Control Hematopoietic Stem Cell Research Articles

PDK1 Controls the Differentiation of Hematopoietic Stem Cells Via Modulating ROS Levels

Hematopoietic stem cells (HSCs) exist as a rare population with two essential properties of self-renewal and differentiation. HSCs can give rise to all hematopoietic progenitor and mature cells. While critical for a full understanding of the hematopoietic process and HSC-related clinical applications, the mechanisms of self-renewal and differentiation of HSCs remain elusive. The PI3K-Akt signaling pathway plays essential roles in the regulation of hematopoiesis. 3-Phosphoinositide-dependent protein kinase 1 (PDK1) activates multiple AGC kinases including Akt and is a pivotal regulator in this pathway. PDK1 phosphorylates Akt at its T308 residue and regulates the functional development of B and T cells during hematopoiesis. However, the role of PDK1 in HSCs has not been fully defined.In this study, we generated PDK1 conditional knockout mice Vav-Cre;PDK1fl/fl (PDK1Δ/Δ) to explore the roles of PDK1 in HSCs. While PDK1Δ/Δ mice have reduced B and T cell counts as previously described, their LT-HSCs and ST-HSCs were significantly increased in comparison with WT mice while MPPs and CMPs were decreased after PDK1 deletion, indicating that the loss of PDK1 perturbed the steady-state hematopoiesis. Furthermore, although deletion of PDK1 increased the frequency of HSCs, PDK1-deficient HSCs fail to reconstitute the hematopoietic system when PDK1-deficient HSCs were used in bone marrow transplantation and competitive transplantation experiments in comparison to the WT HSCs, indicating that PDK1 is vital for hematopoiesis. To explore the mechanisms by which PDK1 regulates HSC function, we examined the cell cycle status and found the percentage of PDK1Δ/Δ HSCs was decreased significantly in G0 stage while increased in G1 and S/G2/M phases. This suggests an increase in HSC exit from a quiescent state.Since MPPs were significantly decreased in bone marrow, we examined the percentage of Annexin V+ DAPI- PDK1Δ/Δ and WT MPPs and found that they are comparable. This indicates that apoptosis did not cause the decrease in MPPs. In addition, a total of 300 LT-HSCs from PDK1Δ/Δ or WT mice and competitor cells were transplanted into lethally irradiated recipient mice to examine whether the decrease in MPPs is due to a defect in HSC differentiation. We found that less than 1% of MPPs arose from PDK1Δ/Δ HSCs 12 weeks after transplantation, indicating that PDK1 is required for the differentiation from LT-HSCs to MPPs. Because the full activation of Akt requires cooperative phosphorylation at its S473 and T308 residues by mTORC2 and PDK1, respectively, we also investigated the function of HSCs in RictorΔ/Δ PDK1Δ/Δ (DKO) mice in conjunction with RictorΔ/Δ or PDK1Δ/Δ mice to explore how mTORC2 and/or PDK1 influence Akt function in HSCs. The flow cytometric analyses of peripheral blood and bone marrow samples revealed very similar parameters of RictorΔ/Δ PDK1Δ/Δ and PDK1Δ/Δ mice. Interestingly, Rictor seemed to exert a minimal impact on HSCs and MPPs. More importantly, in contrast to RictorΔ/Δ, RictorΔ/Δ PDK1Δ/Δ HSCs failed to reconstitute the hematopoietic system after transplantation as PDK1Δ/Δ HSCs, suggesting that PDK1 plays a dominant role in the Akt-mediated regulation of HSC function.To explore the mechanism that leads to the defect in HSCs due to loss of PDK1, we assessed ROS levels in PDK1-deficient HSCs and found that PDK1-deficient LSKs and HSCs exhibit greatly reduced ROS levels when compared with the control HSCs. Treating PDK1-deficient BM cells with BSO in vitro increased cellular ROS levels and the colony counts of PDK1-deficient BM cells significantly. Notably, the recovery effect was only observed with BSO concentrations lower than 0.03 mM. This suggests that ROS levels are precisely controlled in HSCs. Higher or lower ROS levels beyond the normal range are both harmful to normal HSC functions. Since increased SDFα expression is associated with cellular ROS levels in various cells including hematopoietic cells, we also treated PDK1Δ/Δ mice with SDFα and found that it couldpartially rescue the defective differentiation ability of PDK1-deficient HSCs. In addition, we found that PDK1 deletion could significantly prolong the life span and inhibit the leukemia development in murine T-ALL model via altering leukemic cell differentiation and proliferation. Taken together, PDK1 controls HSC differentiation via regulating cellular ROS levels and regulates malignant hematopoiesis. DisclosuresNo relevant conflicts of interest to declare.

Exhaustion in Myeloid Lineage and Very Early Defect in HSPC Pool: An Embryonic Origin of Fanconi Haematological Disorders

Fanconi anemia (FA) is a genetic disorder due to mutations in one of the sixteen FANC genes involved in DNA repair. Many FA patients develop bone marrow failure (BMF) during childhood, and FA strongly predisposes to myelodysplasia syndrome and/or acute myeloid leukaemia. The pathogenesis of the BMF remains uncompletely understood. Low hematopoietic progenitor cell (HPCs) counts observed early in life and preceeding the onset of blood cytopenia in patients led we, and other, to hypothesize that the hematopoietic development might be abnormal in the FA embryo. Indeed, unlike adult hematopoietic stem cells (HSCs) which are quiescent in the BM niche, during embryonic life HSCs are in active proliferation in sites of expansion such as fetal liver and placenta, where they get amplified and acquire properties of adult HSC .We hypothesized that in FA, the FA defect in response to the replicative stress could impair the expension of the HSC pool.In order to investigate this hypothesis, we carried out studies in Fancg-/- knock out mice and in human FA fetuses obtained with informed consent from medical abortion.In Fancg-/- mice, FACS analysis revealed a 1,5- to 3-fold deficiency in hematopoietic stem and progenitor cells (HSPC) very early during embryonic development (i.e 11.5 days of gestation - E11.5) in fetal liver (FL) and placenta (Pl) (p <0.001). In both organs, this defect persists during the whole period of amplification (until E14.5 for FL and E12.5 for Pl). In vitro clonogenic assays also demonstrated a 2- fold defect in granulocyte, erythrocyte and macrophage (GEM) progenitors both in Fancg-/- FL or Pl compared to WT (p <0.001), and 4 to 5- fold defect in more immature mixed GEM progenitors in FL (p <0.001). LTC-IC frequency of the HSC-enriched Lineage- Sca1+ AA4.1+ population (LSA) of E14.5 Fancg-/- FL comforted this later result, since it was 5-fold lower than for WT.In vivo long-term hematopoietic reconstitution (LTR) assays confirmed a deficit of the HSC enriched LSA population of E14.5 Fancg-/- FL. Indeed, although the percentage of mice reconstituted was as good as that obtained with the same number of WT LSA, the CD45 Ly5.2 chimerism was reduced (49±20% vs 84±4% for 1000 LSA injected, and 56±12% vs 87±2% for 5000 LSA). Interestingly, bone marrow analysis of mice reconstituted with Fancg-/- LSA 22 weeks after injection showed a level of CD45 Ly5.2 chimerism 3-fold lower than that found in blood, spleen and thymus, as well as a very low chimerism for myeloid GEM lineages, contrasting with a high chimerism for B and T lymphoid lineages. Moreover, we were able to demonstrate that this deficit is already present at E12.5, both in Fancg-/- FL and Pl. Indeed, no mice reconstituted with 3.105 total Fancg-/- fetal liver cells, while 100% injected with the same number of WT FL cells got reconstituted with a chimerism of 59,5±5%. For Pl, when 500 000 cells were injected, reconstitution was observed in only 1 out of 3 mice for Fancg-/- (29% chimerism), and in 3 out of 3 mice for WT (88±4% chimerism).In human FA FL of 14 weeks of gestation, we also observed a 4-fold defect of HSPC with a total lack of in vitro amplification compared to control, in agreement with the mice data.Taken together, these data demonstrate that a profound deficit of HSCs and progenitors cells is present since the earlier stages of embryonic development in FA. In addition, using organotypic cultures of E11 aortas, we could show that this defect of amplification is already present in HSCs emerging from Fancg-/- aorta, which showed a 2-fold lower rate of amplification compared to WT. More importantly, our results show for the first time exhaustion in myeloid lineage of FA, in agreement with what is observed in children with FA disease. Altogether, our work suggests a role of the FA pathway during the development of the hematopoietic system leading to a deficit of amplification of HSC. Comparison of FA HSC transcriptome with that of control HSC in FL and Pl is in progress. It should allow to identify the key pathways involved in the embryonic HSC amplification that are deregulated in FA, and hopefully getting more insights in the pathogenesis of the BMF and leukemogenesis in FA patients. DisclosuresNo relevant conflicts of interest to declare.

