wild-type recipients were both reduced. To explore whether NPY regulates human stem cells, we treated human cord blood CD34+ cells ex vivo with NPY for 3 days and evaluated cell expansion. Long-term culture assays demonstrated that NPY treatment enhanced the clonal expansion of CD34+ cells. In conclusion, our studies suggests that NPY plays both an intrinsic and extrinsic role in hematopoiesis and that NPY-mediated protection of the sympathetic nervous system within the bone marrow can facilitate stem cell niche regeneration and enhance regenerative hematopoiesis following irradiation/injury. DisclosuresNo relevant conflicts of interest to declare.

Constitutive Expression of Inducible Cyclic Adenosine Monophosphate Early Repressor (ICER) in Cycling Quiescent Hematopoietic Cells: Implications for Aging Hematopoietic Stem Cells.

Despite extensive insights on the interaction between hematopoietic stem cells (HSCs) and the supporting bone marrow (BM) stroma in hematopoietic homeostasis there remains unanswered questions on HSC regulation. We report on the mechanism by which HSCs attain cycling quiescence by addressing a role for inducible cyclic AMP early repressor (ICER). ICER negatively transcriptional regulators of cAMP activators such as CREM and CREB. These activators can be induced by hematopoietic stimulators such as cytokines. We isolated subsets of hematopoietic cells from ten healthy donors: CD34+CD38-/c-kit + (primitive progenitor), CD34+CD38+/c-kitlow (mature progenitor) and CD34-CD38+/-/c-kitlow/- (differentiated lineage-). The relative maturity of the progenitors were verified in long-term culture initiating assay. Immunoprecipitation indicated the highest level of ICER in the nuclear extracts of CD34+/CD38- cells. Phospho (p)-CREM was also present suggesting a balance between ICER and p-CREM in HSC. ICER seems to be responsible for decrease in G1 transition, based on reduced Cdk4 protein, decreased proliferation and functional studies with propidium iodide. There were no marked changes in the cycling inhibitors, p15 and p-Rb, suggesting that ICER may act independently of other cycling inhibitors. The major effects of ICER were validated with BM mononuclear cells (BMNCs) in which ICER was ectopically expressed, and with BMNCs resistant to 5-fluorouracil- or cyclophosphamide. In total, this study ascribes a novel role for ICER in G1 checkpoint regulation in HSCs. These findings are relevant to gene therapy that require engineering of HSCs, age-related disorders that are associated with hematopoietic dysfunction and other hematological disorders.

Signs, Symptoms, and Characteristics Associated With End of Life in People With a Hematologic Malignancy: A Review of the Literature.

Identifying people with hematologic cancer who are at risk of deteriorating and dying is essential to enable open, honest discussions, leading to appropriate decision making and effective end-of-life care. . PubMed, CINAHL®, PsycINFO, and the Cochrane Central Register of Controlled Trials were searched from January 2005 to December 2015 for descriptive observational studies. . Critique of the studies was guided by the Critical Appraisal Skills Programme Cohort Study Checklist. . Twelve studies were included. The majority of studies (n = 8) sampled patients from palliative populations, and most were retrospective (n = 11). A number of signs, symptoms, and characteristics associated with end of life in people with a hematolgic malignancy were identified, including pain, hematopoietic dysfunction, dyspnea, and reduced oral intake. . The studies described a clinical scenario of deterioration, largely in a palliative population. Findings indicate that people with a hematologic malignancy share certain clinical signs of deterioration with other populations and receive a high level of medical interventions at the end of life. . Nurses are well positioned to identify many of the signs, symptoms, and characteristics found in this review and can play a key role in identifying when a person is nearing the end of life.

Generation of TALEN-mediated FH knockout rat model.

