Primitive Hematopoietic Progenitor Research Articles

Quality control measures in cord blood banking in India - critical appraisal and recommendations.

Umbilical cord blood (UCB) is a potential source of primitive hematopoietic stem and progenitor cells (HSPC) available for clinical application to reconstitute the hematopoietic system in patients lacking conventional stem cell donors. In the last decade because the frequency of UCB stem cell transplantation has increased, the quality of UCB available in banks has become an important criterion for the success of any UCB stem cell transplant. Moreover, with the growing numbers of cord blood banks there is not only a need to improvise quality issues but to also develop a more homogenous approach in setting standards and regulations for assessing them. The present article provides an insight on the importance of cord blood banking in India and the impending requirement to provide clarity on certain issues regarding UCB banking. Furthermore, the article discusses about certain practical approaches which could perhaps be referred to as guidelines (and by no means be solely adopted as the only measures) by cord blood banks in India for establishing or maintaining a standard for quality control (QC) measures.

Lineage Tracing of Pf4-Cre Marks Hematopoietic Stem Cells and Their Progeny

The development of a megakaryocyte lineage specific Cre deleter, using the Pf4 (CXCL4) promoter (Pf4-Cre), was a significant step forward in the specific analysis of platelet and megakaryocyte cell biology. However, in the present study we have employed a sensitive reporter-based approach to demonstrate that Pf4-Cre also recombines in a significant proportion of both fetal liver and bone marrow hematopoietic stem cells (HSCs), including the most primitive fraction containing the long-term repopulating HSCs. Consequently, we demonstrate that Pf4-Cre activity is not megakaryocyte lineage-specific but extends to other myeloid and lymphoid lineages at significant levels between 15–60%. Finally, we show for the first time that Pf4 transcripts are present in adult HSCs and primitive hematopoietic progenitor cells. These results have fundamental implications for the use of the Pf4-Cre mouse model and for our understanding of a possible role for Pf4 in the development of the hematopoietic lineage.

Coxsackievirus B3 Infects the Bone Marrow and Diminishes the Restorative Capacity of Erythroid and Lymphoid Progenitors

Coxsackievirus B3 (CVB3) is known to infect stem cells in the neonatal central nervous system. Here, we evaluated the effects of CVB3 infection on the major source and repository of stem cells, the bone marrow (BM). Viral genome was detectable in BM within 24 h of infection, and productive infection of BM cells was evident, peaking at 48 h postinfection (p.i.), when ∼1 to 2% of BM cells produced infectious virus particles. Beginning at 2 to 3 days p.i., a dramatic and persistent loss of immature erythroid cells, B and T lymphocytes, and neutrophils was observed in BM and, by day 3 to 4 p.i., the femoral BM stroma was largely destroyed. Analysis of peripheral blood revealed a modest neutrophilia, a loss of reticulocytes, and a massive lymphopenia. The abundance of multipotent progenitor cells (Lin(-)/c-kit(+)/Flt3(+)) in BM declined ∼10-fold during CVB3 infection and, consistent with a deficiency of primitive hematopoietic progenitors, serum levels of the hematopoietic growth factor Flt3 ligand were dramatically elevated. Therefore, we analyzed the regenerative capacity of BM from CVB3-infected mice. Granulocyte/macrophage progenitors displayed a relatively normal proliferative ability, consistent with the fact that the peripheral blood level of neutrophils-which are very short-lived cells-remained high throughout infection. However, erythroid and lymphoid hematopoietic progenitors in BM from CVB3-infected mice showed a markedly reduced colony-forming capacity, consonant with the observed loss of both lymphocytes and immature erythroid cells/reticulocytes from the BM and peripheral blood. In summary, CVB3 infects the BM and exerts differential effects on the various hematopoietic progenitor populations.

The pharmacological activation of adenosine A1 and A 3 receptors does not modulate the long- or short-term repopulating ability of hematopoietic stem and multipotent progenitor cells in mice.

This study continues our earlier findings on the hematopoiesis-modulating effects of adenosine A1 and A3 receptor agonists that were performed on committed hematopoietic progenitor and precursor cell populations. In the earlier experiments, N (6)-cyclopentyladenosine (CPA), an adenosine A1 receptor agonist, was found to inhibit proliferation in the above-mentioned hematopoietic cell systems, whereas N (6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA), an adenosine A3 receptor agonist, was found to stimulate it. The topic of this study was to evaluate the possibility that the above-mentioned adenosine receptor agonists modulate the behavior of early hematopoietic progenitor cells and hematopoietic stem cells. Flow cytometric analysis of hematopoietic stem cells in mice was employed, as well as a functional test of hematopoietic stem and progenitor cells (HSPCs). These techniques enabled us to study the effect of the agonists on both short-term repopulating ability and long-term repopulating ability, representing multipotent progenitors and hematopoietic stem cells, respectively. In a series of studies, we did not find any significant effect of adenosine agonists on HSPCs in terms of their numbers, proliferation, or functional activity. Thus, it can be concluded that CPA and IB-MECA do not significantly influence the primitive hematopoietic stem and progenitor cell pool and that the hematopoiesis-modulating action of these adenosine receptor agonists is restricted to more mature compartments of hematopoietic progenitor and precursor cells.