The Role of Jarid2 in Leukemic Transformation of Chronic Myeloid Neoplasms

Despite the increasing use of targeted therapies, a subset of patients with myeloproliferative neoplasms (MPN) transform to secondary acute myeloid leukemia (sAML). MPN patients who develop sAML have a dismal outcome, with a median survival of six-months. The mechanisms and pathways that contribute to transformation from MPN to sAML have not been well delineated. The most commonly mutated genes in MPN include JAK2, MPL and CALR and are likely responsible for initiation of the disease. Although these mutations have potential roles in the pathogenesis and for some cases progression to sAML, their role in sustaining the sAML clone is challenged by the finding that some patients with post-MPN sAML who harbor these mutations in their primary MPNs have no evidence of the same mutation in the leukemic blasts. Recent genome sequencing studies identified deletions of JARID2, associated with Polycomb Repressive Complex 2 (PRC2) involved in implementing global H3K27me3, only in leukemic phase of the disease, but not in chronic phase MPNs. This data suggests that JARID2 deletion could be a sAML-specific transforming event by acting as a tumor suppressor in HSCs.We show in 32D cells, Jarid2 pull-down is able to co-immunoprecipitate core PRC2 proteins, Ezh2 and Suz12, and Jarid2 depletion using shRNAs leads to reduction in global H3K27 methylation. These data suggest Jarid2 acts in concert with PRC2 in hematopoietic cells to mediate H3K27 methylation. To examine the function of Jarid2 in vivo, we generated a Jarid2 knockout mouse model (Mx1-CRE:Jarid2fl/fl; Jarid2-KO) in which Jarid2 is conditionally deleted in HSCs. Hematopoiesis in these mice was compromised with a 3-fold reduction in hematopoietic stem cell (HSC) number, defective B-cell generation in the bone marrow (BM), a differentiation block in T-cell development in thymus, and a significant reduction in peripheral blood counts.A competitive transplantation strategy was then employed to assess the potential of Jarid2-KO HSCs. One-hundred phenotypically defined Jarid2-KO HSCs (Lineage- Sca-1+ c-Kit+ CD48- CD150+) from 8-week old mice were transplanted into lethally irradiated recipient mice along with 250,000 whole bone marrow cells from genetically distinguishable wild-type mice. Preliminary analysis of these mice show that the loss of Jarid2 is deleterious for HSC function, leading to reduced lymphoid and enhanced myeloid output and failure to maintain HSC population compared to control HSCs. To further dissect the role of Jarid2 in HSC self-renewal, 18-weeks post-transplant, 100 HSCs were re-purified from the bone marrow of primary recipient mice and transplanted into the secondary recipients along with 250,000 fresh wild-type competitor cells. In this transplant setting, Jarid2-KO HSCs failed to contribute to any PB lineages (myeloid, B-cell and T-cell). Together, these data suggest that Jarid2 is essential for HSC maintenance and is required for HSC self-renewal.To study the tumor suppressor role of Jarid2 we are using mouse models of the MPN mutation FLT3ITD in combination with Jarid2 deletion to assess the function of Jarid2 as a sAML tumor suppresser. We have established a mouse model by crossing Mx1-CRE:Jarid2fl/fl mice with FLT3ITD/+ mice to generate a Mx1-CRE:Jarid2fl/fl FLT3ITD/+ strain. These mice express the germline ITD mutation under control of the endogenous murine FLT3 promoter and develop MPN with a median survival of 10 months. To mimic the genetic progression of chronic stage MPN to sAML, the genetic deletion of Jarid2 is induced in these mice by pIpC injections once MPN is established at 3-months of age. Blood counts of these mice (2 months after Jarid2 deletion, aged 5 months old) started showing the signs of worsening MPN in the absence of Jarid2 such as, high WBC counts and increased neutrophil differentials compared to control (Mx1-CRE: FLT3ITD/+).Our ultimate goal is to understand the genetic processes associated with progression of MPN to sAML, which could eventually improve treatment outcomes for patients who can be identified as at increased risk for undergoing sAML transformation. DisclosuresNo relevant conflicts of interest to declare.

239. Preclinical Studies for the First Hematopoietic Stem Cell (HSC) Gene Editing Trial: Phase 1 Study of Beta-Thalassemia With Autologous Transplantation of Zinc Finger Nuclease-Treated HSC To Upregulate Fetal Hemoglobin

Beta-thalassemia (β-thal) and sickle cell disease (SCD) are monogenic diseases caused by mutations in the adult β-globin gene. Allogeneic hematopoietic cell transplantation (HCT) is the only curative treatment, but its application is limited by: (i) a paucity of HLA-matched donors and (ii) a fixed risk of graft vs host disease (GvHD) and transplant-related mortality. Sustained elevation of fetal hemoglobin (HbF) has a very strong clinical effect in both thalassemia and SCD with curative potential. HbF levels are under genetic control, and alleles of the transcription factor BCL11A that lower its expression result in elevation of HbF. BCL11A is thus an attractive target for gene editing of autologous hematopoietic stem cell (HSC) for HCT in β-hemoglobinopathies.We have designed zinc finger nucleases (ZFNs) that when transiently introduced into stem cells ex vivo can efficiently disrupt the BCL11A gene in hematopoietic stem cells (HSCs) by introduction of mutations following target site cleavage and non-homologous end joining. We have developed a clinical scale GMP process for mRNA electroporation of BC11A ZFNs into HSCs, and show that erythrocytes generated ex vivo from BCL11A-modified HSC have elevated fetal globin mRNA, and over 40% of all Hb is represented by HbF. A similar increase in HbF was seen in modified HSCs from subjects with β-thalassemia. Engraftment and differentiation potential of BCL11a modified and control HSCs in NSG mice were comparable. Furthermore, following engraftment into NSG mice, HSCs placed into erythropoiesis cultures maintained increased HbF levels. Preclinical safety studies included soft agar transformation, karyotyping, and double-strand break quantification following ZFN modification. Both bioinformatics guided and a novel, unbiased, oligonucleotide capture method were used to confirm low levels of off-target activity in BCL11A modified HSC as gauged by deep sequencing. In vivo carcinogenicity was evaluated by engraftment of HSC modified by BCL11A ZFNs in NSG mice. Engraftment and multilineage differentiation were comparable between groups and no evidence for carcinogenicity was found.The primary objective of the Phase I clinical study is to evaluate the safety of autologous BCL11A gene edited HSPC infusion after myeloablative (full dose busulfan) conditioning in subjects with transfusion dependent β-thalassemia major. The secondary objectives are to assess hematologic and immunological reconstitution, and to measure the treatment effects on anemia, RBC transfusion frequency, and iron burden.

Connexins: Intercellular Signal Transmitters in Lymphohematopoietic Tissues.

Life-long hematopoietic demands are met by a pool of hematopoietic stem cells (HSC) with self-renewal and multipotential differentiation ability. Humoral and paracrine signals from the bone marrow (BM) hematopoietic microenvironment control HSC activity. Cell-to-cell communication through connexin (Cx) containing gap junctions (GJs) allows pluricellular coordination and synchronization through transfer of small molecules with messenger activity. Hematopoietic and surrounding nonhematopoietic cells communicate each other through GJs, which regulate fetal and postnatal HSC content and function in hematopoietic tissues. Traffic of HSC between peripheral blood and BM is also dependent on Cx proteins. Cx mutations are associated with human disease and hematopoietic dysfunction and Cx signaling may represent a target for therapeutic intervention. In this review, we illustrate and highlight the importance of Cxs in the regulation of hematopoietic homeostasis under normal and pathological conditions.