Transcription activator-like effector nucleases (TALENs) are valuable tools for precise genome engineering of laboratory animals. Here we utilized this technique for efficient site-specific gene modification to create a fumarate hydratase (FH) gene knockout rat model, in which there was an 11 base-pair deletion in the first exon of the FH gene in 111 rats. 18 live-born targeted mutation offsprings were produced from 80 injected zygotes with 22.5% efficiency, indicating high TALEN knockout success in rat zygots. Only heterozygous deletion was observed in the offsprings. Sixteen pairs of heterozygous FH knockout (FH+/−) rats were arranged for mating experiments for six months without any homozygous KO rat identified. Sequencing from the pregnant rats embryo samples showed no homozygous FH KO, indicating that homozygous FH KO is embryonically lethal. Comparatively, the litter size was decreased in both male and female FH+/− KO rats. There was no behaviour difference between the FH+/− KO and the control rats except that the FH+/− KO male rats showed significantly higher body weight in the 16-week observation period. Clinical haematology and biochemical examinations showed hematopoietic and kidney dysfunction in the FH+/− KO rats. Small foci of anaplastic lesions of tubular epithelial cells around glomeruli were identified in the FH+/− kidney, and these anaplastic cells were comparatively positive for Ki67, p53 and Sox9, and such findings are most probably related to the kidney dysfunction reflected by the biochemical examinations of the rats. In conclusion, we have successfully established an FH+/− KO rat model, which will be useful for further functional FH studies.

CD8+ T cells drive autoimmune hematopoietic stem cell dysfunction and bone marrow failure

Bone marrow (BM) failure syndrome encompasses a group of disorders characterized by BM stem cell dysfunction, resulting in varying degrees of hypoplasia and blood pancytopenia, and in many patients is autoimmune and inflammatory in nature. The important role of T helper 1 (Th1) polarized CD4+ T cells in driving BM failure has been clearly established in several models. However, animal model data demonstrating a functional role for CD8+ T cells in BM dysfunction is largely lacking and our objective was to test the hypothesis that CD8+ T cells play a non-redundant role in driving BM failure. Clinical evidence implicates a detrimental role for CD8+ T cells in BM failure and a beneficial role for Foxp3+ regulatory T cells (Tregs) in maintaining immune tolerance in the BM. We demonstrate that IL-2-deficient mice, which have a deficit in functional Tregs, develop spontaneous BM failure. Furthermore, we demonstrate a critical role for CD8+ T cells in the development of BM failure, which is dependent on the cytokine, IFNγ. CD8+ T cells promote hematopoietic stem cell dysfunction and depletion of myeloid lineage progenitor cells, resulting in anemia. Adoptive transfer experiments demonstrate that CD8+ T cells dramatically expedite disease progression and promote CD4+ T cell accumulation in the BM. Thus, BM dysregulation in IL-2-deficient mice is mediated by a Th1 and IFNγ-producing CD8+ T cell (Tc1) response.

A Molecular Profile of the Endothelial Cell Response to Ionizing Radiation.

Ionizing radiation exposure can cause acute radiation sickness (ARS) by damaging the hematopoietic compartment. Radiation damages quiescent hematopoietic stem cells (HSCs) and proliferating hematopoietic cells, resulting in neutropenia, thrombocytopenia and increased risk for long-term hematopoietic dysfunction and myelodysplasia. While some aspects of the hematopoietic response to radiation injury are intrinsic to hematopoietic cells, the recovery of the HSC pool and overall hematopoiesis is also dependent on signals from bone marrow endothelial cells (BM ECs) within the HSC vascular niche. The precise mechanisms through which BM ECs regulate HSC regeneration remain unclear. Characterization of the altered EC gene expression that occurs in response to radiation could provide a roadmap to the discovery of EC-derived mechanisms that regulate hematopoietic regeneration. Here, we show that 5 Gy total-body irradiation substantially alters the expression of numerous genes in BM ECs within 24 h and this molecular response largely resolves by day 14 postirradiation. Several unique and nonannotated genes, which encode secreted proteins were upregulated and downregulated in ECs in response to radiation. These results highlight the complexity of the molecular response of BM ECs to ionizing radiation and identify several candidate mechanisms that should be prioritized for functional analysis in models of hematopoietic injury and regeneration.

Extracellular vesicle miR-7977 is involved in hematopoietic dysfunction of mesenchymal stromal cells via poly(rC) binding protein 1 reduction in myeloid neoplasms.

