Normal Hematopoietic Stem Cell Function Research Articles

Hypoxic induction of vascular endothelial growth factor regulates erythropoiesis but not hematopoietic stem cell function in the fetal liver

Hypoxia is an important factor in the hematopoietic stem cell (HSC) niche in the bone marrow, but whether it also plays a role in the regulation of fetal liver (FL) HSCs is unclear. Vascular endothelial growth factor A (VEGFA) is essential for adult HSC survival, and hypoxic induction of VEGFA in adult HSCs is required for proper function. Loss of hypoxia-regulated VEGFA expression increases the number of phenotypically defined hematopoietic stem and progenitor cells in the FL, but whether stem cell function is affected in FL HSCs has not, to our knowledge, been assessed. We show that fetal erythropoiesis is severely impaired when hypoxic induction of VEGFA is lacking. FL HSCs deficient for hypoxia-induced VEGFA expression have normal HSC function, arguing against a hypoxic FL HSC niche. However, after adaptation of FL HSCs to the bone marrow microenvironment, FL HSCs lose their function, as measured by serial transplantation.

Culture Of Mobilized Rhesus Macaque CD34+ Cells In Hypoxic Conditions Does Not Improve Lentiviral Transduction Efficiency In Long-Term Repopulating Hematopoietic Stem Cells

Low oxygen levels have been proposed to play a fundamental role in the maintenance of normal hematopoietic stem cells (HSC) function. We investigated whether culture of rhesus macaque mobilized CD34+ cells under low oxygen conditions (1-5% O2) could improve lentiviral transduction efficiency in HSCs compared to culture under atmospheric O2 conditions (21%). Mobilized CD34+ cells from 4 animals were prestimulated for 12 hours in the presence of cytokines and subsequently divided in two equal fractions for transduction with SIN-lentiviral vectors (MOI=100) expressing GFP or TdTomato fluorescent proteins for 48 hours. In 3 animals, cells were transduced with GFP vectors in hypoxia and TdTomato vectors in normoxia. These conditions were reversed in one animal. A portion of the transduced cells was used for phenotypic, cell cycle, clonogenic and apoptosis assays. The remaining cells from both fractions were combined and injected intravenously in lethally irradiated rhesus macaques and in vivo gene marking was measured in peripheral blood granulocytes for up to 21 months after transplantation. The numbers of total live cells and CD34+ cells after 2.5 days in culture were not significantly different compared to baseline for both hypoxic and normoxic conditions (p=0.85 and p=0.84, respectively). Similarly, numbers of CD34+CD38- cells were stable compared to baseline in hypoxia and normoxia (p=0.94). After 2.5 days of culture, the total number of colony-forming cells increased 1.4-fold under both hypoxic and normoxic conditions (p=0.69) compared to freshly isolated CD34+ cells. At baseline, the majority (50.6 + 4.4%) of the CD34+ cells were in the G0 phase of the cell cycle. After 2.5 days in culture under hypoxic or normoxic conditions, the percentages of cells in G0 were 19.9 + 8.8% and 18.6 + 5.9%, respectively (p=0.82). The differences in percentages of cells in the G1 and S/G2/M phases of the cell cycle were also not statistically significant (p=0.175 and p=0.732, respectively). The pO2 had no impact on cell death (18.2 + 5.3% in hypoxia and 16.8 + 4.8% in normoxia, p=0.69) or on the percentages of apoptotic cells (11.1 + 6.7% in hypoxia and 8.4 + 2.3% in normoxia, p=0.47). When considering the bulk of CD34+ cells after transduction with lentiviral vectors, the percentages of GFP-marked cells were consistently higher (range 1.8 to 2.2-fold, mean 2.1-fold) compared to TdTomato-marked cells independently of the transduction conditions used, consistent with a 2.1-fold intrinsic superiority of GFP-based lentiviral vectors compared to TdTomato vectors at equivalent MOI. Taking into account the inherent vector differences, transduction efficiencies were similar in normoxia (70.7 + 11.7%) and hypoxia (68.0 + 16.3%) (p=0.80). When adjusted for the intrinsic superiority of GFP-based vectors, the contribution to long-term in vivo gene marking measured by flow cytometry and quantitative PCR was comparable between cells transduced in 21% pO2 (9.0 + 4.5%) and 5% pO2 conditions (8.4 + 4.9%) (p=0.87). Consistent with these data, GFP and TdTomato-labeled hematopoietic cells were equally visualized, using a confocal/2-photon hybrid microscopy approach, in BM biopsy specimens collected at various time points after transplantation. Given recent evidence suggesting that lower oxygen concentrations (<1.5%) may be required to stabilize HIF-1a, a key sensor of hypoxic conditions, transduction was performed under extreme hypoxic conditions (1% O2) in one animal. While the overall viability of the bulk CD34+ cells after 2.5 days of transduction under extreme hypoxia was comparable to cells cultured in normoxia, their contribution to long-term in vivo marking was negligible (0.3%) compared to cells transduced under normoxic conditions (2.4%), suggesting toxicity of very low oxygen levels on HSCs. Overall, when current methodologies used for the genetic manipulation of HSCs for gene therapy applications were performed under hypoxic conditions, susceptibility to lentiviral transduction of CD34+ cells was not ameliorated compared to cells cultured in 21% O2 conditions in the rhesus macaque transplantation model. The short culture times (2-3 days) used in recent lentivirus-based gene therapy clinical trials and replicated in this study may not be sufficient to impact the phenotype of long-term repopulating HSCs and do not warrant incorporation of hypoxia in current gene therapy protocols. Disclosures:No relevant conflicts of interest to declare.