Single Cell Proteomics Reveals Novel Cytokine-Producing Function of Hematopoietic Stem and Progenitor Cells

AbstractAbstract 26During infection, hematopoietic stem and progenitor cells (HSPCs) are called upon to proliferate and differentiate to produce more innate and adaptive immune cells to combat infection. Traditionally, HSPCs are thought to respond to depletion of downstream hematopoietic cells during infection. More recent evidence suggests that HSPCs may respond directly to infection and pro-inflammatory cytokines. However, little is known about the direct immune response of HSPCs and the molecular signaling regulating this response upon sensing an infection. In this study, we have combined transgenic and genetic knockout mouse models with a novel single cell barcode proteomics microchip technology to tackle these questions. We show that although long-term hematopoietic stem cells (HSCs) (defined by Lineage-cKit+Sca1+CD150+CD48-) do not secrete cytokines upon toll-like receptor (TLR) stimulation, short-term HSCs and multipotent progenitor cells (MPPs) (defined by Lineage-cKit+Sca1+, referred to as LKS thereafter) can produce copious amounts of cytokines upon direct TLR-4 and TLR-2 stimulation, indicating that LKS cells can directly participate in an immune response by producing a myriad of cytokines, upon a bacterial infection. Within the population of LKS cells we detect multiple functional subsets of cells, specialized in producing myeloid-like, lymphoid-like or both types of cytokines. Moreover, we show that the cytokine production by LKS cells is regulated by the NF-κB activity, as p50-deficient LKS cells show reduced cytokine production while microRNA-146a (miR-146a)-deficient LKS cells show significantly increased cytokine production.As long-term HSCs differentiate, they start to gain effector immune function much earlier than we had originally anticipated. In light of this finding, we should start to view the stepwise differentiation scheme of HSCs, and perhaps all other stem cells, as a strategy to sequentially gain functional capacity, instead of simply losing stemness and self-renewal ability. The remarkable ability of LKS cells to produce copious amounts of cytokines in response to bacteria may provide some protective immunity during severe neutropenia and lymphopenia or in the early stage of HSC transplantation. This study further extends the functions of NF-κB to include the regulation of primitive hematopoietic stem and progenitor cells and provides direct evidence of the bacteria-responding ability of HSPCs through the TLR/NF-κB axis. The single cell barcode proteomics technology can be widely applied to study proteomics of other rare cells or heterogeneous cell population at a single cell level. Disclosures:No relevant conflicts of interest to declare.

Depletion of Naïve Lymphocytes with Fas Ligand Ex Vivo Prevents Graft-versus-Host Disease without Impairing T Cell Support of Engraftment or Graft-versus-Tumor Activity

Graft-versus-host disease (GVHD) can be prevented by Fas-mediated selective depletion of host-sensitized donor lymphocytes ex vivo. We tested the hypothesis that Fas-mediated depletion of lymphocytes in the absence of host-specific antigenic stimulation can alleviate GVHD. Brief exposure (24 hours) of unstimulated donor lymphocytes to Fas ligand (FasL) ex vivo results in balanced apoptosis of CD8+ and CD4+ subsets with preferential depletion of CD62L and CD69, increased T regulatory fractions, and sustained responses to stimulation. This procedure ameliorates weight loss and improves the clinical and histologic score of skin and gastrointestinal GVHD with and without concurrent transplantation of hematopoietic progenitors and irrespective of conditioning-induced tissue injury. Although FasL-resistant donor T cells are less potent effectors of GVHD, they facilitate hematopoietic progenitor engraftment when infused with or after the graft and retain the potential to elaborate graft-versus-tumor reactions. These findings in a preclinical model together with the known trophic effects of FasL on primitive hematopoietic progenitors suggest that brief ex vivo incubation of hematopoietic grafts with FasL may improve the outcome and safety of clinical T cell–replete allogeneic and haploidentical transplants.