Enforced Differentiation of Dnmt3a-Null Bone Marrow Leads to Failure with c-Kit Mutations Driving Leukemic Transformation

Genome sequencing studies of patient samples have implicated the involvement of various components of the epigenetic machinery in myeloid diseases, including the de novo DNA methyltransferase DNMT3A (Cancer Genome Atlas Research, N Engl J Med, 2013). We have recently shown that Dnmt3a is essential for normal hematopoietic stem cell (HSC) differentiation. Genetic ablation of Dnmt3a resulted in HSCs that showed diminished capacity for peripheral blood generation after serial transplantation (on a per-HSC basis), while phenotypically-defined HSCs accumulated in the bone marrow (Challen et al., Nature Genetics, 2012). Although this differentiation arrest was insufficient to cause overt disease, in these competitive transplants the presence of wild-type whole bone marrow may have suppressed malignant transformation of the mutant HSCs. Dnmt3a-null HSCs were less proliferative than counterpart control HSCs in this transplantation setting, suggesting that the cellular turnover threshold necessary to generate additional genetic and/or epigenetic lesions required for leukemogenesis was not achieved. To further understand the contribution of Dnmt3a loss-of-function in hematopoiesis, we performed non-competitive transplantation of Dnmt3a-null bone marrow. This forces the mutant HSCs to divide in vivoto regenerate the hematopoietic system following lethal irradiation, and should uncover any predispositions to transformation.Mice transplanted with Dnmt3a-null bone marrow in the absence of wild-type support cells succumbed principally to bone marrow failure (median survival 328 days) characteristic of myelodysplastic syndromes (MDS) with symptoms including anemia, neutropenia, bone marrow hypercellularity and splenomegaly with myeloid infiltration. 2/25 mice developed myeloid leukemia with >20% blasts in the blood and bone marrow. 4/25 primary mice succumbed to myeloproliferative disorders, some of which progressed to secondary leukemia after long latency. Exome sequencing was performed to identify co-operating mutations that drove leukemic transformation, and revealed c-Kit mutations found only in the Dnmt3a-null AML samples. As DNMT3A and KIT mutations can co-occur in AML and mastocytosis, we tested whether these two pathways could co-operate in vivo by ectopic introduction of c-Kit variants into hematopoietic progenitors followed by bone marrow transplantation (Figure 1). As previously reported, expression of c-KitD814V in wild-type cells lead to development of B-cell acute lymphoblastic leukemia (B-ALL). However, expression of c-KitD814V in a Dnmt3a-null background lead to acute leukemia with a much shorter latency (median survival 67 days), implicating a synergism between these pathways in vivo. Moreover, the absence of Dnmt3a also distorted the spectrum of leukemia resulting from enforced c-Kit signaling. While some of the mice transplanted with Dnmt3a-null c-KitD814V cells also succumbed to a B-ALL, 4/13 (31%) developed mastocytosis with involvement of myeloid blasts, and 4/13 (31%) mice developed a T-cell acute lymphoblastic leukemia (T-ALL). We show for the first time that these pathways can co-operate to accelerate transformation in vivo. This Dnmt3a/c-Kit disease model resembles the classical “two-hit” model of leukemogenesis in which one mutation in a hematopoietic progenitor cell inhibits differentiation (Dnmt3a loss-of-function), whilst another drives proliferation (c-Kit gain-of-function). Such mouse models present a unique opportunity to study the sequence of early events leading to HSC transformation following Dnmt3a-inactivation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Keap1‐Nrf2 system regulates cell fate determination of hematopoietic stem cells

Nrf2 is a major transcriptional activator of cytoprotective genes against oxidative/electrophilic stress, and Keap1 negatively regulates Nrf2. Emerging works have also suggested a role for Nrf2 as a regulator of differentiation in various cells, but the contribution of Nrf2 to the differentiation of hematopoietic stem cells (HSCs) remains elusive. Clarifying this point is important to understand Nrf2 functions in the development and/or resolution of inflammation. Here, we established two transgenic reporter mouse lines that allowed us to examine Nrf2 expression precisely in HSCs. Nrf2 was abundantly transcribed in HSCs, but its activity was maintained at low levels due to the Keap1-mediated degradation of Nrf2 protein. When we characterized Keap1-deficient mice, their bone marrow cells showed enhanced granulocyte-monocyte differentiation at the expense of erythroid and lymphoid differentiation. Importantly, Keap1-null HSCs showed lower expression of erythroid and lymphoid genes than did control HSCs, suggesting granulocyte-monocyte lineage priming in Keap1-null HSCs. This abnormal lineage commitment was restored by a concomitant deletion of Nrf2, demonstrating the Nrf2-dependency of the skewing. Analysis of Nrf2-deficient mice revealed that the physiological level of Nrf2 is sufficient to contribute to the lineage commitment. This study unequivocally shows that the Keap1-Nrf2 system regulates the cell fate determination of HSCs.

Dnmt3a Deletion Predisposes Hematopoietic Stem Cells To Malignant Transformation

DNA methyltransferase 3A (DNMT3A), a de novo DNA methyltransferase, is mutated in various hematological malignancies affecting both myeloid (20%), mixed (50%), and lymphoid (18%) malignancies and is associated with poor prognosis. The most frequently reported DNMT3A mutation is R882 in acute myeloid leukemia (AML), which results in altered enzyme activity, but various missense and nonsense mutations have also been found throughout the gene, suggesting that loss-of-function mutations in DNMT3A may also contribute to leukemogenesis. Our group recently showed that transplantation of HSCs from Dnmt3a knock-out (KO) mice led to increased hematopoietic stem cell (HSC) self-renewal and inhibition of differentiation, but was insufficient to cause transformation. However, in these experiments, Dnmt3a-KO HSCs were transplanted alongside wild-type whole bone marrow to quantitate HSC function, potentially protecting against malignant transformation.We hypothesized that if Dnmt3a-KO HSCs were transplanted alone, a predisposition to transformation would be uncovered. We established a large non-competitive transplantation cohort receiving 500 control or Dnmt3a-KO HSCs and monitored the mice closely for disease. Strikingly, mice with Dnmt3a-KO HSCs had significantly shorter survival (246d vs 467d, p<0.0001, Figure 1). As mice succumbed to disease, we analyzed histological changes in hematopoietic organs and performed CBCs and immunophenotyping to diagnose the diseases. We identified multiple disease classes within the Dnmt3a-KO recipients, including T-cell acute lymphoblastic leukemia, myeloproliferative disease (MPD), myelofibrosis (MF), and myelodysplastic syndromes (MDS). The relatively long disease latency suggests that acquisition of secondary hits promotes disease; identification of these secondary mutations is ongoing. [Display omitted] Here, we show that Dnmt3a deletion in noncompetitive transplanted HSCs leads to an array of hematologic disorders that models the spectrum of disorders seen in human malignancies. Since DNMT3A mutations are known early genetic lesions in leukemia development, mutations that cooperate with DNMT3A might influence the type of disease developed. This mouse model serves to validate an important role for Dnmt3a in the development of hematologic malignancies, and is also valuable for the study of future targeted therapies.Mice transplanted with Dnmt3a-KO HSCs died significantly earlier than mice transplanted with control HSCs (median survival 246 days and 467 days, p<0.0001). Fifty and 20 female mice were transplanted with 500 Dnmt3a-KO or control HSCs respectively, all at 6-8 weeks of age. Censored points indicate mice that were euthanized for unrelated reasons. Disclosures:No relevant conflicts of interest to declare.