The failure of normal hematopoiesis is observed in myeloid neoplasms. However, the precise mechanisms governing the replacement of normal hematopoietic stem cells in their niche by myeloid neoplasm stem cells have not yet been clarified. Primary acute myeloid leukemia and myelodysplastic syndrome cells induced aberrant expression of multiple hematopoietic factors including Jagged-1, stem cell factor and angiopoietin-1 in mesenchymal stem cells even in non-contact conditions, and this abnormality was reverted by extracellular vesicle inhibition. Importantly, the transfer of myeloid neoplasm-derived extracellular vesicles reduced the hematopoietic supportive capacity of mesenchymal stem cells. Analysis of extracellular vesicle microRNA indicated that several species, including miR-7977 from acute myeloid leukemia cells, were higher than those from normal CD34(+)cells. Remarkably, the copy number of miR-7977 in bone marrow interstitial fluid was elevated not only in acute myeloid leukemia, but also in myelodysplastic syndrome, as compared with lymphoma without bone marrow localization. The transfection of the miR-7977 mimic reduced the expression of the posttranscriptional regulator, poly(rC) binding protein 1, in mesenchymal stem cells. Moreover, the miR-7977 mimic induced aberrant reduction of hematopoietic growth factors in mesenchymal stem cells, resulting in decreased hematopoietic-supporting capacity of bone marrow CD34(+)cells. Furthermore, the reduction of hematopoietic growth factors including Jagged-1, stem cell factor and angiopoietin-1 were reverted by target protection of poly(rC) binding protein 1, suggesting that poly(rC) binding protein 1 could be involved in the stabilization of several growth factors. Thus, miR-7977 in extracellular vesicles may be a critical factor that induces failure of normal hematopoiesis via poly(rC) binding protein 1 suppression.

Repression of p53-target gene Bbc3/PUMA by MYSM1 is essential for the survival of hematopoietic multipotent progenitors and contributes to stem cell maintenance.

p53 is a central mediator of cellular stress responses, and its precise regulation is essential for the normal progression of hematopoiesis. MYSM1 is an epigenetic regulator essential for the maintenance of hematopoietic stem cell (HSC) function, hematopoietic progenitor survival, and lymphocyte development. We recently demonstrated that all developmental and hematopoietic phenotypes of Mysm1 deficiency are p53-mediated and rescued in the Mysm1−/−p53−/− mouse model. However, the mechanisms triggering p53 activation in Mysm1−/− HSPCs, and the pathways downstream of p53 driving different aspects of the Mysm1−/− phenotype remain unknown. Here we show the transcriptional activation of p53 stress responses in Mysm1−/− HSPCs. Mechanistically, we find that the MYSM1 protein associates with p53 and colocalizes to promoters of classical p53-target genes Bbc3/PUMA (p53 upregulated modulator of apoptosis) and Cdkn1a/p21. Furthermore, it antagonizes their p53-driven expression by modulating local histone modifications (H3K27ac and H3K4me3) and p53 recruitment. Using double-knockout mouse models, we establish that PUMA, but not p21, is an important mediator of p53-driven Mysm1−/− hematopoietic dysfunction. Specifically, Mysm1−/−Puma−/− mice show full rescue of multipotent progenitor (MPP) viability, partial rescue of HSC quiescence and function, but persistent lymphopenia. Through transcriptome analysis of Mysm1−/−Puma−/− MPPs, we demonstrate strong upregulation of other p53-induced mediators of apoptosis and cell-cycle arrest. The full viability of Mysm1−/−Puma−/− MPPs, despite strong upregulation of many other pro-apoptotic mediators, establishes PUMA as the essential non-redundant effector of p53-induced MPP apoptosis. Furthermore, we identify potential mediators of p53-dependent but PUMA-independent Mysm1−/−hematopoietic deficiency phenotypes. Overall, our study provides novel insight into the cell-type-specific roles of p53 and its downstream effectors in hematopoiesis using unique models of p53 hyperactivity induced by endogenous stress. We conclude that MYSM1 is a critical negative regulator of p53 transcriptional programs in hematopoiesis, and that its repression of Bbc3/PUMA expression is essential for MPP survival, and partly contributes to maintaining HSC function.