Targeting SHP2 phosphatase in Myeloproliferative Neoplasms

Targeting SHP2 phosphatase in Myeloproliferative Neoplasms

Teleost growth factor independence (gfi) genes differentially regulate successive waves of hematopoiesis

Growth Factor Independence (Gfi) transcription factors play essential roles in hematopoiesis, differentially activating and repressing transcriptional programs required for hematopoietic stem/progenitor cell (HSPC) development and lineage specification. In mammals, Gfi1a regulates hematopoietic stem cells (HSC), myeloid and lymphoid populations, while its paralog, Gfi1b, regulates HSC, megakaryocyte and erythroid development. In zebrafish, gfi1aa is essential for primitive hematopoiesis; however, little is known about the role of gfi1aa in definitive hematopoiesis or about additional gfi factors in zebrafish. Here, we report the isolation and characterization of an additional hematopoietic gfi factor, gfi1b. We show that gfi1aa and gfi1b are expressed in the primitive and definitive sites of hematopoiesis in zebrafish. Our functional analyses demonstrate that gfi1aa and gfi1b have distinct roles in regulating primitive and definitive hematopoietic progenitors, respectively. Loss of gfi1aa silences markers of early primitive progenitors, scl and gata1. Conversely, loss of gfi1b silences runx-1, c-myb, ikaros and cd41, indicating that gfi1b is required for definitive hematopoiesis. We determine the epistatic relationships between the gfi factors and key hematopoietic transcription factors, demonstrating that gfi1aa and gfi1b join lmo2, scl, runx-1 and c-myb as critical regulators of teleost HSPC. Our studies establish a comparative paradigm for the regulation of hematopoietic lineages by gfi transcription factors.

RUNX1 Mutations Are Associated With Poor Outcome in Younger and Older Patients With Cytogenetically Normal Acute Myeloid Leukemia and With Distinct Gene and MicroRNA Expression Signatures

To determine the association of RUNX1 mutations with therapeutic outcome in younger and older patients with primary cytogenetically normal acute myeloid leukemia (CN-AML) and with gene/microRNA expression signatures. Younger (< 60 years; n = 175) and older (≥ 60 years; n = 225) patients with CN-AML treated with intensive cytarabine/anthracycline-based first-line therapy on Cancer and Leukemia Group B protocols were centrally analyzed for RUNX1 mutations by polymerase chain reaction and direct sequencing and for established prognostic gene mutations. Gene/microRNA expression profiles were derived using microarrays. RUNX1 mutations were found in 8% and 16% of younger and older patients, respectively (P = .02). They were associated with ASXL1 mutations (P < .001) and inversely associated with NPM1 (P < .001) and CEBPA (P = .06) mutations. RUNX1-mutated patients had lower complete remission rates (P = .005 in younger; P = .006 in older) and shorter disease-free survival (P = .058 in younger; P < .001 in older), overall survival (P = .003 in younger; P < .001 in older), and event-free survival (P < .001 for younger and older) than RUNX1 wild-type patients. Because RUNX1 mutations were more common in older patients and almost never coexisted with NPM1 mutations, RUNX1 mutation-associated expression signatures were derived in older, NPM1 wild-type patients and featured upregulation of genes normally expressed in primitive hematopoietic cells and B-cell progenitors, including DNTT, BAALC, BLNK, CD109, RBPMS, and FLT3, and downregulation of promoters of myelopoiesis, including CEBPA and miR-223. RUNX1 mutations are twice as common in older than younger patients with CN-AML and negatively impact outcome in both age groups. RUNX1-mutated blasts have molecular features of primitive hematopoietic and lymphoid progenitors, potentially leading to novel therapeutic approaches.

DNA-Damage-Induced Differentiation in Hematopoietic Stem Cells

Aging of hematopoietic stem cells (HSCs) is accompanied by diminished functional potential. Wang et al. now provide evidence for an HSC-specific differentiation checkpoint mediated by the transcription factor BATF, which limits self-renewal of HSCs in response to the accumulation of DNA damage.

Ex vivo fucosylation improves human cord blood engraftment in NOD-SCID IL-2Rγnull mice

Delayed engraftment remains a major hurdle after cord blood (CB) transplantation. It may be due, at least in part, to low fucosylation of cell surface molecules important for homing to the bone marrow microenvironment. Because fucosylation of specific cell surface ligands is required before effective interaction with selectins expressed by the bone marrow microvasculature can occur, a simple 30-minute ex vivo incubation of CB hematopoietic progenitor cells with fucosyltransferase-VI and its substrate (GDP-fucose) was performed to increase levels of fucosylation. The physiologic impact of CB hematopoietic progenitor cell hypofucosylation was investigated in vivo in NOD-SCID interleukin (IL)-2Rγ(null) (NSG) mice. By isolating fucosylated and nonfucosylated CD34(+) cells from CB, we showed that only fucosylated CD34(+) cells are responsible for engraftment in NSG mice. In addition, because the proportion of CD34(+) cells that are fucosylated in CB is significantly less than in bone marrow and peripheral blood, we hypothesize that these combined observations might explain, at least in part, the delayed engraftment observed after CB transplantation. Because engraftment appears to be correlated with the fucosylation of CD34(+) cells, we hypothesized that increasing the proportion of CD34(+) cells that are fucosylated would improve CB engraftment. Ex vivo treatment with fucosyltransferase-VI significantly increases the levels of CD34(+) fucosylation and, as hypothesized, this was associated with improved engraftment. Ex vivo fucosylation did not alter the biodistribution of engrafting cells or pattern of long-term, multilineage, multi-tissue engraftment. We propose that ex vivo fucosylation will similarly improve the rate and magnitude of engraftment for CB transplant recipients in a clinical setting.