Dnmt3b Has Few Specific Functions In Adult Hematopoietic Stem Cells But Shows Abnormal Activity In The Absence Of Dnmt3a

DNA methylation is one of the major epigenetic modifications in the vertebrate genome and is important for development, stem cell differentiation, and malignant transformation. DNA methylation is catalyzed by the DNA methyltransferase enzymes Dnmt1, Dnmt3a, and Dnmt3b. We have recently shown that Dnmt3a is essential for hematopoietic stem cell (HSC) differentiation. Ablation of Dnmt3a in hematopoietic cells (Mx1-CRE; Dnmt3a-KO) resulted in HSCs that could not sustain peripheral blood generation after serial transplantation, while phenotypically defined HSCs accumulated in the bone marrow. Recurrent somatic mutations in DNTM3A have been discovered in patients with a wide range of hematopoietic malignancies (AML, MDS, MPN, CML, T-ALL, T-cell lymphoma) suggesting a critical role for de novo DNA methylation in normal and leukemic hematopoiesis. As Dnmt3b is also highly expressed in HSCs and congenital mutations in DNMT3B can cause ICF (immunodeficiency, centromeric instability, and facial anomalies) syndrome, in this study we used a mouse model to investigate if Dnmt3b had distinct roles in HSCs.We conditionally inactivated Dnmt3b in HSCs using the Mx1-CRE system (Dnmt3b-KO) and performed serial competitive transplantation. Loss of Dnmt3b had minimal functional consequences for adult HSC function even after three rounds of transplantation. However, combinatorial deletion of both Dnmt3a and Dnmt3b (Dnmt3ab-dKO) exacerbated the differentiation defect seen in Dnmt3a-KO HSCs, leading to a dramatic accumulation of mutant HSCs in the bone marrow (>50-fold), suggesting a synergistic effect resulting from simultaneous ablation of both de novo DNA methyltransferases. The accumulation of Dnmt3ab-dKO HSCs cannot be attributed to altered proliferation or apoptosis, but is due to an imbalance between self-renewal and differentiation. RNA-SEQ of the mutant HSCs revealed loss of transcriptional integrity in Dnmt3ab-dKO HSCs including increased expression of repetitive elements, inappropriate mRNA splicing, and over-expression of HSC-specific genes.To examine the impact of loss of Dnmt3a and -3b on DNA methylation in HSCs, we performed Whole Genome Bisulfite Sequencing (WGBS). Ablation of both enzymes resulted in loss of DNA methylation that was much more extensive than that seen in the absence of Dnmt3a alone, while loss of Dnmt3b alone showed only minimal changes in DNA methylation compared to control HSCs. One puzzling finding was the observation that a subset of promoter CpG islands (CGIs) actually gained DNA methylation in Dnmt3a-KO HSCs. This CGI hypermethylation is a cancer methylome phenotype and was specific to Dnmt3a-KO HSCs (Figure 1A). The HSC transplant experiments suggest that Dnmt3a can compensate for Dnmt3b in HSCs, but Dnmt3b cannot reciprocate in the reverse situation. An explanation for increases in DNA methylation is that in the absence of Dnmt3a, abnormal function of Dnmt3b may lead to aberrant CGI hypermethylation as the hypermethylation was lost when both enzymes were conditionally inactivated. To confirm the mechanism, post-transplant Dnmt3ab-dKO HSCs were transduced with a retroviral vector encoding ectopic expression of Dnmt3b (MIG-Dnmt3b) or a control empty vector (MIG) and assessed for DNA methylation by bisulfite PCR. Using the promoter CGI of Praf2 as an example, enforced expression of Dnmt3b in Dnmt3ab-dKO HSCs resulted in increased DNA methylation at this loci compared to Dnmt3ab-dKO HSCs transduced with a control empty vector (MIG), control HSCs transduced with either MIG or MIG-Dnmt3b and untransduced HSCs (Figure 1B). It is possible that when Dnmt3b tries to compensate for Dnmt3a, the locus-specificity for targets is reduced, leading to aberrant DNA methylation patterns. Promoter CGI hypermethylation is a cancer phenotype observed in a wide range of tumors, including hematopoietic neoplasms driven by mutation in DNMT3A. Targeting DNMT3B in DNMT3A-mutation hematopoietic pathologies may be a therapeutic option for restoring normal DNA methylation and gene expression patterns. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Long Non-Coding RNAs Control Hematopoietic Stem Cells (HSC) Function

The mammalian genome encodes a significant number of long non-coding RNAs (lncRNAs). The functions of some lncRNAs have been determined in biological processes, such as cancer progression, cell-cycle regulation and embryonic stem cell (ESC) pluripotency. However, our understanding of the basic function of lncRNAs in hematopoietic stem cell (HSC) is still limited. Here, we aim to identify the full complement of lncRNAs expressed in mouse HSCs and to determine whether they control HSC function. To uncover lncRNAs expressed in HSC across different ages, we performed RNA-seq on highly purified HSCs (SP-KSL-CD150+) from 4 month (m04), 12 month (m12) and 24 month (m24) old mice. With two biological replicates for each age, deep sequencing generated 368, 311 and 293 million mapped reads for m04, m12 and m24 HSC, respectively. After combining these datasets, assembly of over 1 billion mapped reads for the HSC transcriptome reconstructed 3,104 novel transcripts, which do not correspond to any UCSC, RefSeq or Ensemble known genes. Among them, 2,853 transcripts have multiple assembled exons and a total length &gt;200 bp, representing potential lncRNAs. It has been shown that lncRNAs usually exhibit stage- or cell type-specific expression. To identify lncRNAs specifically expressed in HSC, we further performed RNA-seq on differentiated lymphoid lineage B cells (B220+) and myeloid lineage Granulocytes (Gr1+). Comparison of the expression of these 2,853 transcripts in the three cell types revealed that 173 transcripts are specifically expressed in HSC. As epigenetic mechanisms play critical roles to regulate gene transcription, we checked the chromatin map associated with those novel transcripts by ChIP-seq for H3K4me3, H3K27me3 and H3K36me3 in purified HSCs. Like protein-coding genes, these HSC specific novel transcripts typically contain the H3K4me3 mark at their transcriptional start site (TSS) and H3K36me3 along the gene body. Remarkably, one fifth of those 173 transcripts showed altered expression with HSC aging. Given that HSC function declines with aging, we hypothesize that those transcripts contribute to control HSC homeostasis. We selected three of these transcripts for further validation: LncHSC-1, LncHSC-2 and LncHSC-3. RT-PCR confirmed that they were highly expressed in stem and progenitor populations (KSL), but not in differentiated lineages (B220, CD4, CD8, Mac1, Gr1 and Ter119). Next, we generated retrovirally expressed-miRNA constructs to knockdown these transcripts. In vitro methocult colony forming assay showed that knockdown of LncHSC-1 in HSC significantly increased the colony number after second plating. Lineage analysis revealed that the majority of those cells are c-Kit+, and exhibit similar morphology, possibly representing expanded myeloid progenitors. To confirm our in vitro findings, we further examined their functions in vivo by HSC transplantation. Progenitors in which LncHSC-3 was knocked down failed to contribute to long-term hematopoietic reconstitution, as revealed by loss of retrovirally transduced population in the peripheral blood and bone marrow. In contrast, progenitors in which LncHSC-1 was knocked down resulted in augmented myeloid differentiation, consistent with in vitro CFU results that knockdown increased myeloid colony number. To understand the molecular mechanism through which lncRNAs influence hematopoiesis, we checked gene expression changes upon knockdown of specific transcripts in KSL cells. Overall, 80-100 genes were significantly changed after knockdown of specific transcripts, including cell cycle regulators and chromatin modification enzymes. For example, after LncHSC-3 knockdown, cell cycle regulator Cdkn1a (p21) expression increased, possibly contributing to the inhibition of hematopoietic reconstitution. In summary, here we carried out a comprehensive lncRNAs analysis in HSC and determined HSC specific novel transcripts. Loss-of-function experiments demonstrated that these transcripts may play important roles for HSC self-renewal and differentiation. These findings provide a useful resource to study lncRNA functions in normal hematopoiesis and disease progression. Disclosures: No relevant conflicts of interest to declare.