Increased Levels of Reactive Oxygen Species and Exhaustion of the Quiescent CD34+ Cells May Operate in Poor Graft Function after Allogeneic Hematopoietic Stem Cell Transplantation

Background: Poor graft function (PGF) is a serious complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, the mechanisms underlying PGF remain to be elucidated, which increases the difficulty of managing PGF. In murine study, effective cross-talk between hematopoietic stem cells (HSCs) and bone marrow (BM) micro-environment plays an important role in hematopoiesis. HSCs occupy a hypoxic BM micro-environment to protect them from oxidative stress, whereas excessive reactive oxygen species (ROS) could inhibit HSCs self-renewal and induce HSCs exhaustion resulting in hematopoietic dysfunction. We recently reported that the impaired BM micro-environment may contribute to the occurrence of PGF post-HSCT using a prospective nested case-control study (Kong Y, et al. Biol Blood Marrow Transplant. 2013;19:1465-1473). Nevertheless, it is largely unknown whether the quantitatively and functionally impaired HSCs pre- and post-HSCT operate in the occurrence of PGF in allotransplants patients.Aims: To investigate whether the quantitative and functional abnormalities of the donor BM CD34+ cells pre- and post-HSCT are involved in the pathogenesis of PGF.Methods: The hematopoietic reconstitution activities of the CD34+ cells, sorted from the donors' BM of PGF and good graft function (GGF) patients, were evaluated in xenografted NOD-Prkdcscid IL2rgnull mice as an indicator of donors' HSCs function. To further investigate the effect of oxidative stress on normal hematopoiesis post-HSCT, the BM CD34+ cells of GGF allotransplant patients were treated of hydrogen peroxide (H2O2) with or without antioxidant N-acetyl-L-cysteine (NAC) in vitro. Subsequently, a prospective nested case-control study was performed enrolling 15 patients with PGF, 30 matched patients with GGF after allo-HSCT and their healthy donors. Quantification of the frequency, intracellular ROS levels, and cell cycle status of the BM CD34+ cells were analyzed by flow cytometry pre- and post-HSCT. Colony-forming capacity was investigated in CD34+ cells post-HSCT in vitro. The study was approved by the Ethics Committee of Peking University People's Hospital and written informed consent was obtained from all subjects.Results: The hematopoietic reconstitution activity of the BM CD34+ cells in NOD-Prkdcscid IL2rgnull mice demonstrated no significant differences between the donors of PGF and GGF patients. In the subsequent in vitro study, increased ROS were found to play an important role in the exhaustion of the quiescent BM CD34+ cells of GGF patients, whereas treatment of ROS-abrogated CD34+ cells with the antioxidant NAC could partially, but significantly restore the exhaustion and colony-forming capacity of CD34+ cells. In the prospective nested case-control study, all patient- and therapy-related variables were similar between patients with PGF and GGF. Polymerase chain reaction DNA fingerprinting of the STRs confirmed 100% donor chimerism in these patients. The frequency, intracellular ROS levels and cell cycle status of the transplanted donor BM CD34+ cells showed no remarkable differences pre-HSCT. Nevertheless, the percentage of CD34+ cells post-HSCT and their colony-forming capacity, especially the quiescent CD34+ CD38low fraction, decreased remarkably in PGF patients when compared to that in GGF patients. Notably, significantly increased ROS levels were observed in CD34+ and CD34+ CD38low fractions of PGF patients post-HSCT.Summary/Conclusion: Although the frequency and function of the transplanted donor BM CD34+ cells of PGF were demonstrated normal pre-HSCT, the increased levels of ROS and exhaustion of the quiescent CD34+ cells may operate in PGF post-HSCT. Our preliminary data indicate that an impaired BM micro-environment which may hamper the hematopoietic reconstitution of the donor HSCs in the recipients, rather than the defective donor HSCs, was involved in the occurrence of PGF. Therefore, novel therapeutic approaches, such as antioxidative therapy to maintain hypoxia BM micro-environment, promise to facilitate hematopoietic recovery in PGF.Acknowledgment: Supported by the National Natural Science Foundation of China (grant nos. 81370638&81230013), and the Beijing Municipal Science and Technology Program (grant nos. Z141100000214011& Z151100004015164& Z151100001615020). DisclosuresNo relevant conflicts of interest to declare.