Karyotypic findings in chronic myeloid leukemia cases undergoing treatment

BACKGROUND:Chronic myeloid leukemia (CML) is a clonal myeloproliferative expansion of primitive hematopoietic progenitor cells.MATERIALS AND METHODS:In the present study, CML samples were collected from various hospitals in Amritsar, Jalandhar and Ludhiana.RESULTS:Chromosomal alterations seen in peripheral blood lymphocytes of these treated and untreated cases of CML were satellite associations, double minutes, random loss, gain of C group chromosomes and presence of marker chromosome. No aberrations were observed in control samples. Karyotypic abnormalities have also been noted in the Ph-negative cells of some patients in disease remission.CONCLUSION:This is a novel phenomenon whose prognostic implications require thorough and systematic evaluation.

Umbilical cord blood CD34+cell–derived progeny produces human leukocyte antigen–G molecules with immuno-modulatory functions

Human umbilical cord blood units (UCBs) are an alternative source in allogeneic-stem-cell transplantation. Human leukocyte antigen (HLA)-G is a tolerogenic molecule with a possible implication in UCB immunoregulatory effect. HLA-G expression was observed in UCB myeloid and plasmacytoid dendritic cells; in contrast, CD34(+) cells did not produce this molecule. CD34(+) cells are primitive hematopoietic progenitor cells that are present in UCB and are necessary for long-term engraftment via production of immunoregulatory molecules and a hematopoietic progeny that supports cellular recovery. The role of these cells in UCB transplantation needs further evaluation of HLA-G expression in CD34(+) cells and their hematopoietic progeny. We confirmed the absence of HLA-G expression in CD34(+) cells, whereas CD34(+)-derived progeny secreted HLA-G molecules and expressed HLA-G mRNA in in vitro cultures. Furthermore, soluble HLA (sHLA)-G molecules purified from the culture supernatants of CD34(+)-derived progeny were able to suppress lymphoproliferative response in an HLA-G dose-dependent manner. Overall these results identify CD34(+)-derived hematopoietic progeny as producers of HLA-G molecules and support a role of this antigen as an immuno-modulatory factor in UCB.

Targeted Gene Correction of Glanzmann Thrombasthenia Induced Pluripotent Stem Cells Restores Surface Expression and Fibrinogen Binding of Integrin αIIbβ3,

Abstract 4173Glanzmann Thrombasthenia (GT) is a rare, autosomal recessive disorder resulting from an absence of functional platelet integrin αIIbβ3, leading to impaired platelet aggregation and clinically presenting with severe bleeding. It is a model of an inherited platelet disorder that might benefit from corrective gene therapy. Treatment options for GT are limited and largely supportive. They include anti-fibrinolytics, activated factor VII, platelet transfusions, and bone marrow transplantation. Recent gene therapy research in a canine model for GT demonstrated that lentiviral transduction of mobilized hematopoietic stem cells could restore 6% αIIbβ3 receptors in thrombasthenic canine platelets relative to wild type (WT) canine platelets. As an alternative gene therapy strategy, we generated induced pluripotent stem (iPS) cell lines from the peripheral blood of two patients with GT and examined whether a megakaryocyte-specific promoter driving αIIb cDNA expression within the AAVS1 safe harbor locus could ameliorate the GT phenotype in iPS cell-derived megakaryocytes. Patient 1 is a compound heterozygote for αIIb with the following two missense mutations: exon 2 c.331T>C (p.L100P) and exon 5 c.607G>A (p.S192N). Patient 2 is homozygous for a c.818G>A (p.G273D) mutation adjacent to the first calcium-binding domain of αIIb, leading to impaired intracellular transport of αIIbβ3. Both patients express <5% αIIbβ3 on the surface of their platelets. Peripheral blood mononuclear cells from both GT patients and WT controls were efficiently reprogrammed to pluripotency using a doxycycline-inducible polycistronic lentivirus containing OCT4, KLF4, SOX2, and CMYC. Transgene constructs using a murine GPIbα promoter driving either a green fluorescent protein (GFP) reporter or αIIb cDNA were inserted into a gene-targeting vector specific for the first intron of AAVS1, a locus amenable to gene targeting and resistant to transgene silencing in human iPS cells. The GPIbα-driven GFP transgene was efficiently targeted into AAVS1 in WT iPS cells using zinc finger nuclease-mediated homologous recombination, as was the αIIb construct into GT iPS cell lines. PCR and Southern blot analyses confirmed single, non-random, transgene integrations. The iPS cells were differentiated into megakaryocytes using a feeder-free/serum-free adherent monolayer protocol and analyzed by flow cytometry. GFP, along with endogenous CD41 (αIIb), was initially expressed in primitive WT hematopoietic progenitor cells. GFP expression was lost in erythrocytes and myeloid cells, but maintained in CD41+/CD42+ megakaryocytes, demonstrating that this transgenic construct mirrors endogenous CD41 expression. The GT phenotype was confirmed in megakaryocytes derived from patient iPS cells, showing loss of αIIbβ3 expression. When compared to WT iPS cell-derived megakaryocytes, gene-corrected GT iPS cell-derived megakaryocytes showed >50% and >70% αIIbβ3 surface expression for patients 1 and 2, respectively. Both patients’ iPS cell-derived megakaryocytes also demonstrated fibrinogen binding upon thrombin activation. This is the first report of the generation and genetic correction of iPS cell lines from patients with a disease affecting platelet function. These findings suggest that this GPIbα-promoter construct targeted to the AAVS1 locus drives megakaryocyte-specific expression at a therapeutically significant level, which offers the possibility of correcting severe inherited platelet disorders beginning with iPS cells derived from these affected individuals. Disclosures:Lambert:Cangene: Honoraria.