Growth Factor Independence 1b (Gfi1b) Regulates The Commitment, Differentiation and Expansion Of Hematopoietic Stem Cells

The efficacy of bone marrow stem cell transplantation is the therapy of choice for many hematopoietic diseases, in particular leukemia and lymphoma. This therapy is critically dependent on the transfer of sufficient numbers of hematopoietic stem cells (HSCs), which possess the capacity for self-renewal and can fully reconstitute the hematopoietic system. As such, the development of techniques for the expansion of fully functional HSCs is of significant clinical interest. By transiently manipulating the factors that govern HSC homeostasis it has been proposed that HSCs can be expanded without the loss of essential stem cell characteristics. Previously we have observed that ablation of the gene encoding the transcription factor Gfi1b in-vivo results in a dramatic expansion and mobilization of hematopoietic stem cells in the bone marrow and periphery. More recent data suggest that the blood mobilization of Gfi1b deficient HSCs is very likely mediated by a deregulation of the integrin expression. These data led us to hypothesize that Gfi1b could be a potential target for ex-vivo treatment and expansion of HSCs. Indeed, when deletion of Gfi1b was induced in whole bone marrow ex-vivo, HSCs showed a significant expansion in both in absolute number and in terms of proportion of bone marrow.We followed HSCs in ex-vivo expansion cultures from mouse bone marrow by tracking expression of the surface marker CD48, which indicates whether an HSC has transitioned to a differentiation committed multi-potent progenitor. We observed that Gfi1b null HSCs expanded without up-regulating CD48 in contrast to wt HSCs. This suggests that Gf11b deficient HSCs underwent symmetric self-renewal type cell divisions at a significantly increased frequency, when compared to wt HSCs. We had previously shown that HSCs lacking Gfi1b cycle at a faster rate than control HSCs. The combination of increased cell division and preferential self-renewal of Gfi1b-/- HSCs indicates that inhibition of Gfi1b may be the ideal strategy for ex-vivo HSC expansion. As well, in accordance with this preference for self-renewal, Gfi1b null HSCs that were cultured under myeloid differentiation conditions remained primarily in an undifferentiated state as defined by a lack of the myeloid surface markers Gr1 and Mac1. These cultures also demonstrated increased long term colony forming capacity versus controls, further supporting an undifferentiated phenotype in Gfi1b-/- cells.Because the stem cell niche is a highly complex and heterogeneous environment we also investigated whether bone marrow in which Gfi1b has been deleted exerts paracrine effects that contributed to HSC expansion. Co-Culture assays demonstrated that Gfi1b-/- bone marrow was able to induce an expansion of progenitors in wild-type bone marrow of more than 10 fold compared to Gfi1b-/+ bone marrow. Interestingly cells co-cultured with Gfi1b null bone marrow also exhibited an overall proliferation advantage after short-term cultures. This suggests that not only does Gfi1b deletion induce HSC expansion via cell intrinsic mechanisms, but also points to the possibility that this occurs through paracrine factors that alter bone marrow homeostasis. Disclosures:No relevant conflicts of interest to declare.

Inhibition Of CXCR2 As a Therapeutic Strategy In AML and MDS

Acute Myeloid Leukemia (AML) and Myelodysplastic syndrome (MDS) arise from accumulation of multiple stepwise genetic and epigenetic changes in hematopoietic stem cells (HSC) and/or committed progenitors. A series of transforming events can initially give rise to pre-leukemia stem cells (pre-LSC) as well as fully transformed leukemia stem cells (LSC), both of which need to be targeted in strategies aimed at curing these diseases. We conducted parallel transcriptional analysis of multiple, highly fractionated stem and progenitor populations in individual patients of MDS and AML (N=16) and identified candidate genes that are consistently dysregulated at multiple immature stem and progenitor cell stages. Interleukin 8 (IL8), was one of the most consistently overexpressed genes in MDS/AML Hematolpoetic Stem Cells (HSCs) and progenitors when compared to healthy control HSCs and progenitors. IL8 is a pro-inflammatory chemokine, which is able to activate multiple intracellular signaling pathways after binding to its surface receptor CXCR2. Even though increased IL8-CXCR2 signaling has been shown to promote angiogenesis, metastasis and chemotherapy resistance in many solid tumors, its role in AML and MDS is not well elucidated. We further analyzed gene expression profiles of CD34+ cells from 183 MDS patients and found significant increased expression of CXCR2 in MDS when compared to healthy controls (FDR<0.1). Most importantly, analysis of The Cancer Genome Atlas (TCGA) AML (n=200) dataset showed that CXCR2 expression was predictive of significantly adverse prognosis (log rank P value=0.0182; median survival of 245 days in cxcr2 high vs 607 days in cxcr2 low) in patients, further pointing to a critical role of IL8-CXCR2 signaling in AML/MDS.Next, we studied the functional role of IL8 and CXCR2 in AML. A panel of leukemic cell lines (THP-1, U937, KG-1, MOLM13, HL-60, K532) were screened for CXCR2 expression and revealed significantly higher expression when compared to healthy CD34+ control cells. SB-332235, a specific inhibitor of CXCR2 was used for functional studies. CXCR2 inhibition led to significant, (p<0.05) reduction in proliferation in all 6 cell lines tested and an effect was seen as early as 24 hrs of exposure. CXCR2 inhibition was found to lead to G0/G1 cell cycle arrest and trigged apoptosis in THP-1 and U937 cells (p-value 0.004 and 0.02 respectively). Incubation of primary AML/MDS bone marrow samples with SB-332235 similarly lead to significantly reduced proliferation at 24hrs, when compared to healthy CD34+ cells. Selective, and highly significant inhibition of leukemic cell growth was also seen in colony assays from primary MDS/AML samples (mean leukemic colonies in AML/MDS= 73 vs 313 in controls, P < 0.001). Interestingly, inhibition of CXCR2 in primary AML marrow samples led to induction of apoptosis in immature CD34+/CD38- cells when compared to healthy controls. Lastly, xenografting studies with THP-1 leukemic cells revealed that CXCR2 inhibitor treatment led to decreased leukemic burden and organ infiltration when compared to placebo controls in vivo.In summary we have found significantly increased expression of IL8 and its receptor CXCR2 in sorted HSCs and progenitors from AML and MDS patients. High CXCR2 expression was a marker of adverse prognosis in a large cohort of AML patients. Most importantly, in vitro and in vivo functional studies showed that CXCR2 is a potential therapeutic target in AML/MDS and is able to selectively target immature, LSC-enriched cell fractions in AML. Disclosures:No relevant conflicts of interest to declare.

Osteolineage Connexin 43 Expression Is Required for Stromal Support of Leukemic Stem Cells in Vitro and in Vivo