Physiologic Hematopoiesis Is Dependent on Stromal Expression of Laminin-γ1

OBJECTIVE: The microenvironment of the bone marrow hematopoietic niche includes: 1) blood vessels; 2) adjacent stromal cells; 3) hematopoietic cells; and 4) cytokines, growth factors, and structural support molecules. Injury to the bone marrow niche affects hematopoietic cells through loss of either physical contact with stromal cells or niche-derived growth factors. Extracellular matrix factors required to maintain the steady-state bone marrow perivascular niche and hematopoiesis have not been fully identified. Here, we examine the role of the extracellular matrix protein laminin-γ1 in adult bone marrow for maintaining the perivascular hematopoietic niche and hematopoiesis.METHODS: A global and inducible laminin-γ1 deficient mouse, hereafter referred to as mutant, was generated in which LAMC1 gene recombination could be monitored by a fluorescent reporter transgene. Tamoxifen was used to induce LAMC1 gene recombination and knock-down of laminin-γ1 protein expression in 8-12 week old mice. Tamoxifen-treated mice lacking the inducible Cre transgene were used as controls for all experiments. Analysis of mutant mice was performed 17-24 days following the first dose of tamoxifen. Bone marrow samples were examined by immunohistochemistry for the presence of laminin-1 protein, and hematopoietic tissues, including bone marrow, peripheral blood, spleen, thymus and lymph node were analyzed by flow cytometry for hematopoietic stem and progenitor cells, and mature hematopoietic cell populations, and by ex vivo hematopoietic progenitor cell colony forming assays. To examine the stromal cell contribution to laminin-γ1 mediated hematopoietic dysfunction, control and mutant mice that had not yet undergone LAMC1 gene recombination were then transplanted with wild type bone marrow. After full hematopoietic reconstitution, chimeric mice were induced with tamoxifen and hematopoietic stem and progenitor cells and mature hematopoietic cell populations were examined.RESULTS: LAMC1 gene recombination in the bone marrow of tamoxifen-treated mutant animals varied between 20-45%. With LAMC1 gene recombination, laminin-protein in the bone marrow is rapidly depleted. Bone marrow blood vessels dilate, and the bone marrow becomes hypocellular. Hematopoietic stem and progenitor cells are reduced in number, as are bone marrow B-cell progenitors, and thymic double-positive T-cells. Erythrocyte and thrombocyte numbers are not changed, nor were NK cell populations, the majority of myeloid cell subtypes, or mature B cells in the bone marrow and blood. CD8+ T-cells were increased within the bone marrow, as were CD11b+, Ly6Clo Ly6G+ myeloid cells in the peripheral blood. Examination of gene recombination within each of these cell types was not consistent with cell-intrinsic mechanism of hematopoietic alterations. Analysis of bone marrow chimeric animals identified loss of stromal cell-produced laminin-γ1 as a significant mediator of these hematopoietic alterations.CONCLUSIONS: The rapid depletion of laminin-γ1 from the bone marrow indicates a high basal turnover rate for the extracellular matrix in the bone marrow. The attendant hematopoietic dysfunction that follows loss of laminin-γ1 production in the bone marrow is specific for immature hematopoietic cell populations, and is dependent on stromal cell production of laminin-γ1. This study identifies laminin-γ1 as a niche-dependent regulator of hematopoietic stem and progenitor cell populations, and suggests that that regulation of its production and degradation may be new targets for the study of the hematopoietic niche. DisclosuresNo relevant conflicts of interest to declare.

Loss of Tifab, a del(5q) MDS gene, alters hematopoiesis through derepression of Toll-like receptor-TRAF6 signaling.