High Throughput Sequencing Following Cross-Linked Immune Precipitation (HITS-CLIP) of Argonaute (AGO) Identifies Mir-9 As a Regulator of MMP2 in the Marrow Microenvironment (ME)

Abstract 2392There is increasing recognition of the role of small noncoding RNAs in post-transcriptional regulation of gene expression in diverse tissues of eukaryotic organisms including vertebrates. MicroRNAs (miRNAs) are the best studied amongst these small RNAs and are thought to act by binding to the 3' untranslated regions (3' UTRs) of mature mRNAs in a sequence-specific fashion and preventing the initiation of peptide translation and/ or initiating mRNA degradation. Recent evidence suggests that miRNA-based regulation might involve binding to regions other than 3' UTRs including coding regions. Current approaches to defining miRNA-mRNA interactions are mostly restricted to those based on bio-informatic prediction, protein down-regulation following in-vitro transfection of miRNA precursors and luciferase assays to determine binding to 3' UTRs. None of these methods however show direct interaction between a specific miRNA and its purported target RNA. Bio-informatics-based approaches are also prone to false positive and negative results given the short length of sequence matching, and reliance on heuristics and cross-species conservation. Newer genome-wide approaches like HITS-CLIP (High Throughput Sequencing following Cross Linked Immuno Precipitation, or CLIP-Seq) overcome some of these limitations by directly isolating the miRNA-mRNA interactome bound to argonaute (AGO), a critical component of the rna-induced silencing complex (RISC)1. HITS-CLIP utilizes the ability of ultraviolet (UV) light to cross-link RNAs to proteins in their close proximity. The crosslinked miRNA-mRNA-Ago complexes are then isolated and the RNA reverse transcribed to cDNA libraries and sequenced by next generation sequencing (NGS).Given the widespread role of miRNAs in several vertebrate tissues, we hypothesized that miRNA-regulation of gene expression is operant in the hematopoietic microenvironment (ME) and thus contributes to regulation of hematopoiesis. We hence used HITS-CLIP to analyze the miRNA-mRNA interactome of three key cellular components of the ME: stromal cells, endothelium and macrophages. We have previously reported on the use of the stromal cell lines Hs27a and Hs5 to define specific functional niches within the ME. Hs27a can functionally support primitive hematopoietic stem and progenitor cells (HSPC) in cobblestone areas (CSAs) and express high levels of factors known to support HSPC such as SDF1, Jagged1 and Angiopoietin1. In contrast, Hs5 drives HSPC to mature lineages and secretes high levels of cytokines like IL1, IL6 and GCSF. Human umbilical vein endothelial cells (HUVECs) and MCSF-treated CD14+ cells were utilized for the endothelial and macrophage cultures respectively. The HITS-CLIP datasets from each of these populations were enriched for a putative binding site for miR-9 in the coding region of Matrix Metalloproteinase 2 (MMP2) mRNA. MMP2 belongs to a family of endopeptidases critical in the remodeling of extracellular matrix in several tissues and in the egress/ homing of HSPC to their functional niches in the ME. Functional binding of miR-9 to MMP2 was validated by Western-blotting of stromal cells transfected with miR-9 which revealed > 50% reduction of protein levels when compared to control-transfected cells. This was also confirmed by gelatin zymography which showed significantly reduced MMP2 activity in stromal cells transfected with miR-9. Finally, to confirm direct binding of miR-9 to the putative binding region on the MMP2 transcript, we cloned this microRNA responsive region (MRE) downstream of the Renilla luciferase gene and assayed its activity by luciferase assays. MiR-9 transfection down-regulated luciferase activity > 50% confirming direct binding to the MRE. Our results show that genome-wide approaches such as HITS-CLIP can be used to define in vivo miRNA-mRNA interactions in the ME and should be considered in studies that define such interactions given the significant false-positive and false negative results associated with approaches based on bio-informatics alone. The approach can also define specific interactions between miRNAs and mRNAs such as MMP2, of relevance to regulation of the hematopoietic ME. Disclosures:No relevant conflicts of interest to declare.