Abstract 4090Leukemic stem cells (LSC) are believed to be responsible for disease maintenance and/or propagation. A hallmark example is chronic myelogenous leukemia (CML). CML is a lethal malignancy that results from hematopoietic stem cell (HSC) transformation by the oncogene BCR-ABL. BCR-ABL tyrosine kinase inhibitors (TKI) are highly effective drugs to induce disease remission in CML patients and prolong their survival, but do not eliminate the LSC population and are required to be maintained throughout the patient’s life to prevent relapse. Therefore, there is a strong rationale to develop strategies to target residual LSC to enable cessation of TKI treatment without leukemia relapse. Growing evidence indicates that signals from the bone marrow (BM) microenvironment are crucial for the maintenance of LSC and it has been suggested that LSC and HSC may locate in different niches where distinct microenvironment generated signals arise. We have previously demonstrated that Connexin-43 (Cx43) regulates HSC activity by preventing chemotherapy-induced senescence/apoptosis by allowing stromal-mediated ROS scavenging (Taniguchi-Ishikawa E et al., PNAS 2012), and allows homing/engraftment of HSC through stromal mediated trans-stromal migration (Gonzalez-Nieto D et al., Blood 2012). Here, we analyzed whether the microenvironment Cx43 function is required for BCR-ABL LSC maintenance or BCR-ABL LSC are independent of microenvironment Cx43-mediated signals. For this purpose, we utilized binary transgenic mice which express p210-BCR-ABL driven by inducible (Tet-off) expression of Scl in HSC. An enriched fraction of LSC (1,000 Lin−/c-kit+/Sca1+ cells) from Dox-off Scl-tTA × TRE-BCR-ABL (SclTg) mice (CD45.2+) was competitively transplanted with 3×106 CD45.1+ BM cells as previously described by our group (Sengupta A et al., Blood 2010 & Blood 2012) into lethally-irradiated Col1α1-WT (OB/P WT) or Col1α1-Cx43f/f (OB/P Cx43Δ/Δ) mice. HSC/P BM chimera was analyzed at 18 weeks post-transplantation by measuring the BM content of CD45.2+ BM cells, CD45.2+ Lin−/c-kit+/Sca1+ (HSC/multipotential progenitors) and CD45.2+ Lin−/c-kit+/Sca1− (progenitors) BM cells. Chimera of equivalent CD45.1+ populations was used as an internal control of normal HSC engraftment. Interestingly, the content of CD45.2+ or CD45.1+ Lin−/c-kit+/Sca1+ was not significantly modified by the deficiency of Cx43 in the osteolineage microenvironment (average ∼ 0.50% of BM cellularity). However, the content of progenitors from SclTg mice was ∼85% decreased in OB/P Cx43Δ/Δ mice while the content of competitor progenitors was also decreased albeit at a lower level (∼30% reduction), indicating that the hematopoietic ability of LSC and HSC were significantly impaired by the deficiency of Cx43 in the osteolineage microenvironment. To determine whether this defect was exclusively dependent on osteolineage deficient BM stroma, we performed long-term BM cultures of Scl-tTA × TRE-BCR-ABL (or non-Tg control from littermates) LSK cells onto WT or Cx43-deficient irradiated stroma obtained from OB/P Cx43Δ/Δ mice (Gonzalez-Nieto D et al., Blood 2012). At the end of 4 weeks of culture, we observed an average reduction in the number of CFUs of 84% (vs 86% for Non-Tg control HSC) when LSC were cultured on OB/P Cx43-deficient stroma. Altogether, these data indicate that the osteolineage deficiency of Cx43 impairs the hematopoietic activity of both LSC and HSC. BCR-ABL activity in LSC does not confer independence of Cx43-mediated signals from the BM osteolineage microenvironment. Disclosures:No relevant conflicts of interest to declare.

Disseminated Mycobacterium Avium Infection Accelerates Hematopoietic Stem Cell Exhaustion,

Abstract 2365 Background:Anemia, leucopenia, or pancytopenia is a common presenting sign in a number of chronic infectious diseases such as HIV and atypical mycobacterial infection. In order to understand the pathophysiology of pancytopenia as a complication of chronic infection, we have investigated the responses of hematopoietic stem cells (HSCs) to mycobacterial infection. We previously found that HSCs proliferate in the setting of a Mycobacterium avium infection. We hypothesized that sustained infection impairs self-renewal in HSCs and leads to premature exhaustion of the stem cell compartment. Method:Testing HSC self-renewal is nontrivial and requires serial bone marrow transplantation to measure HSC totipotency over time. Three successive rounds of bone marrow transplantation were conducted using HSCs from mice with M. avium infection. Results:HSCs from infected mice were significantly less capable of sustaining trilineage peripheral blood production after serial transplantation compared to control HSCs. These findings demonstrate that self-renewal of HSCs is compromised during chronic infection. It follows that the total number of HSCs should decline with time during sustained infection. To test this, serial monthly infection with M. avium was conducted over an 8-month period. Repeatedly infected animals showed a gradual decrease in the number of committed lymphoid and myeloid progenitors and a significant depletion of HSCs in the bone marrow by 6 months following initial infection. Transcriptional profiling of HSCs from M. avium-infected animals by RNAseq revealed previously uncharacterized potential regulators of HSC self-renewal. Conclusion:Our results demonstrate that HSC self-renewal is compromised during chronic infection, leading to exhaustion of the stem cell compartment. These findings elucidate both the basis for and possible therapeutic approaches towards hematologic complications in patients with atypical mycobacterial infection, tuberculosis, HIV, and other chronic inflammatory conditions. Disclosures:No relevant conflicts of interest to declare.

Connexin-43 prevents hematopoietic stem cell senescence through transfer of reactive oxygen species to bone marrow stromal cells

Hematopoietic stem cell (HSC) aging has become a concern in chemotherapy of older patients. Humoral and paracrine signals from the bone marrow (BM) hematopoietic microenvironment (HM) control HSC activity during regenerative hematopoiesis. Connexin-43 (Cx43), a connexin constituent of gap junctions (GJs) is expressed in HSCs, down-regulated during differentiation, and postulated to be a self-renewal gene. Our studies, however, reveal that hematopoietic-specific Cx43 deficiency does not result in significant long-term competitive repopulation deficiency. Instead, hematopoietic Cx43 (H-Cx43) deficiency delays hematopoietic recovery after myeloablation with 5-fluorouracil (5-FU). 5-FU-treated H-Cx43-deficient HSC and progenitors (HSC/P) cells display decreased survival and fail to enter the cell cycle to proliferate. Cell cycle quiescence is associated with down-regulation of cyclin D1, up-regulation of the cyclin-dependent kinase inhibitors, p21(cip1.) and p16(INK4a), and Forkhead transcriptional factor 1 (Foxo1), and activation of p38 mitogen-activated protein kinase (MAPK), indicating that H-Cx43-deficient HSCs are prone to senescence. The mechanism of increased senescence in H-Cx43-deficient HSC/P cells depends on their inability to transfer reactive oxygen species (ROS) to the HM, leading to accumulation of ROS within HSCs. In vivo antioxidant administration prevents the defective hematopoietic regeneration, as well as exogenous expression of Cx43 in HSC/P cells. Furthermore, ROS transfer from HSC/P cells to BM stromal cells is also rescued by reexpression of Cx43 in HSC/P. Finally, the deficiency of Cx43 in the HM phenocopies the hematopoietic defect in vivo. These results indicate that Cx43 exerts a protective role and regulates the HSC/P ROS content through ROS transfer to the HM, resulting in HSC protection during stress hematopoietic regeneration.

Deletion of Hoxa Genes in Adult Mice Affects Hematopoietic Stem Cell and B Cell Progenitors

Abstract 2359 BACKGROUND AND OBJECTIVE:Functional compensation between homeodomain proteins has hindered the ability to unravel their role in hematopoiesis using single gene knock-outs. Although several Hox genes can expand hematopoietic stem cells (HSC) when overexpressed, it remains unclear whether these genes are required for proper adult hematopoiesis. Moreover, it has been shown that HoxB genes are dispensable for hematopoiesis, and that expression of most HoxA genes is ten-fold superior to genes from other Hox clusters in HSC enriched fetal liver populations (Bijl, 2006). Using a haploinsufficient mice for the entire HoxA cluster (HoxA+/−), we have shown that adult HSCs and progenitors are particularly sensitive to HoxA gene levels (Lebert-Ghali, 2010). Thus, we hypothesize that HoxA genes have a crucial function in definitive hematopoiesis. MATERIALS AND METHODS:To assess the role of HoxA genes in definitive hematopoiesis, we used a conditional mutant mouse model for the entire HoxA cluster in combination with an inducible Mx-Cre model. The functional effect of HoxA cluster deletion on hematopoietic cells was analysed by culture and repopulation assays. RESULTS:Highly efficient excision of HoxA cluster was achieved by 7 doses of poly(I):poly(C) treatment (91–100%). Mice (control, n=3 and Mx-CreHoxAflox/flox, n=3) were sacrificed and analysed three days after the last injection. Immunophenotyping showed a 3 to 4 fold increase of CD150+/CD48-/CD244-/Sca+/c-kit+/Lin- hematopoietic stem cells. Despite the enhancement of the HoxA−/− HSC population, single cell cultures showed that their proliferative potential in response to growth factors was significantly reduced (p=0.036) as growth was observed only for 16.6 ± 14.4% of HoxA−/− compared to 42.4 ±10.3% of control HSCs after 3-weeks of culturing. In contrast, the number of multipotent progenitor (MPP) cells (CD34+/CD135+/Sca+/c-kit+/Lin-) was reduced, indicating a partial block from the short-term HSC (CD34+/CD135-/Sca+/c-kit+/Lin-) to the MPP transition. Colony forming cell assays showed a dramatic decrease of B-cell progenitors in the bone marrow (BM) (10-fold, p=0.0079), while myeloid progenitors were not affected by the deletion. Transplantation assays demonstrated that grafts composed of > 91% HoxA−/− HSCs have slower repopulation kinetics compared to control HSCs and strongly reduced long-term engraftment (37 ± 28% and 92 ± 6% for HoxA−/− and control, respectively, 20 weeks post-transplantation). Genotyping of engrafted donor cells is currently analyzed to confirm repopulation by HoxA−/− cells. Consistent with the observations in primary mice, peripheral blood analysis revealed also a dramatic reduction of B220+ B-cell population in mice transplanted with HoxA−/− BM cells compared to control (7.1 ± 8.5% and 56.2 ±4.8% respectively p=0,000002) Altogether, in vitro assays and transplantation assays revealed that the functions of HoxA−/− HSC seem to be impaired. CONCLUSION:Together, these results show that HoxA cluster genes are required for both HSC function and B cell development, indicating that these genes are important regulators of adult hematopoiesis. Disclosures:No relevant conflicts of interest to declare.