The Short Lef-1 Isoform, Lacking The β-Catenin Binding Domain, Is Not Acting As a Dominant Negative Lef-1 Variant, But Is a Hematopoietic Active Protein With Unique DNA Binding Properties

Studies in the hematopoietic system of human and mice have demonstrated that the Wnt signaling pathway is essential for maintenance, activation and proliferation of normal hematopoietic stem cells. Lef-1 is a member of the Lef-1/T-cell-factor (Tcf) family of transcription factors regulated by the canonical Wnt signaling pathway and generally directly interacts with β-catenin in transcriptional complexes to induce expression of target genes, including the cell cycle regulators cyclin D1 and c-myc. We have previously shown that aberrant expression of Lef-1 perturbs normal hematopoietic stem cell (HSC) and progenitor function and induces acute myeloid leukemia in the murine bone marrow transplantation model and that it is a novel prognostic independent factor in patients with normal karyotype AML (Petropoulos et al. JEM, 2008; Metzeler et al., Blood 2012). Furthermore, we could recently demonstrate that shRNA mediated depletion of Lef-1 drastically compromises the function of long-term repopulating stem cells (Edmaier et al., Leukemia, in press), indicating a vital function of Lef-1 at the level of the most primitive stem cell compartment. So far, all these Lef-1 functions are ascribed to the full-length transcript of Lef-1, which acts as transcriptional mediator of Wnt signaling via its β-catenin binding domain, whereas the shorter Lef-1 isoform, deriving from an intronic promoter is thought to function as a dominant negative variant. To dissect the role of the ‘dominant negative' Lef-1 isoform we first retrovirally engineered primary murine bone marrow cells to express the N-terminal deleted Lef-1 (Lef-1d56), lacking the β-catenin binding domain in comparison to the full-length Lef-1 (Lef-1WT) and the empty retroviral EGFP control. Loss of binding to β-catenin was validated by Co-IP for Lef-1d56. Of note, Lef-1d56 induced a 2,05fold and significant increase at the level of clonogenic cells in vitro compared to the control (n=3; p<0,05) and did not differ substantially from the activity of Lef-1 WT. β-catenin binding did not impact Lef-1 activity at the level of the short-term repopulating stem cells as documented in the CFU-S assay with almost comparable CFU-S frequencies between the wild-type and the mutant Lef-1 (d56) (73 CFU-S/1x105 and 64 CFU-S/1x105 , respectively, compared to 38 CFU-S/1x105 for the EGFP control; p<0.05). In clear contrast, loss of β-catenin interaction reduced the CRU frequency dramatically (1:597.197(Lef-1 WT) and 1:1.675.238 (Lef-1 d56), respectively and 1: 1.233.152 for the EGFP control; p=0,066), indicating the necessity of Lef-1 to collaborate with β-catenin at the level of the long-term repopulating stem cell. Retroviral expression of Lef-1 d56 in murine bone marrow stem and progenitor cells for 48h induced a distinct gene expression profile and deregulated more genes than Lef-1WT compared to the EGFP control. Differentially expressed genes between Lef-1d56 and Lef-1WT comprised known hematopoietic factors such as like Gata2, Ets1 and genes associated to the G-protein coupled receptor protein signaling. ChIP-Seq in hematopoietic murine cells revealed unique binding sites for Lef-1d56 compared to Lef-1WT with binding of the Lef-1d56 to promoters of Wnt5a and CD81. Taken together, our data clearly indicate that loss of the β-catenin binding site does not convert Lef-1 into an overall dominant negative variant, but creates a ‘neomorphic' isoform with distinct biological and DNA binding properties. Analyses are ongoing which quantify the expression of LEF-1WT and the β-catenin domain lacking LEF-1 isoform in over 100 patients with normal karyotype AML in comparison to normal human stem and progenitor cells to test potential differences between normal and leukemic cells as well as associations of the two LEF-1 transcripts with the mutational status, gene expression and treatment outcome in this AML patient group. Disclosures:No relevant conflicts of interest to declare.

Polycomb Repressive Complex 2 Regulates Normal Hematopoietic Stem Cell Function in a Developmental-Stage-Specific Manner

Recent studies point to a pivotal role of Polycomb repressive complex 2 (PRC2) in stem cell function and cancer. Loss-of-function approaches targeting individual PRC2 subunits have, however, generated findings that are difficult to reconcile. Here, we prevent assembly of both Ezh1- and Ezh2-containing PRC2 complexes by conditional deletion of Eed, a core subunit, and assess hematopoiesis. We find that deletion of Eed exhausts adult bone marrow hematopoietic stem cells (HSCs), although fetal liver HSCs are produced in normal numbers. Eed-null neonatal HSCs express HSC signature genes but are defective in maintenance and differentiation. Comparative gene expression profiling revealed that neonatal and adult HSCs lacking Eed upregulated gene sets of conflicting pathways. Deletion of Cdkn2a, a PRC2 target gene, in Eed-null mice enhances hematopoietic stem/progenitor cell (HSPC) survival but fails to restore HSC functions. Taken together, our findings define developmental-stage-specific requirements for canonical PRC2 complexes in normal HSC function.

