Dnmt3a-Deletion Accelerates FLT3-ITD Malignancies In Mice By Hypomethylation Of Enhancer Sites and Activating Stem Cell Programs; Implications For Therapy

Abstract The de novo DNA methyltransferase (DNMT) 3A is mutated in 50% of patients with mixed phenotype acute leukemia, 20% with acute myeloid leukemia (AML) and 18% with T-cell acute lymphoblastic leukemia (T-ALL). The mechanisms through which mutant DNMT3A contributes to hematologic malignancy are poorly understood. In mice, deletion of Dnmt3a in hematopoietic stem cells (HSCs) leads to abnormal DNA methylation and inhibition of differentiation, but is insufficient for leukemic transformation. To study the role of Dnmt3a in leukemia, we combined Dnmt3a-deletion with the activated FLT3 proto-oncogene (FLT3-ITD), a frequent co-mutation with DNMT3A in AML patients, to establish a murine model of Dnmt3a-associated malignancy. In mice transplanted with Dnmt3a-knockout (KO) or wild-type (WT) bone marrow cells transduced with a FLT3-ITD retrovirus, Dnmt3a-loss dramatically impacted the disease phenotype. Dnmt3aKO/ITD transplanted mice had significantly shortened survival (79 days vs. 116 days) and increased rate of acute leukemia compared to mice with ITD alone. The mice developed CD4+CD8+ Notch activation-associated T-ALL or myeloproliferative disease (MPD), or concurrently both, consistent with previous studies of FLT3-ITD in mice. To determine the leukemia-initiating population, we transplanted sorted HSC, myeloid, and lymphoid progenitors transduced with FLT3-ITD. All mice transplanted with HSC and myeloid progenitors succumbed to both malignancies. To uncover the mechanisms by which Dnmt3a-deletion accelerated acute leukemia, we analyzed changes in DNA methylation in T-ALL blasts by whole genome bisulfite sequencing. Compared to Dnmt3aWT/ITD, Dnmt3aKO/ITD blasts exhibited global hypomethylation, particularly at distal enhancer sites. These hypomethylated enhancer sites were associated with genes in signaling pathways, transcription regulators, and metabolic pathways in cancer (KEGG and GO Analysis). Transcriptome analysis showed that relative to Dnmt3aWT/ITD, the Dnmt3aKO/ITD blasts had 1577 significantly differentially expressed genes positively related to cancer, cellular growth, and proliferation, and negatively to apoptosis by Ingenuity Pathway Analysis (IPA). Surprisingly, we observed increased expression of genes related to HSCs and myeloid function and decreased expression of genes related to lymphocyte function. Human AML signature genes (Oncomine) were also upregulated in our mouse model. Predicted activated pathways include Myc, Nfe2l2, Eif4e, E2f1, Csf2, Cebpb, Vegf, Rxra, Ezh2, and Brd4 and inhibited pathways include tumor suppressors Rb, let7, Cdkn2a, and Tob1 (IPA). We did not observe changes in genomic copy number variation by chromosomal comparative hybridization (cCGH). To test whether Dnmt3a-deletion could functionally bestow stem cell properties on pre-leukemic cells, we examined self-renewal capabilities of malignant cells of Flt3+/ITD knock-in mouse (an ITD mutation knocked in to the endogenous murine Flt3 allele causing MPD). Remarkably, when Dnmt3aKO; Flt3+/ITD bone marrow cells were serially transplanted, MPD was seen in all recipients, compared to none in Dnmt3aWT; Flt3+/ITD transplanted mice (n=7). Further, we transplanted sorted CLP, CMP, GMP, MPP, ST-HSC, LT-HSC populations and observed myeloproliferation in transplanted non-stem (CMP, GMP, ST-HSC) and stem cell (LT-HSC) populations. This strongly suggests that Dnmt3aKO synergized with Flt3-ITD to confer stem cell self-renewal abilities to transformed progenitor and stem cells. Increasingly, decitabine is being used to treat patients with AML and MDS, but whether patients with DNMT3A mutations could benefit is unclear, so we examined the impact of decitabine treatment on the retroviral transduced Dnmt3aKO/ITD mice. Monthly treatment led to significantly increased survival of Dnmt3aKO/ITD mice from T-ALL and MPD and reduced presence of ITD-transduced KO cells. Together, we demonstrate that Dnmt3aKO accelerated malignancies induced by FLT3-ITD in mouse and may shed light on how DNMT3A mutations contribute to lymphoid and myeloid disease in patients. Dnmt3a deletion ignited multilineage and stem cell programs at the expense of lymphoid programs to accelerate disease, but was extinguishable by decitabine therapy. The findings from our mouse model can be used for the development and testing of targeted epigenetic therapy for DNMT3A-associated malignancies. Disclosures: No relevant conflicts of interest to declare.