Results of a Phase I Open-Label Study of Decitabine in Combination with Midostaurin (PKC412) for Elderly (Age ≥ 60) Newly Diagnosed or Relapsed/Refractory Adult Patients with Acute Myeloid Leukemia,

Abstract 3610The activated FMS-like tyrosine kinase 3 (FLT3) receptor promotes proliferation, survival and differentiation of primitive hematopoietic progenitor cells, with downstream targets including the Raf/MEK/ERK pathway. Methylation-associated silencing inactivates certain tumor suppressor genes as effectively as some mutations potentially acting as one of the cancer predisposing hits of the two-hit = leukemogenesis hypothesis. Methods:We present data from a phase I, single-center study designed to determine the MTD and recommended Phase II dose of midostaurin (PKC412), a FLT3 inhibitor, combined either sequentially (days 8–21) or concurrently (days 1–28) with decitabine 20 mg/m2 days 1–5 in AML patients. Additional endpoints: tolerability; exploratory activity assessments. Adult AML patients with relapsed or refractory (R/R) disease or elderly (≥ 60 years) patients unable to receive standard induction chemotherapy, with ECOG PS ≤2, were eligible and received treatment for 3 cycles or until disease progression. Previous hypomethylating agents were allowed. Results:Enrollment is complete with 16 patients (Table 1) with 15 evaluable for safety (1 pt withdrew prior to receiving any midostaurin). One patient in cohort 1 remains on study in cycle 14. Median number of cycles completed was 2 with 5 patients receiving 3 or more cycles. Median age was 68 years (range 48–81) with 9 males. The number of newly diagnosed and R/R patients was evenly split at 8 each. Of note, 3 of 8 patients (38%) with R/R disease had relapsed subsequent to allogeneic hematopoietic stem cell transplant (HSCT). Only 2 of 16 patients (13%) had FLT3 ITD mutations and no patient had KIT mutations. Additionally, 44% of patients had received prior therapy with either azacitidine or decitabine.Table 1Age and GenderCohortAML StatusMutation StatusMidostaurin DoseDecitabine DoseNumber of Cycles completedPrior hypomethylating therapyBest Response (CR, PR, SD, PD)Reason for Study Withdrawal81/M1NewFLT3 WT; KIT-4NoCRVoluntary74/F1R/RFLT3 WT; KIT-25 mg bid (d 8–21)2YesSDPoor QOL71/M1R/RFLT3 WT; KIT-14NoPRStill on study66/F2R/RFLT3 WT; KIT-—YesPDProgression63/F2NewFLT3 MUT; KIT-—NoNot evaluableDLT (QTc > 500)70/F2NewFLT3 WT; KIT-—NoNot evaluableVoluntary48/M2R/RFLT3 MUT; KIT-4NoCRHSCT64/M2NewFLT3 WT; KIT-50 mg bid (d 8–21)20mg/m2 IV (d 1–5)2YesCRiHSCT74/M2R/RFLT3 WT; KIT-7YesSDProgression68/M2NewFLT3 WT; KIT-1NoPRFungal Pneumonia65/F2R/R (HSCT)FLT3 WT; KIT-1NoSDViral Pneumonia54/F2R/R (HSCT)FLT3 WT; KIT-1NoSDFungal Pneumonia66/M2NewFLT3 WT; KIT-3NoSDProgression68/F3R/R (HSCT)FLT3 WT; KIT-1YesPDProgression76/M3NewFLT3 WT; KIT-50 mg bid (d 1–28)—YesNot evaluableDLT (Pulmonary)80/M3NewFLT3 WT; KIT-—YesNot evaluableDLT (Pulmonary) Safety profile:All patients whom received treatment (15) experienced adverse events (AE), the majority of which were Grade 1/2 in severity and included fatigue, nausea, mouth sores, and insomnia. Most AEs were hematologic, including anemia, thrombocytopenia, and neutropenia as expected in this population. Three patients developed dose limiting toxicities: 2 patients in cohort 3 developed pulmonary edema requiring mechanical ventilation and 1 patient in cohort 2 developed a prolonged QTc > 500 msec. All but two of 15 patients required admission for neutropenic fever. Three patients developed fungal (2) or viral pneumonia (1) and expired. However, most study treatments were given in the clinic allowing patients to spend a majority of the time as an outpatient. Response:Of twelve patients evaluable for response, 10 (83%) achieved stable disease (SD) or better while on trial. Three of the 12 patients (25%) had a complete response (CR or CRi) after 1 cycle (28 days) and 2 of these patients were able to undergo allogeneic HSCT. Of the two patients with FLT3 ITD mutations, one patient was evaluable for response and achieved a CR (cohort 2). Pharmacokinetic analysis is ongoing. Conclusion:These data indicate that the combination of decitabine with sequential midostaurin is possible without significant unexpected toxicity. The MTD for this combination was determined to be decitabine 20mg/m2 IV daily × 5 days (days 1–5) followed by midostaurin 50mg po bid days 8–21 given every 4 weeks. In addition, the combination shows promising clinical activity in R/R and untreated AML in elderly patients. Disclosures:No relevant conflicts of interest to declare.