FLVCR is necessary for erythroid maturation, may contribute to platelet maturation, but is dispensable for normal hematopoietic stem cell function

AbstractHeme is a pleiotropic molecule that is important for oxygen and oxidative metabolism, most notably as the prosthetic group of hemoglobin and cytochromes. Because excess free intracellular heme is toxic, organisms have developed mechanisms to tightly regulate its concentration. One mechanism is through active heme export by the group C feline leukemia virus receptor (FLVCR). Previously, we have shown that FLVCR is necessary for embryonic and postnatal erythropoiesis. However, FLVCR is also expressed in numerous other tissues, including hematopoietic stem cells (HSCs). To explore a possible role for FLVCR in HSC function, we performed serial, competitive repopulation transplant experiments using FLVCR-deleted and control bone marrow cells, along with wild-type competitor cells. Loss of FLVCR did not impact HSC function under steady-state or myelotoxic stress conditions (such as arsenic or radiation exposure), nor did FLVCR deletion result in alterations in the various progenitor compartments. However, even when 95% of the donor bone marrow cells lacked FLVCR, all red cells in recipient mice were wild type. This is due to the increased apoptosis of FLVCR-deleted proerythroblasts. Also, remarkably, loss of FLVCR increased megakaryocyte ploidy. Together, these findings show FLVCR is redundant in stem cells but has critical and contrasting stage-specific roles in discrete hematopoietic lineages.

The MYB proto-oncogene suppresses monocytic differentiation of acute myeloid leukemia cells via transcriptional activation of its target gene GFI1

The MYB gene is a master regulator of hematopoiesis and contributes to leukemogenesis in several species including humans. Although it is clear that MYB can promote proliferation, suppress apoptosis and block differentiation, the identities of the MYB target genes that mediate these effects have only been partially elucidated. Several studies, including our own, have collectively identified substantial numbers of MYB target genes, including candidates for each of these activities; however, functional validation, particularly in the case of differentiation suppression, has lagged well behind. Here we show that GFI1, which encodes an important regulator of hematopoietic stem cell (HSC) function and granulocytic differentiation, is a direct target of MYB in myeloid leukemia cells. Chromatin immunoprecipitation and reporter studies identified a functional MYB-binding site in the promoter region of GFI, whereas ectopic expression and small hairpin RNA-mediated knockdown of MYB resulted in concomitant increases and decreases, respectively, in GFI1 expression. We also demonstrate that GFI1, like MYB, can block the induced monocytic differentiation of a human acute myeloid leukemia cell line, and most importantly, that GFI1 is essential for MYB's ability to block monocytic differentiation. Thus, we have identified a target of MYB that is a likely mediator of its myeloid differentiation-blocking activity, and which may also be involved in MYB's activities in regulating normal HSC function and myeloid differentiation.

MLL fusion protein–driven AML is selectively inhibited by targeted disruption of the MLL-PAFc interaction

AbstractMLL rearrangements are common in leukemia and considered an adverse risk factor. Through interactions with the polymerase-associated factor complex (PAFc), mixed lineage leukemia (MLL) fusion proteins activate genes critical for blocking differentiation, such as HOXA9. Here we investigate whether the MLL-PAFc interaction can be exploited therapeutically using both genetic and biochemical approaches. We tested the genetic requirement of the PAFc in acute myeloid leukemia (AML) using a conditional allele of the PAFc subunit, Cdc73. We show that the PAFc is indiscriminately necessary for the proliferation of AML cells through the epigenetic regulation of proleukemogenic target genes, such as MEIS1 and Bcl2. To investigate the therapeutic potential of targeting the MLL-PAFc interaction, we engineered a dominant negative fragment of MLL capable of binding to the PAFc. Disruption of the MLL-PAFc interaction selectively inhibits the proliferation of MLL leukemic cells without affecting cells transformed by an unrelated E2A-HLF fusion protein. Using in vivo hematopoietic reconstitution assays, we demonstrate that disruption of the MLL-PAFc does not alter normal hematopoietic stem cell function. Together, our data show a selective growth inhibition of MLL-associated leukemic cells and tolerance of normal hematopoiesis to disruption of the MLL-PAFc interaction establishing the MLL-PAFc interaction as an attractive therapeutic target.

Expression of the lymphoid enhancer factor 1 is required for normal hematopoietic stem and progenitor cell function

Expression of the lymphoid enhancer factor 1 is required for normal hematopoietic stem and progenitor cell function

Hypoxic induction of vascular endothelial growth factor regulates erythropoiesis but not hematopoietic stem cell function in the fetal liver