Dnmt3a Is Essential for Hematopoietic Stem Cell Differentiation

Abstract 386Aberrant genomic DNA methylation patterns are widely reported in human cancers but the prognostic value and pathological consequences of these marks remain uncertain. CpG methylation is catalyzed by a family of DNA methyltransferase enzymes comprised of three members – Dnmt1, Dnmt3a and Dnmt3b. Mutations in the de novo DNA methyltransferase enzyme DNMT3A have now been reported in over 20% of adult acute myeloid leukemia (AML) and 10–15% of myelodysplastic syndrome (MDS) patients. However, analysis of promoter methylation and gene expression in these patients has thus far failed to yield any mechanistic insight into the pathology of DNMT3A mutation-driven leukemia. In this study, we have used a conditional knockout mouse model to study the role of Dnmt3a in normal hematopoiesis. Hematopoietic stem cells (HSCs) from Mx1-Cre:Dnmt3afl/fl mice were serially transplanted into lethally irradiated recipient mice to study the effect of loss of Dnmt3a on HSC self-renewal and differentiation. We show that loss of Dnmt3a progressively impedes HSC differentiation over four-rounds of serial transplantation, while simultaneously expanding HSC numbers in the bone marrow. Examination of the bone marrow post-transplant revealed that control HSCs showed a gradual decline in their ability to regenerate the HSC pool at each successive round of transplantation, while in contrast Dnmt3a-KO HSCs show a remarkably robust capacity for amplification, generating 40,000 – 100,000 HSCs per mouse. Quantification of peripheral blood differentiation on a per HSC basis demonstrated in the absence of Dnmt3a, a cell division is more likely to result in a self-renewal rather than differentiation fate (Figure 1). Using semi-global reduced representation bisulfite sequencing (RRBS), we show that Dnmt3a-KO HSCs manifest both increased and decreased methylation at distinct loci, including dramatic CpG island hypermethylation. Global transcriptional analysis by microarray revealed that Dnmt3a-KO HSCs show upregulation of HSC multipotency genes coupled with simultaneous downregulation of early differentiation factors (e.g. Flt3, PU.1, Mef2c), likely inhibiting the initial stages of HSC differentiation. Upregulation of key HSC regulators including Runx1, Gata3 and Nr4a2 was associated with gene-body hypomethylation and activated chromatin marks (H3K4me3) in Dnmt3a-KO HSCs. Finally, we show that Dnmt3a-KO HSCs are unable to methylate and transcriptionally repress these key HSC multipotency genes in response to chemotherapeutic ablation of the hematopoietic system, leading to inefficient differentiation and manifesting hypomethylation and incomplete repression of HSC-specific genes in their limited differentiated progeny. In conclusion, we show that Dnmt3a plays a specific role in permitting HSC differentiation, as in its absence, phenotypically normal but impotent stem cells accumulate and differentiation capacity is progressively lost. This differentiation-deficit phenotype is reminiscent of Dnmt3a/Dnmt3b-null embryonic stem (ES) cells while markedly distinct from that of Dnmt1-KO HSCs which show premature HSC exhaustion and lymphoid-deficient differentiation, demonstrating distinct roles for the different DNA methyltransferase enzymes in HSCs. In light of the recently-identified DNMT3A mutations in AML and MDS patients, these studies are the first biological models linking mutation of Dnmt3a with inhibition of HSC differentiation which may be one of the first pathogenic steps occuring in such patients. [Display omitted] Disclosures:Issa:Novartis: Honoraria; GSK: Consultancy; SYNDAX: Consultancy; Merck: Research Funding; Eisai: Research Funding; Celgene: Research Funding; Celgene: Honoraria; J&J: Honoraria.

Gene Expression Profiling of Murine HOXB4-Transduced HSCs Shows Multiple Counter-Regulatory Signals That May Control HSC Pool Size

Abstract 2361HOXB4 is a homeobox transcription factor that can induce hematopoietic stem cell (HSC) expansion both in vivo and in vitro. An interesting feature of HOXB4-induced HSC expansion is that HSC numbers do not exceed normal levels in vivo due to an unexplained physiological capping mechanism. To gain further insight into HOXB4 regulatory signals, we transplanted mice with bone marrow cells that had been transduced with a MSCV-HOXB4-ires-YFP vector and analyzed gene expression profiles in HSC-enriched populations 20 weeks after transplant, a time point at which HSC numbers have expanded to normal levels but no longer increasing beyond physiologic levels. We used Affymetrix arrays to analyze gene expression profiles in bone marrow cells sorted for a Lin−Sca-1+c-Kit+ (LSK), YFP+ phenotype. Using ANOVA, we identified1985 probe sets with >2 fold difference in expression (FDR<, 0.1) relative to a control vector-transduced LSK cells. A cohort of genes was identified that were known positive regulators of HSC self-renewal and proliferation. Hemgn, which we identified in a previous screen as a positive regulator of expansion and a direct transcriptional target of HOXB4, was 3.5 fold up-regulated in HOXB4 transduced LSKs. Other genes known to be important for HSCs survival, self-renewal and differentiation were upregulated to significant levels including N-myc, Meis1, Hoxa9, Hoxa10 and GATA2. Microarray data for selected genes was validated by quantitative real-time PCR on HOXB4 transduced CD34low LSK cells, a highly purified HSC population, obtained from another set of transplanted mice at the 20 week time point.In contrast, other gene expression changes were noted that would potentially limit or decrease stem cell numbers. PRDM16, a set domain transcription factor critical for HSC maintenance and associated with clonal hematopoietic expansions when inadvertently activated as a result of retroviral insertion, was dramatically down-regulated on the expression array and 7.6 fold decreased in the real time PCR assay of CD34low LSK cells. TFG-beta signaling is a well defined inhibitor HSC proliferation and utilize Smad proteins as downstream effectors. Expression of Smad1 and Smad7 were significantly upregulated on the LSK expression array and 8.1 and 3.5 fold up-regulated by qPCR in CD34low LSK cells. Another potential counter-regulatory signal was down regulation of Bcl3 mRNA, a potential anti-apoptotic effector in HSCs. We hypothesize that the HOXB4 expansion program involves activation of genes that lead to increased HSC numbers with later activation of counter-regulatory signals that limit expansion to physiologic numbers of HSCs in vivo. We are now examining how this program changes at various time points after transplantation and hypothesize the capping limits are set at relatively later time points during reconstitution. We also are studying the functional effects of these gene expression changes, and in particular, whether enforced expression of HOXB4 and PRMD16 will result in uncontrolled HSC proliferation and/or leukemia. Disclosures:No relevant conflicts of interest to declare.