β-Arrestin-1 Regulates Epigenetics of Whole Genome in Chronic Myeloid Leukemia Cells

Abstract 4636Chronic myeloid leukemia (CML) is a clonal myeloproliferative expansion of transformed, primitive hematopoietic progenitor cells, which is an incidence of 1 to 2 cases per 100,000 people per year and accounts for 15 percent of adult leukemias. Although the-philadelphia (Ph) chromosome and the BCR-ABL fusion gene are known as the featured molecular characteristic, the pathogenetic mechanism of CML is still not fully clear. In recent years, epigenetics, the heritable changes of gene expression without change in DNA sequence, has rapid development in the area of regulating gene transcription and expression. The epigenetic modification includes the types of DNA methylation, histone acetylation, protein phosphorylation, ubiquitination, chromatin remodeling, and so on. β-arrestin1, one key member of arrestin family that are multifunctional scaffold proteins, was reported to regulate histone acetylation and gene transcription in GPCR signaling. However, little is known about the role of β-arrestin1 in mediating the whole genome epigenetics, especially in CML. Here, we firstly knocked down β-arrestin1 expression in K562 cells, the typical cell lines for CML, and then measured and analyzed the alterations of whole genome epigenetics by combinedly using chromatin immunoprecipitation(ChIP)-on-chip and methylation chip. The data from ChIP-on-chip showed that β-arrestin-1 mediated 7498 genes histone H4 acetylation, mainly afffected the genes of cell part, cell vability and prolieration when β-arrestin1 was knocked down in CML cells. Clustering analysis showed that these affected genes of histone H4 acetylation were involved in MAPK signaling. Moreover, the data from methylation chip indicated that the methylative level of the whole genome were decreased, and genome hypermethylated was more obvious than hypomethylated, when β-arrestin1 knockout. Further analysis found that the differences of CpG island number primarily located in 500–1000bp promoter region. And methylative modifications of influenced CpG island number easily occurred in chromosome 1, 4, 13, 14, 19 and X, compared with other chromosomes. Most interestingly, the distinct changes of acetylation and methylation for some cell part genes (ZNF259, IL23A, COPS6 and COASY) were simultaneously regulated by β-arrestin1 with GO analysis, which could shift the epigenetic alterations by β-arrestin1 in CML cells. These common genes both involved in these two kinds of epigenetics, and function analysis are further study. Taken together, our study shed new light on β-arrestin1 in regulating epigenetics of whole genome in chronic myeloid leukemia cells. Disclosures:No relevant conflicts of interest to declare.

RhoA Is An Essential Regulator of Mitosis and Survival During Hematopoietic Stem Cell Differentiation to Multipotent Progenitors