Hypoxia is an important factor in the hematopoietic stem cell (HSC) niche in the bone marrow (BM) but whether it also plays a role in the regulation of fetal liver (FL) HSCs is unclear. Definitive HSCs first arise in the AGM region and the placenta during development and later migrate to the fetal liver (FL). FL HSCs undergo extensive proliferation in the liver and migrate to the bone marrow (BM) shortly before birth, where they become quiescent. The molecular mechanisms behind this switch in proliferation remain elusive. While many components of the BM stem cell niche have been identified, relatively little is known about the FL HSC niche. Vascular endothelial growth factor A (VEGFA) is essential for adult HSC survival and hypoxic induction of Vegfa in adult HSC is required for proper function. Mice with defective hypoxia-induced Vegfa expression (Vegfaδ/δ) show a partial embryonic lethality and increases the numbers of phenotypically defined hematopoietic stem and progenitor cells in the FL, but whether stem cell function is affected in FL HSCs has not been examined. To investigate whether hypoxia-induced Vegfa is involved in regulation of fetal HSC function and to identify the reason for the embryonic lethality we have examined FL hematopoiesis in the Vegfaδ/δ model. We show that fetal erythropoiesis is severely impaired when hypoxic induction of Vegfa is lacking. FL HSCs deficient for hypoxia-induced Vegfa expression have normal HSC function arguing against a hypoxic FL HSC niche. However, after adaptation of FL HSCs to the BM microenvironment, FL HSCs lose their function as measured by serial transplantation. Hypoxia is an important factor in the hematopoietic stem cell (HSC) niche in the bone marrow (BM) but whether it also plays a role in the regulation of fetal liver (FL) HSCs is unclear. Definitive HSCs first arise in the AGM region and the placenta during development and later migrate to the fetal liver (FL). FL HSCs undergo extensive proliferation in the liver and migrate to the bone marrow (BM) shortly before birth, where they become quiescent. The molecular mechanisms behind this switch in proliferation remain elusive. While many components of the BM stem cell niche have been identified, relatively little is known about the FL HSC niche. Vascular endothelial growth factor A (VEGFA) is essential for adult HSC survival and hypoxic induction of Vegfa in adult HSC is required for proper function. Mice with defective hypoxia-induced Vegfa expression (Vegfaδ/δ) show a partial embryonic lethality and increases the numbers of phenotypically defined hematopoietic stem and progenitor cells in the FL, but whether stem cell function is affected in FL HSCs has not been examined. To investigate whether hypoxia-induced Vegfa is involved in regulation of fetal HSC function and to identify the reason for the embryonic lethality we have examined FL hematopoiesis in the Vegfaδ/δ model. We show that fetal erythropoiesis is severely impaired when hypoxic induction of Vegfa is lacking. FL HSCs deficient for hypoxia-induced Vegfa expression have normal HSC function arguing against a hypoxic FL HSC niche. However, after adaptation of FL HSCs to the BM microenvironment, FL HSCs lose their function as measured by serial transplantation.

Hypoxia and Metabolic Properties of Hematopoietic Stem Cells

The effect of redox signaling on hematopoietic stem cell (HSC) function is not clearly understood. A growing body of evidence suggests that adult HSCs reside in the hypoxic bone marrow microenvironment or niche during homeostasis. It was recently shown that primitive HSCs in the bone marrow prefer to utilize anaerobic glycolysis to meet their energy demands and have lower rates of oxygen consumption and lower ATP levels. Hypoxia-inducible factor-α (Hif-1α) is a master regulator of cellular metabolism. With hundreds of downstream target genes and crosstalk with other signaling pathways, it regulates various aspects of metabolism from the oxidative stress response to glycolysis and mitochondrial respiration. Hif-1α is highly expressed in HSCs, where it regulates their function and metabolic phenotype. However, the regulation of Hif-1α in HSCs is not entirely understood. The homeobox transcription factor myeloid ecotropic viral integration site 1 (Meis1) is expressed in the most primitive HSCs populations, and it is required for primitive hematopoiesis. Recent reports suggest that Meis1 is required for normal adult HSC function by regulating the metabolism and redox state of HSCs transcriptionally through Hif-1α and Hif-2α. Given the profound effect of redox status on HSC function, it is critical to fully characterize the intrinsic, and microenvironment-related mechanisms of metabolic and redox regulation in HSCs. Future studies will be needed to elucidate the link between HSC metabolism and HSC fates, including quiescence, self-renewal, differentiation, apoptosis, and migration.

The Ubiquitin Ligase FBXW7 Modulates Leukemia-Initiating Cell Activity by Regulating MYC Stability

Sequencing efforts led to the identification of somatic mutations that could affect the self-renewal and differentiation of cancer-initiating cells. One such recurrent mutation targets the binding pocket of the ubiquitin ligase Fbxw7. Missense FBXW7 mutations are prevalent in various tumors, including Tcell acute lymphoblastic leukemia (T-ALL). To study the effects of such lesions, we generated animals carrying regulatable Fbxw7 mutant alleles. Here, we show that these mutations specifically bolster cancer-initiating cell activity in collaboration with Notch1 oncogenes but spare normal hematopoietic stem cell function. We were also able to show that FBXW7 mutations specifically affect the ubiquitylation and half-life of c-Myc protein, a key T-ALL oncogene. Using animals carrying c-Myc fusion alleles, we connected Fbxw7 function to c-Myc abundance and correlated c-Myc expression to leukemia-initiating activity. Finally, we demonstrated that small-molecule-mediated suppression of MYC activity leads to T-ALL remission, suggesting an effective therapeutic strategy.

Mixed lineage leukemia protein in normal and leukemic stem cells.

Transcription factors critical for normal hematopoietic stem cell functions are frequently mutated in acute leukemia leading to an aberrant re-programming of normal hematopoietic progenitor/stem cells into leukemic stem cells. Among them, re-arrangements of the mixed lineage leukemia gene (MLL), including chimeric fusion, partial tandem duplication (PTD), amplification and internal exonic deletion, represent one of the most common recurring oncogenic events and associate with very poor prognosis in human leukemias. Extensive research on wild type MLL and MLL-fusions has significant advanced our knowledge about their functions in normal and malignant hematopoiesis, which also provides a framework for the underlying pathogenic role of MLL re-arrangements in human leukemias. In contrast, research progress on MLL-PTD, MLL amplification and internal exonic deletion remains stagnant, in particular for the last two abnormalities where mouse model is not yet available. In this article, we will review the key features of both wild-type and re-arranged MLL proteins with particular focuses on MLL-PTD and MLL amplification for their roles in normal and malignant hematopoiesis.