Ott1(Rbm15)-Deficient Hematopoietic Stem Cells Are Unable to Maintain Quiescence During Replicative Stress and Display Features of Premature Aging,

Abstract 3433With age, hematopoietic stem cells (HSCs) have numerical expansion, skewing towards myeloid development, loss of lymphoid potential, an underlying pro-inflammatory state and loss of self-renewal potential thus severely limiting responses to hematopoietic stress, ultimately leading to bone marrow failure. The mechanisms and pathways responsible for these changes in aged HSCs are incompletely understood. Using a conditional allele of Ott1, a gene originally isolated as the 5’ fusion partner in t(1;22) acute megakaryocytic leukemia, we previously found a global regulatory role for the gene in hematopoiesis. Deletion of Ott1 in adult mice utilizing Mx1-cre recapitulated certain aspects of aging hematopoiesis including increased Lin−Sca1+c-Kit+ (LSK) population, myeloid expansion and decreased lymphopoiesis. The LSK compartment was further characterized using SLAM and CD34/Flk2 markers and demonstrated normal levels of LT-HSCs and increased ST-HSCs. Despite sufficient LT-HSC numbers, Ott1-deleted bone marrow was unable to competitively or non-competitively repopulate irradiated recipients. To exclude a homing or engraftment effect, Ott1flox/null Mx1-cre bone marrow was transplanted with competitor then excised post-engraftment. The rapid loss of the Ott1-deficient graft demonstrated Ott1 is required for maintenance under competitive stress. In contrast, primary mice undergoing Ott1 excision lived a normal lifespan and were able to maintain sufficient hematopoiesis although with a partial reduction in bone marrow clonagenicity showing loss of Ott1 is not limiting under steady state conditions. To test the HSC requirement for Ott1 under replicative stress, Ott1 knockout mice were challenged with 5-fluorouracil (5-FU). Ott1-deleted mice treated with 5-FU displayed delayed peripheral blood neutrophil recovery and showed accelerated bone marrow failure. Cell cycle analysis of steady state Ott1 knockout HSCs showed a similar profile to wild type controls, however, after 5-FU treatment, the G0 fraction was dramatically reduced. The G0 fraction is associated with the quiescent, self-renewing HSC population, therefore, Ott1 is required for maintaining HSC quiescence during replicative stress but not steady state hematopoiesis.To more specifically assess whether the functional hematopoietic changes seen after loss of Ott1 were accompanied by alterations in known aging-associated pathways, Gene Set Enrichment Analysis comparing Ott1-deleted HSCs in steady state to aged HSCs was performed and showed a highly enriched gene expression signature (NES 2.02 p<0.0001). Physiologic sequelae of HSC aging were observed after Ott1 excision including activation of NFκβ, elevation of reactive oxygen species (ROS), increase in DNA damage (γH2A.X levels) and activation of p38Mapk. Although ROS was elevated under steady state conditions, neither apoptosis, senescence or proliferation was significantly different from wild type control HSCs. Furthermore, anti-oxidant treatment with N-acetyl-cysteine was unable to rescue the HSC maintenance defect of the Ott1 knockout, signifying additional requirements in HSCs for Ott1 beyond regulation of ROS. An observed increase of mitochondrial mass in Ott1-deleted HSCs suggests an upstream function for Ott1 in metabolic control, potentially contributing to ROS generation or degradation. In summary, we have demonstrated an essential role for Ott1 in maintaining HSC quiescence during replicative stress and shown loss of Ott1 leads to the acquisition of key gene expression patterns and pathophysiologic changes associated with aging. These data suggest Ott1 functions in part to oppose specific consequences of aging in the hematopoietic compartment. Ott1 and Ott1-dependent pathways therefore represent a potential therapeutic target to prevent the morbidity and mortality arising from age-related defects in hematopoiesis. Disclosures:No relevant conflicts of interest to declare.

A Functional In Vivo RNAi screen Involving Jumonji C Domain Containing Candidates Unravels Kdm5b As a Negative Modulator of Hematopoietic Stem Cell Self-Renewal

Abstract 385Epigenetic modifications influence chromatin accessibility, impacting on cell fate decisions, such as stem cell self-renewal and differentiation, in both normal and leukemic stem cells (LSC). To investigate the putative role of histone demethylases (HDM) in modulating primary hematopoietic stem cell (HSC) fate, an in vivo functional screen was performed, using an RNAi based strategy, involving 25 members of the Jumonji (JmjC) domain protein family.As a first step, expression profile studies of these gene candidates were undertaken. Transcripts of all these enzymes were detected in isolated HSC populations (frequency 1:2) from fetal liver (n=1) and bone marrow (n=2), except for Hairless. As compared to unsorted bone marrow (BM), stem cells harboured higher expression of Jarid1b (relative-fold enrichment (RQ) of 3.9±1.7), Jmjd2d (RQ3.8±1.9), and Jhdm1b (3.1±1.7). Next, 5shRNA were designed against each of the 25 JmjC containing proteins, and cloned into a retroviral LMP vector encoding GFP to permit tracking of transduced cells in vivo. HSC-enriched CD150+CD48−Lin−cells (∼60 LT-HSC) were infected over 5 days by co-culture with retroviral producer cells in an arrayed 96-well format, with one shRNA per well. Directly after infection, the in vivo reconstituting potential of ¼ of each well was evaluated through duplicate competitive repopulation assays involving the co-transplantation of 1.5 × 105 congenic BM competitor cells into irradiated recipients. The remaining cell fraction served to asses gene transfer by GFP epifluorescence measurements, and RNA isolated from sorted GFP+ cells was used to evaluate gene knockdown levels by Q-RT-PCR analysis. Blood reconstitution was evaluated at an early (4wks) and late time point (16–20wks), tracking the contribution of the donor CD45.1+ transduced (GFP+) cells to recipient hematopoiesis over time. As baseline references, sh-RNA to Luciferase (no effect) and the histone acetyl transferase Myst3 (stem cell loss) were used, as well as Hoxb4 over-expression (stem cell expansion). The primary screen, followed by validation experiments, unravelled one positive (Jhdm1f/Phf8) and two negative (Jarid1b, Hif1an) regulators of HSC activity. The strongest impact was seen with Jarid1b knockdown, and the resulting gain in HSC activity. As a confirmation step, cells were kept in culture for one week, to better contrast an increase in HSC activity, compared to control HSC. After 7 days in vitro, 1/8 equivalents of single well cultures were transplanted into 3 mice, and blood reconstitution levels serially assessed. Cells transduced with sh-RNA against Jarid1b contributed more significantly to host hematopoiesis than sh-RNA Luciferase transduced cells (58±16% vs 26±3% GFP), or Hoxb4 over-expressing cells (37±2% GFP), at comparable gene transfer rates, at the 16 week time point and beyond (3 independent experiments). Long-term HSC frequencies were evaluated from these cultures, and found to be 6–10 fold increased in shJari1d1b-cell cultures. In long-term recipients, differentiation potential of these cells was preserved, as evidenced by CD4+CD8+ thymic cells, B220+ splenic cells and CD11b+ bone marrow cells in the GFP positive contingent. Clonality studies on DNA isolated from these sorted populations confirmed oligoclonality of the stem cell expansion, and HSC pluripotency. There were no cases of leukemic transformation in all of the transplant recipients (n>30). As assessed by Q-RT-PCR, levels of HoxA5, HoxA9, HoxA10 and CxCl5 were increased in day7 sh3Jarib1b-cells (vs ctl), while the levels of the tumor suppressors Cav1, Sash1 and Egr1 were decreased. A more detailed assessment of the HoxA cluster revealed predominant expression of 5’ cluster genes in expanding shJarib1b-cells, from HoxA5 to HoxA11, with a concomitant increase in the level of H3K4 tri-methylation, as assessed by ChIP-CHIP.In conclusion, HDM of the JmjC family can modulate HSC activity, both positively and negatively. These data suggest that the H3K4 demethylase Jarid1b (KDM5b) restrains stem cell self-renewal, acting as a co-repressor, possibly via epigenetic regulation of the HoxA gene cluster, among other target genes. This observation could be further exploited as an HSC expansion strategy. Disclosures:No relevant conflicts of interest to declare.