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DNMT3b’s Role in Hematopoietic Stem Cell Self-Renewal.

Blood ◽

10.1182/blood.v112.11.1406.1406 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1406-1406

Author(s):

Matthew J Boyer ◽

Feng Xu ◽

Hui Yu ◽

Tao Cheng

Keyword(s):

Dna Methylation ◽

Bone Marrow ◽

Stem Cell ◽

Bone Marrow Transplantation ◽

Peripheral Blood ◽

Marrow Transplantation ◽

Bone Marrow Cells ◽

Self Renewal ◽

Hematopoietic Stem ◽

Marrow Cells

Abstract DNA methylation is an epigenetic means of gene regulation and is carried out by a family of methyltransferases of which DNMT1 acts to maintain methylation marks following DNA replication and DNMT3a and DNMT3b methylate DNA de novo. DNMT3b has been shown to be essential for mammalian development and necessary for differentiation of germline and neural progenitor cells. Mutations of DNMT3b in humans lead to a rare autosomal recessive disorder characterized by immunodeficiency, centromeric instability, and facial abnormalities. We have shown by real-time, RT-PCR that DNMT3b mRNA is uniquely over-expressed by approximately 30-fold in immunophenotypically-defined longterm repopulating hematopoietic stem cells (HSCs) that are CD34−lineage−c-kit+Sca-1+ as compared to progenitor and differentiated cell types within the bone marrow and with respect to the other members of the DNMT family, namely DNMT1 and DNMT3a. To determine DNMT3b’s function in HSCs competitive bone marrow transplantation was undertaken. Isolated lineage− enriched bone marrow cells were transduced with a retroviral backbone based on the Murine Stem Cell Virus (MSCV) carrying either GFP and a short, hairpin RNA (shRNA) targeting DNMT3b or GFP alone. Following transduction 1×105 GFP+ cells along with 1×105 competitor cells were transplanted into 9.5 Gray irradiated congenic recipients. Two months following transplantation mice receiving bone marrow cells transduced with DNMT3b shRNA showed a significantly lower engraftment of donor cells as a percentage of total competitor cell engraftment in the peripheral blood as compared to those receiving cells transduced with GFP alone (24.8 vs 3.7, p<0.05) which persisted at 3 months (22.8 vs 1.5, p<0.05). Similarly, within the donor derviced cells in the peripheral blood there was a lower percentage of myeloid (CD11b+) cells at 2 and 3 months in the recipients of DNMT3b shRNA transduced cells as compared to controls. However there was no observed difference in the percentage of peripheral B (CD45R+) or T (CD3+) cells within the donor-derived cells. To determine the mechanism behind the observed engraftment defect with DNMT3b knockdown we cultured GFP+ transduced bone marrow cells in vitro with minimal cytokine support. As a control for our targeting methodology we also transduced bone marrow cells from mice harboring two floxed DNMT3b alleles with a MSCV carrying Cre recombinase and GFP. While lineage− bone marrow cells transduced with GFP alone increased 10-fold in number over two weeks of culture, cells in which DNMT3b was down regulated by shRNA or Cre-mediated recombination only doubled. Culture of lineage− bone marrow cells in methylcellulose medium by the colony-forming cell (CFC) assay revealed increases in the granulocytic and total number of colonies with DNMT3b knockdown or Cre-mediated recombination of DNMT3b similar to the increased myeloid engraftment of DNMT3b shRNA transduced cells observed 1 month following competitive bone marrow transplantation. However when 5,000 of these cells from the first CFC assay were sub-cultured there was a significant loss of colony forming ability within all lineages when DNMT3b was targeted by shRNA or Cre-mediated recombination. Taken together with the decreased engraftment of DNMT3b shRNA cells following competitive bone marrow transplantation, the observed limited proliferation in liquid culture and loss of colony forming ability during serial CFC assays is suggestive of a self-renewal defect of HSCs in the absence of DNMT3b, that was previously only reported in the absence of both DNMT3a and DNMT3b. Further elucidation of this proposed self-renewal defect is being undertaken and results of ongoing studies including long-term culture initiating cell (LTC-IC) assays and identification of genomic sites of DNA methylation within different hematopoietic subsets will also be presented.