HSC Aging Epigenome: Widespread Alterations in DNA Methylation and Transcription.

AbstractAbstract 2329Age is the most important risk factor for myelodysplastic syndrome (MDS), a premalignant state that transforms into acute myelogenous leukemia in one third of cases. Indeed with normal aging, hematopoietic stem cell (HSC) regenerative potential diminishes and differentiation skews from lymphopoiesis toward myelopoiesis. The expansion in the HSC pool with aging provides sufficient but abnormal blood production, and animals experience a decline in immune function. Previous studies from our lab established that the DNA methyltransferase 3a (Dnmt3a) enables efficient differentiation by critically regulating epigenetic silencing of HSC genes (Challen et al. 2012) Interestingly, Dnmt3a expression is decreased in old HSCs, leading us to hypothesize that epigenetic changes in old HSCs may partially mimic the changes seen in Dnmt3a mutant HSCs. We propose that revealing the genome-wide DNA methylation and transcriptome signatures will lead to a greater understanding of HSC aging and MDS, which is characterized by frequent epigenetic abnormalities.In this study, we investigated genome-wide DNA methylation and transcripts by whole genome bisulfite sequencing (WGBS) and transcriptome sequencing (mRNA-seq)in young and old HSCs. For WGBS, we generated ∼600M raw reads resulting in ∼ 60 raw Gb of paired-end sequence data and aligned them to either strand of the reference genome (mm9), providing an average 40-fold sequencing depth. Globally, there was a 1.1% difference in the DNA methylation between young and old HSCs. Of these differences, 38% (172,609) of the CpG dinucleotides were hypo-methylated, and 62% (275,557) were hyper-methylated in old HSCs. To understand where the methylation changes predominantly occurred, the genome was subdivided into 77 features. Among these features, SINEs, especially Alu elements, exhibited the highest level of DNA methylation (90.94% in young HSCs, and 91.87% in old HSCs). CpG islands (CGIs) adjacent to the transcription start sites (TSS) exhibited the lowest level of DNA methylation (2.02% in young HSCs, and 2.11% in old HSCs). Interestingly strong hypo-methylation was observed in ribosomal RNA regions (68.04% in young HSCs, 59.04% in old HSCs), and hyper-methylation was observed in LINEL1 repetitive elements (88.62% in young HSCs, 90.12% in old HSCs). Moreover, the examination of differentially methylated promoters identified enrichment of developmentally important transcription factors such as Gata2, Runx1, Gfi1b, Erg, Tal1 Eto2, Cebpa and Pu.1. Additionally, we compare our ∼10,000 differentially methylation regions (DMRs, regions with clustered DNA methylation changes) with a chip-seq data set containing binding of 160 ChIP-seq analyses of hematopoietic transcription factors in different hematopoietic cells. We found significant overlaps between DMRs and transcription factor binding regions. We found DMRs which were hypermethylated showed association with differentiation-promoting Ets factors, in particular Pu.1 from a range of different blood cell types. In contrast, hypomethylated DMRs showed associations with HSC-associated transcription factors such as Scl and Gata2. Further examination of the differentially methylated gene bodies, intragenic and intergenic DMRs identified some previously noted targets for epigenetic silencing or alteration in AML and also novel transcripts including long non-coding RNAs (lincRNA) and upstream regulatory elements (URE). We found significant correlation between RNA-seq expression and DMRs within +1kb upstream of TSS. RNA-sequencing provided complementary and distinct information about HSC aging. We identified differentially expressed genes, novel RNA transcripts, differential promoter, coding sequence, and splice variant usage with age. Gene set enrichment analysis of up- and down- regulated genes, revealed ribosomal protein and RNA metabolism as critical contributors to HSC aging.In conclusion, our study marks a milestone in the mouse HSC epigenome, reporting the first complete methylome and transcriptome of pure HSC using whole-genome bisulfite sequencing and RNA-seq. These provide novel information about the magnitude and specificity of age-related epigenetic changes in a well-defined HSC population. Understanding the roles of DNA methylation and transcription in normal HSC function will allow for greater therapeutic exploitation of HSCs in the clinic. Disclosures:No relevant conflicts of interest to declare.

Alcam Mediated Interaction Regulates Normal Hematopoietic Stem Cell Function and Cbfβ-SMMHC Induced Leukemogenesis