TRAF-interacting protein with forkhead-associated domain B (TIFAB) is a haploinsufficient gene in del(5q) myelodysplastic syndrome (MDS). Deletion of Tifab results in progressive bone marrow (BM) and blood defects, including skewed hematopoietic stem/progenitor cell (HSPC) proportions and altered myeloid differentiation. A subset of mice transplanted with Tifab knockout (KO) HSPCs develop a BM failure with neutrophil dysplasia and cytopenia. In competitive transplants, Tifab KO HSPCs are out-competed by wild-type (WT) cells, suggesting a cell-intrinsic defect. Gene expression analysis of Tifab KO HSPCs identified dysregulation of immune-related signatures, and hypersensitivity to TLR4 stimulation. TIFAB forms a complex with TRAF6, a mediator of immune signaling, and reduces TRAF6 protein stability by a lysosome-dependent mechanism. In contrast, TIFAB loss increases TRAF6 protein and the dynamic range of TLR4 signaling, contributing to ineffective hematopoiesis. Moreover, combined deletion of TIFAB and miR-146a, two genes associated with del(5q) MDS/AML, results in a cooperative increase in TRAF6 expression and hematopoietic dysfunction. Re-expression of TIFAB in del(5q) MDS/AML cells results in attenuated TLR4 signaling and reduced viability. These findings underscore the importance of efficient regulation of innate immune/TRAF6 signaling within HSPCs by TIFAB, and its cooperation with miR-146a as it relates to the pathogenesis of hematopoietic malignancies, such as del(5q) MDS/AML.

Understanding chronic neutropenia: life is short.

The pathophysiological mechanisms underlying chronic neutropenia are extensive, varying from haematopoietic stem cell disorders resulting in defective neutrophil production, to accelerated apoptosis of neutrophil progenitors or circulating mature neutrophils. While the knowledge concerning genetic defects associated with congenital neutropenia or bone marrow failure is increasing rapidly, the functional role and consequences of these genetic alterations is often not well understood. In addition, there is a large group of diseases, including primary immunodeficiencies and metabolic diseases, in which chronic neutropenia is one of the symptoms, while there is no clear bone marrow pathology or haematopoietic stem cell dysfunction. Altogether, these disease entities illustrate the complexity of normal neutrophil development, the functional role of the (bone marrow) microenvironment and the increased propensity to undergo apoptosis, which is typical for neutrophils. The large variety of disorders associated with chronic neutropenia makes classification almost impossible and possibly not desirable, based on the clinical phenotypes. However, a better understanding of the regulation of normal myeloid differentiation and neutrophil development is of great importance in the diagnostic evaluation of unexplained chronic neutropenia. In this review we propose insights in the pathophysiology of chronic neutropenia in the context of the functional role of key players during normal neutrophil development, neutrophil release and neutrophil survival.

Wolf-Hirschhorn syndrome: A review and update.

Since 4p- was first described in 1961, significant progress has been made in our understanding of this classic deletion disorder. We have been able to establish a more complete picture of the WHS phenotype associated with distal 4p monosomy, and we are working to delineate the phenotypic effects when each gene on distal 4p is hemizygous. Our aim is to provide genotype-specific anticipatory guidance and recommendations to families of individuals with a diagnosis of WHS. In addition, establishing the molecular underpinnings of the disorder will potentially suggest targets for molecular treatments. Thus, the next step is to determine the precise effects of specific gene deletions. As we look forward to deepening our understanding of distal 4p deletion, our focus will continue to be on the establishment of robust genotype-phenotype correlations and the penetrance of these phenotypes. We will continue to follow our WHS cohort closely as they age to determine the presence or absence of some of these comorbidities, including hepatic neoplasms, hematopoietic dysfunction, and recurrence of seizures. We will also continue to refine the critical regions for other phenotypes as we enroll additional (hopefully informative) participants into the research study and as the mechanisms of the genes in these regions are elucidated. New animal models will also be developed to further our understanding of the effects of hemizygosity as well as to serve as models for treatment development.