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The identification in adult bone marrow of pluripotent and restricted stem cells of the myeloid and lymphoid systems

Journal of Experimental Medicine ◽

10.1084/jem.145.6.1567 ◽

1977 ◽

Vol 145 (6) ◽

pp. 1567-1579 ◽

Cited By ~ 348

Author(s):

S Abramson ◽

RG Miller ◽

RA Phillips

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

T Lymphocytes ◽

Chromosome Aberrations ◽

Bone Marrow Cells ◽

Hemopoietic Stem Cell ◽

Lymphoid System ◽

Unique Chromosome ◽

Marrow Cells

The precise relationship between the stem cells for the lymphoid system and those for the blood-forming system is unclear. While it is generally assumed that the hemopoietic stem cell, the spleen colony-forming unit (CFU-S), is also the stem cell for the lymphoid system, there is little evidence for this hypothesis. To investigate the stem cells in these two systems, we irradiated bone marrow cells to induce unique chromosome aberrations in the stem cell population and injected them at limiting dilution into stem cell-deficient recipients. Several months (between 3 and 11) were allowed for the injected cells to repopulate the hemopoietic system. At that time, the bone marrow, spleen, and thymus were examined for a high frequency of cells having the same unique chromosome aberration. The presence of such markers shows that the marker was induced in a cell with extensive proliferative capacity, i.e., a stem cell. In addition, the splenic lymphocytes were stimulated with phytohemagglutinin (PHA) or lipopolysaccharide (LPS) to search for unique chromosomes in dividing T and B cells, respectively. Finally, bone marrow cells were injected into secondary irradiated recipients to determine if the marker occurred in CFU-S and to determine whether or not the same tissue distributions of marked cells could be propogated by bone marrow cells in a second recipient. After examination of 28 primary recipients, it was possible to identify three unique patterns of stem cell regeneration. In one set of mice, a unique chromosome marker was observed in CFU-S and in PHA- and LPS-stimulated cultures. These mice provide direct evidence for a pluripotent stem cell in bone marrow. In addition, two restricted stem cells were identified by this analysis. In three recipients, abnormal karyotypes were found only in myeloid cells and not in B and T lymphocytes. These mice presumably received a marked stem cell restricted to differentiate only into myeloid progeny. In three other recipients, chromosome aberrations were found only in PHA-stimulated cells; CFU-S and cells from LPS cultures did not have cells with the unique chromosome. This pattern suggests that bone marrow contains cells committed to differentiation only into T lymphocytes. For each of the three types of stem cells, secondary recipients had the same cellular distribution of marked cells as the primary recipients. This observation provides further evidence that unique markers can be induced in both pluripotent and restricted stem cells.

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Long-term monoclonal reconstitution of erythropoiesis in genetically anemic W/Wv mice by injection of 5-fluorouracil-treated bone marrow cells of Pgk-1b/Pgk-1a mice

Blood ◽

10.1182/blood.v70.6.1758.1758 ◽

1987 ◽

Vol 70 (6) ◽

pp. 1758-1763 ◽

Cited By ~ 12

Author(s):