Abstract 4091The inv(16) acute myeloid leukemia (AML)-associated CBFβ-SMMHC fusion protein impairs hematopoietic differentiation and predisposes to leukemic transformation. Alcam, which encodes the activated leukocyte cell adhesion molecule (CD166), is a cell surface immunoglobulin superfamily member mediating homophilic adhesion as well as heterotypic interactions with CD6. We found that Alcam expression marks long-term repopulating HSCs (LT-HSC), multipotent progenitors (MPP), a subset of granulocyte-macrophage progenitors (GMP), and that Alcam expression is lost or reduced in subsets of pre-leukemic and leukemic progenitors expressing the Cbfβ-SMMHC fusion protein. We characterized the role of Alcam in HSC differentiation and self-renewal using an Alcam-null (Alcam−/−) mouse model (Weiner et al. 2004 Mol Cell Neurosci 27:1, 59–69). We show that Alcam is highly expressed in LT-HSCs where its level progressively increases with age. Young adult Alcam−/− mice had normal homeostatic hematopoiesis, and normal numbers of phenotypic HSCs. However, Alcam−/− HSCs had reduced long-term replating capacity in vitro and reduced long-term engraftment potential upon transplantation. We show that Alcam−/− BM contain a markedly lower frequency of long-term repopulating cells than wild type (WT). Further, the long-term repopulating potential and engraftment efficiency of Alcam−/− LT-HSCs was greatly compromised despite a progressive increase in phenotypic LT-HSC numbers during long-term serial transplantation. In addition, an age-associated increase in phenotypic LT-HSC cellularity was observed in Alcam−/− mice. This increase was predominately within the CD150hi fraction, and was accompanied by significantly reduced leukocyte output. Moreover, Alcam−/− LT-HSCs display premature elevation of Selp expression, a hallmark of HSC aging. To understand the role of Alcam in leukemic transformation, we generated conditional Cbfb-MYH11 knock-in (Cbfb56M/+/Mx1-Cre), Alcam-deficient (Alcam−/−) mice. Interestingly, we found that loss of Alcam drastically delayed or reduced leukemia incidence. Transplantation of Alcam−/−/Cbfb56M/+/Mx1-Cre pre-leukemic bone marrow cells into WT recipients also led to delayed and reduced incidence of leukemia development. These results suggest that Alcam contributes to leukemia transformation in a cell-intrinsic manner. Collectively, our study reveals that Alcam regulates the functional integrity and self-renewal of LT-HSCs, and contributes to leukemia initiation induced by CBFβ-SMMHC. Disclosures:No relevant conflicts of interest to declare.

SOCS2 is dispensable for BCR/ABL1-induced chronic myeloid leukemia-like disease and for normal hematopoietic stem cell function

Suppressor of cytokine signaling 2 (SOCS2) is known as a feedback inhibitor of cytokine signaling and is highly expressed in primary bone marrow (BM) cells from patients with chronic myeloid leukemia (CML). However, it has not been established whether SOCS2 is involved in CML, caused by the BCR/ABL1 fusion gene, or important for normal hematopoietic stem cell (HSC) function. In this study, we demonstrate that although Socs2 was found to be preferentially expressed in long-term HSCs, Socs2-deficient HSCs were indistinguishable from wild-type HSCs when challenged in competitive BM transplantation experiments. Furthermore, by using a retroviral BCR/ABL1-induced mouse model of CML, we demonstrate that SOCS2 is dispensable for the induction and propagation of the disease, suggesting that the SOCS2-mediated feedback regulation of the JAK/STAT pathway is deficient in BCR/ABL1-induced CML.

Pyruvate kinase M2 plays distinct roles in hematopoietic stem cells and leukemia (111.19)

Abstract It has been realized that different cell states employ different metabolic modes. Whether metabolism is a determinant of cell state is unclear, but studies in cancer suggest that altering metabolic activity can modify cellular function. Cancer cells metabolize glucose through glycolysis even in the presence of oxygen, a phenomenon called Warburg effect. Normal somatic cells thought to also preferentially use glycolytic metabolism are tissue stem cells, particularly the hematopoietic stem cells (HSC) resident in the hypoxic microenvironment of the bone marrow. Recently the molecular basis for Warburg effect in cancer cells has been identified as due to a specific isoform of pyruvate kinase M2 (PKM2). Interestingly, HSC also express PKM2. Using a mouse strain that allows conditional deletion of PKM2, we observed that PKM2 depletion enhances normal HSC function as revealed by a competitive transplantation assay. The altered cellular function is accompanied with decreased glycolysis and increased oxidative phosphorylation specifically in the primitive stem/progenitor cell population. Notably however, PKM2 deletion markedly disadvantages disease development in mouse models of both acute and chronic myeloid leukemia. Such a distinction in response between normal and malignant cells is unprecedented to our knowledge and may offer a highly unique opportunity to advantage normal while disadvantaging malignant cells of the hematopoietic lineage by targeting a single metabolic enzyme.

Normal Hematopoietic Stem Cell Function in Mice with Enforced Expression of the Hippo Signaling Effector YAP1

The Hippo pathway has recently been implicated in the regulation of organ size and stem cells in multiple tissues. The transcriptional cofactor yes-associated protein 1 (Yap1) is the most downstream effector of Hippo signaling and is functionally repressed by the upstream components of the pathway. Overexpression of YAP1 stimulates proliferation of stem and progenitor cells in many tissues, consistent with inhibition of Hippo signaling. To study the role of Hippo signaling in hematopoietic stem cells (HSCs), we created a transgenic model with inducible YAP1 expression exclusively within the hematopoietic system. Following 3 months induction, examination of blood and bone marrow in the induced mice revealed no changes in the distribution of the hematopoietic lineages compared to control mice. Moreover, the progenitor cell compartment was unaltered as determined by colony forming assays and immunophenotyping. To address whether YAP1 affects the quantity and function of HSCs we performed competitive transplantation experiments. We show that ectopic YAP1 expression does not influence HSC function neither during steady state nor in situations of hematopoietic stress. This is in sharp contrast to effects seen on stem- and progenitor cells in other organs and suggests highly tissue specific functions of the Hippo pathway in regulation of stem cells.