Abstract 1273The maintenance and differentiation of hematopoietic stem cells (HSC) are critical for blood cell homeostasis, which is tightly regulated by a variety of factors. In spite of extensive investigation of HSC biology, however, the mechanism of regulation of HSC and progenitor cell division, particularly the unique molecular events controlling the mitosis process during HSC differentiation, remains unclear. RhoA GTPase is a critical intracellular signaling nodal that has been implicated in signal transduction from cytokines, chemokines, wnt/notch/shh, and adhesion molecules to impact on cell adhesion, migration, cell cycle progression, survival and gene expression. Recent mouse genetic studies in keratinocytes and embryonic fibroblast cells showed that RhoA is a key regulator of mitosis. By using an interferon-inducible RhoA conditional knockout mouse model (Mx-cre;RhoAlox/lox), we have made the discovery that RhoA plays an indispensible role in primitive hematopoietic progenitor differentiation through the regulation of mitosis and survival. RhoA deficient mice die at ∼10 days because of hematopoietic failure, as evidenced by a loss of bone marrow, splenocyte and PB blood cells. Syngenic as well as reverse transplant experiments demonstrate that these effects are intrinsic to the hematopoietic compartment. RhoA loss results in pancytopenia associated with a rapid exhaustion of the lin−c-kit+ (LK) phenotypic progenitor population (within 4 days after two polyI:C injections). Meanwhile, the lin−c-kit+sca1+ (LSK) primitive cell compartment is transiently increased in BM after RhoA deletion due to a compensatory loss of quiescence and increased cell cycle. Interestingly, we find that within the LSK population, there is a significant accumulation of LSKCD34+Flt2− short-term HSCs (ST-HSC) and a corresponding decrease in frequency of LSKCD34+Flt2+ multipotent progenitors (MPPs). Consistent with these phenotypes, the LK and more differentiated hematopoietic cell populations of RhoA knockout mice show an increased apoptosis while the survival activities of LSK and more primitive compartments of WT and RhoA KO mice remain comparable. These data suggest that RhoA plays an indispensible role in the step of ST-HSCs differentiation to MPP cells, possibly through the regulation of MPP cell survival. This hypothesis is further supported by a competitive transplantation experiment. Deletion of RhoA in a competitive transplantation model causes an extinction of donor derived (CD45.2+) differentiated cells (myeloid, erythroid, T and B cells) in the peripheral blood. Interestingly, bone marrow CD45.2+ LSK cells are only marginally affected by deletion of RhoA and RhoA−/− LSK cells are able to engraft into 2nd recipient, whereas CD45.2+ LK and more differentiated cells are mostly eliminated after RhoA deletion. This effect is associated with a decrease in the survival of CD45.2+ RhoA−/− LK, but not LSK cells. Further in vitro culture of isolated lin− progenitors demonstrates that RhoA deficiency results in a failure of cytokinesis, causing an accumulation of multinucleated cells, further suggesting that RhoA is essential for the cytokinesis of hematopoietic progenitors. Surprisingly, the well-defined Rho downstream target, actomyosin machinery, does not appear to be affected by RhoA knockout. We are further exploring the mechanism of RhoA contribution to the differentiation of HSCs by dissecting the signaling and functional relationship of RhoA regulated survival activity and cell cycle mitosis in early hematopoietic progenitors. Disclosures:No relevant conflicts of interest to declare.

P-selectin glycoprotein ligand-1 positive microparticles in allogeneic stem cell transplantation of hematologic malignancies

P-selectin and its receptor P-selectin glycoprotein ligand-1 (PSGL-1) mediate adhesion between leukocytes, tumor cells (including leukemias and lymphomas), and platelets, and play an important role in hematopoiesis, T cell activation, and cancer growth and metastasis. As microparticles (MPs) are released from activated or apoptotic cells, there should be significant numbers of circulating PSGL-1-bearing MPs in the blood of patients undergoing allogeneic stem cell transplantation (alloSCT). We enumerated PSGL-1-expressing MPs in plasma samples from 30 consecutive patients with hematologic disorders at different time points during the course of alloSCT by flow cytometry and analyzed their relation to cell counts, patient characteristics, and clinical outcome. Median follow-up time of surviving patients was 1,772 days (range 1272-1981 days). Nineteen patients (63.3%) died, 10 due to progression of disease (33.3%). The PSGL-1 MPs significantly declined during conditioning therapy but increased again after transfusion of donor cells and even more at the time of engraftment. Numbers >250/μL after graft transfusion were associated with a shorter time to engraftment for patients receiving fresh peripheral stem cell grafts (median, 15 vs. 21days; p = 0.049). Furthermore, low PSGL-1 MP values at those two time points were associated with a higher risk of progress/relapse in univariate analysis (p = 0.008-0.014; hazard ratio [HR] = 15.0-42.0) with cumulative incidences at 5 years of 81.8% versus 28.6% and 85.7% versus 20.0%, respectively. In conclusion, PSGL-1 microparticles show a characteristic course during alloSCT and their possible association with relapse/progress requires further evaluation of the PSGL-1/P-selectin interaction in leukemias and lymphomas.

Les cellules de la microglie

Microglia are the resident macrophage population of the central nervous system (CNS). Adequate microglia function is crucial for the homeostasis of the CNS in health and disease, as they represent the first line of defence against pathogens, contributing to immune responses, but are also involved in tissue repair and remodeling. It is therefore crucial to better understand microglia origin and homeostasis. Much controversy remains regarding the nature of microglial progenitors, as the exact contribution and persistence of embryonic and post-natal hematopoietic progenitors to the adult microglial pool in the steady state remained unclear. In this study, we show that post-natal hematopoietic progenitors do not significantly contribute to microglia homeostasis in the adult brain in mice. In vivo lineage tracing studies established that adult microglia derives from primitive hematopoietic progenitors that arise before embryonic day 8. These results identify microglia as an ontogenically distinct population in the mononuclear phagocyte system and have implications for the use of embryonically-derived microglial progenitors for the treatment of various brain disorders.