Socs2 Is Dispensable for BCR/ABL1-Induced Chronic Myeloid Leukemia-Like Disease in Mice and for Normal Hematopoietic Stem Cell Function,

Abstract 3743 Background:The P210 BCR/ABL1 fusion protein initiates signaling through several down stream pathways such as STAT5, PI3K, AKT, RAS, and WNT. However, only few down stream mediators have so far been thoroughly studied in vivo in the context of BCR/ABL1-mediated induction of CML-like disorder in mice deficient for the specific genes. Suppressor of cytokine signaling 2 (SOCS2) is known as a feedback inhibitor of cytokine signaling, a negative regulator of the STAT5 pathway, and is markedly upregulated in primary bone marrow cells from patients with chronic myeloid leukemia (CML). However, it has not been clear whether SOCS2 is involved in BCR/ABL1 induced cell transformation or whether it is important for normal hematopoietic stem cell (HSC) function. Methods:To evaluate the stem cell function of Socs2, HSCs from wild type and Socs2 deficient mice were competitively transplanted into lethally irradiated recipients. Blood chimerism and lineage distribution were analyzed by flow cytometry at 4 and 18 weeks after transplantation. To investigate the potential role of Socs2 in CML, c-kit enriched Socs2(−/−) and wild type cells were transduced with a BCR/ABL1 expressing retroviral vector and transplanted into lethally irradiated recipients. The in vivo development of malignant disease was analyzed by peripheral blood cell counts and, upon euthanization, by flow cytometry and histopathology. Socs family gene expression was assessed by Q-RT/PCR and Stat5 phosphorylation by Western blotting on BCR-ABL1 expressing cells. Results:Although Socs2 was previously found to be upregulated in long-term repopulating HSCs by gene expression arrays, Socs2 deficient HSCs were indistinguishable from wild type HSCs when challenged in competitive bone marrow transplantation. Furthermore, when expressing BCR/ABL1, both Socs2 deficient and wild type cells induced a CML-like disease with an average survival of three weeks after transplantation. The leukemic mice suffered from elevated white blood cell counts and splenomegaly. To investigate if other Socs family members were upregulated to compensate for the Socs2 deficiency, we compared the expression of the Socs gene family members in Socs2(−/−) with wild type, leukemic cells. The expression levels of all other Socs genes were similar between Socs2(−/−) and Socs2 wt cells, suggesting that compensatory mechanisms of other Socs genes do not account for the lack of Socs2 function. In addition, the finding that the phosphorylation of Stat5 was unaffected in Socs2(−/−) cells indicates that BCR/ABL1-induced Stat5-phosphorylation is insensitive to Socs2 levels. Conclusions:Collectively, our results clarify that Socs2 is dispensable for normal HSC function and for BCR/ABL1-induced CML-like disease. Disclosures:No relevant conflicts of interest to declare.

Hijacking the Niche: The Role of Stromal Osteopontin in the Induction of Leukemic Dormancy within the Bone Marrow Microenvironment

Abstract 754 Introduction:Acute lymphoblastic leukemia (ALL) is a treatable malignancy where initial induction chemotherapy achieves clinical remission in the majority of patients. Relapsed disease, however, occurs in many patients and is significantly more difficult to treat. The majority of relapsed cases are a result of minimal residual disease (MRD) that persists within the bone marrow (BM) after initial chemotherapy. Our evolving knowledge of the importance of the host microenvironment in tumor progression suggests that the stromal microenvironment can protect leukemic cells from chemotherapeutic assault, and that inhibiting the supportive relationship between leukemic blasts and the bone marrow microenvironment (BMM) will provide novel therapeutic opportunities. We therefore aimed to identify and characterize novel stromal signaling mechanisms that retain and support blasts within the malignant BMM. Our preliminary data suggest that osteopontin (OPN), normally secreted by osteoblasts within the marrow, is one such signaling chemokine that is highly upregulated in the leukemic niche. OPN has well-defined roles in both solid tumor metastasis and normal hematopoietic stem cell function within the BMM. Specifically, OPN expression at the endosteal bone surface functions to recruit hematopoietic progenitors to bone where they are induced to become quiescent and maintain long term repopulating potential. We hypothesized that a similar relationship exists between leukemia and OPN resulting in a quiescent population of chemoresistant leukemic blasts at the BM endosteum. Here, we demonstrate that stromal OPN negatively regulates leukemia cell proliferation in the BMM. Methods:A GFP expressing clone of the pre-B ALL cell line Nalm-6 (10 × 106 cells) was engrafted into SCID hosts (6-8 weeks old) via tail vein injection. In vivo imaging was accomplished in live anesthetized mice by reflecting the scalp and imaging the calvarial marrow compartment using real-time multi-photon confocal microscopy. OPN expression in the malignant marrow was imaged by injecting engrafted mice with fluorescently conjugated anti-OPN antibodies 18hrs prior to imaging. For OPN neutralizing experiments, engrafted mice were injected with a cocktail of anti-mouse and anti-human OPN antibodies at a dose of 3 mg/kg. Results:Using PCR, Western blot and ELISA assays, we show that the ALL cell line Nalm-6 expresses OPN and secretes large quantities of OPN into conditioned media. Flow cytometric analysis demonstrates that Nalm-6 also express the cell surface OPN receptors VLA-4 and VLA-5. Furthermore, Nalm-6 cells specifically adhere to OPN in vitro via specific engagement of these integrin receptors. In vivo imaging demonstrates that OPN expression in the BM increases as leukemia progresses and that OPN is highly expressed adjacent to areas of high tumor burden. Specifically, a significant amount of OPN is detected in bony tunnels surrounding the vasculature at the invading tumor front. Using Q-PCR and western analysis, we demonstrate that both host-derived and leukemia-derived OPN are upregulated in malignant BM. In vivo inhibition of the OPN signaling axis in the Nalm-6 xenograft model using neutralizing antibodies directed at both human and murine OPN increased overall tumor burden two-fold as measured by flow cytometry and in vivo imaging (p=0.02, N=7) while simultaneously increasing the Ki-67 positive proliferative tumor population (p=0.029, N=4). Furthermore, IHC analysis of a panel of diagnostic bone marrow biopsies from a diverse cohort of ALL patients demonstrated high OPN expression in the marrow of these patients. Conclusion:Leukemic blasts that have hijacked normal stromal interactions to become quiescent may represent a major source of MRD and patient relapse in ALL. Our data demonstrate that the interaction of leukemic blasts with OPN in the stromal microenvironment reduces the number of cycling blasts and constrains tumor proliferation within the marrow. Current investigations are aimed at combining OPN neutralization with an in vivo model of MRD to determine whether OPN neutralization induces cycling of quiescent blasts, ultimately rendering them sensitive to chemotherapy. The ultimate goal of this work is the development of clinically relevant therapies designed to render leukemic cells more susceptible to chemotherapy by disengaging them from protective interactions with the BM microenvironment. Disclosures:No relevant conflicts of interest to declare.

FLVCR Deletion Does Not Impact Normal Hematopoietic Stem Cell Function but Is Necessary for Erythroid and T-Cell Maturation and Enhances Platelet Formation

Abstract 2363Hematopoietic stem cells (HSCs) are mainly in a quiescent state, but activate and proliferate in response to stress. To maintain quiescence, as well as respond to activation signals, HSCs must coordinately regulate cell cycle status versus metabolic demand. Mismatch in these pathways can lead to HSC exhaustion, failure or malignant transformation. One metabolic pathway that is essential to all cells is heme biosynthesis. Heme can act directly as a signaling molecule by modulating the function of transcriptional (Bach1) or translational (eIFa2 kinase) regulators. In addition, heme can act indirectly through the action of hemoproteins (hemoglobin, oxidases, peroxidases, cytochromes). However, excess free intracellular heme is toxic, therefore organisms have developed “safety valve” pathways to regulate intracellular heme concentrations. The most well studied mechanism is free heme degradation by the heme-oxygenase pathway. We have recently described a second pathway to prevent intracellular heme excess, namely heme export by the cytoplasmic transporter FLVCR. Previously we have shown that FLVCR is essential for erythropoiesis, as FLVCR−/− mice die of anemia (Keel et. al., Science 319: 825, 2008). FLVCR is expressed in many other tissues and blood cells, including HSCs (Lucas et. al., Blood 106:51, 2005). This suggests that FLVCR may be important in HSC function or the development of other lineages, besides red cells. We used a competitive transplantation model to determine the effect of FLVCR deletion on HSC function and lineage maturation. FLVCR−/− or wild type (WT) donor cells and equal numbers of competitor cells were transplanted into lethally irradiated, syngeneic recipients. FLVCR−/− marrow cells reconstitute the granulocyte compartment equally as well as WT cells in serial transplantation (up to tertiary transplants). Equal contributions also remain after myelotoxic stress (radiation or arsenic trioxide). Therefore, FLVCR may be dispensable for normal HSC function under steady-state and stress conditions. As anticipated, FLVCR is required for proerythroblast survival and maturation. Eight weeks after competitive transplantation with FLVCR−/− donor cells, 100% of red cells are derived from competitor cells. This is also seen when the ratio of FLVCR−/−:competitor cells is increased to 95:5 from 50:50. FLVCR−/− cells fail to mature past the proerythroblast stage (CD71pos lin-Ter119neg or lo) and undergo significantly more apoptosis than WT donor cells (88% vs. 36%, p<0.001). FLVCR deletion also blocks T-cell development. Due to biological variability in our experimental model, T-cell numbers were normalized to granulocytes in each determination, in each mouse and expressed as a percentage of the expected value. At 5 weeks post-transplant, the percentage of FLVCR−/− derived T-cells was 65.8% of the percentage of FLVCR−/− derived granulocytes. The percentage of WT derived T-cells was 93.4% of the percentage of WT derived granulocytes (p<0.02). Over time, FLVCR−/− derived T-cells steadily decreased, dropping to 22% of expected values by week 24, and remaining low, whereas T-cells derived from control, WT donor cells remained stable (74–98% of expected values). FLVCR−/− T-cells are arrested at the double positive (CD4+CD8+) stage (Philip et. al., Blood (ASH Abstracts) 114:913, 2009). Surprisingly, loss of FLVCR improves platelet production. FLVCR−/− donor cells make up ∼60% of all platelets (avg. 1.71 x expected, when normalized to granulocytes). In the separate control cohort competitively transplanted with WT donor cells, ∼40% of the platelet population was derived from WT cells (avg. 0.91 x expected, when normalized to granulocytes). Our studies suggest that the increased platelet production likely results from increased megakaryocyte ploidy (e.g. avg. 14.2N in FLVCR−/− vs 5.2N in control) and not from increased megakaryocyte progenitors. Of note, FLVCR−/− deleted mice have thrombocytosis (2221/μL vs 1008/μL, p<0.001 in littermate controls), a finding we initially dismissed as a stress response to severe anemia. Together, these findings reveal stage specific roles for FLVCR in discrete hematopoietic lineages and support the broader hypothesis that heme excess might impair cell division, leading to decreased red and T-cell production, but improved endomitosis and increased platelet production. Disclosures:No relevant conflicts of interest to declare.