T Nakano ◽

N Waki ◽

H Asai ◽

Y Kitamura

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Bone Marrow Cells ◽

Electrophoretic Pattern ◽

Phosphoglycerate Kinase ◽

X Chromosomes ◽

Hematopoietic Stem ◽

Colony Forming Units ◽

Marrow Cells

Abstract The spleen colony-forming assay does not represent the number of hematopoietic stem cells with extensive self-maintaining capacity because five to 50 spleen colony-forming units (CFU-S) are necessary to rescue a genetically anemic (WB X C57BL/6)F1-W/Wv(WBB6F1-W/Wv) mouse. We investigated which is more important for the reconstitution of erythropoiesis, the transplantation of multiple CFU-S or that of a single stem cell with extensive self-maintaining potential. The electrophoretic pattern of hemoglobin was used as a marker of reconstitution and that of phosphoglycerate kinase (PGK), an X chromosome-linked enzyme, as a tool for estimating the number of stem cells. For this purpose, we developed the C57BL/6 congeneic strain with the Pgk-1a gene. Bone marrow cells were harvested after injection of 5- fluorouracil from C57BL/6-Pgk-1b/Pgk-1a female mice in which each stem cell had either A-type PGK or B-type PGK due to the random inactivation of one or two X chromosomes. When a relatively small number of bone marrow cells (ie, 10(3) or 3 X 10(3] were injected into 200-rad- irradiated WBB6F1-W/Wv mice, the hemoglobin pattern changed from the recipient type (Hbbd/Hbbs) to the donor type (Hbbs/Hbbs) in seven of 150 mice for at least 8 weeks. Erythrocytes of all these WBB6F1-W/Wv mice showed either A-type PGK alone or B-type PGK alone during the time of reconstitution, which suggests that a single stem cell with extensive self-maintaining potential may sustain the whole erythropoiesis of a mouse for at least 8 weeks.

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Enforced P-glycoprotein pump function in murine bone marrow cells results in expansion of side population stem cells in vitro and repopulating cells in vivo

Blood ◽

10.1182/blood.v96.3.902 ◽

2000 ◽

Vol 96 (3) ◽

pp. 902-909 ◽

Cited By ~ 126

Author(s):

Kevin D. Bunting ◽

Sheng Zhou ◽

Taihe Lu ◽

Brian P. Sorrentino

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Bone Marrow Cells ◽

Side Population ◽

Murine Bone Marrow ◽

Stem Cell Expansion ◽

Myeloproliferative Syndrome ◽

P Glycoprotein ◽

Marrow Cells

Abstract The human multidrug resistance-1 (MDR1) gene product, P-glycoprotein (P-gp), is well known for its ability to confer drug resistance; however, recent evidence suggests that P-gp expression can have more general effects on cellular development. In support of this idea, it was previously shown that retroviral-mediated MDR1 expression in murine bone marrow cells resulted in the expansion of stem cells in culture and in the development of a myeloproliferative syndrome in transplanted mice. It is now reported that MDR1-mediated stem cell expansion is associated with an increase in side population (SP) stem cells, defined by Hoechst dye staining. Transduction of murine bone marrow cells with an MDR1 retroviral vector resulted in an almost 2 log increase in SP cell numbers over 12 days in culture, whereas there was a rapid loss of SP cells from control cultures. Stem cell amplification was not limited to ex vivo expansion cultures but was also evident when MDR1-transduced cells were directly transplanted into irradiated mice. In these cases, stem cell expansion was associated with relatively high vector copy numbers in stem cell clones. As previously reported, some cases were associated with a characteristic myeloproliferative syndrome. A functionally inactive MDR1 mutant cDNA was used to show that P-gp pump function was required both for amplification of phenotypically defined SP cells and functionally defined repopulating cells. These studies further support the concept that ABC transporter function can have important effects on hematopoietic stem cell development.

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Loss of P-Selectin Promotes the Function of Aged Hematopoietic and Leukemic Stem Cells.

Blood ◽

10.1182/blood.v116.21.1577.1577 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1577-1577

Author(s):

Yaoyu Chen ◽

Sullivan Con ◽

Yiguo Hu ◽

Linghong Kong ◽

Cong Peng ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Flow Cytometry ◽

Stem Cell ◽

Bone Marrow Cells ◽

Leukemic Stem Cells ◽

Wild Type ◽

Post Transplant ◽

Marrow Cells

Abstract Abstract 1577 Hematopoiesis is a tightly regulated biological process that relies upon complicated interactions between the blood cells and their microenvironment. Adhesion molecules like P-selectin are essential to hematopoiesis, and their dysregulation has been implicated in leukemogenesis. We have previously shown a role for P-selectin in chronic myeloid leukemia and demonstrated that in its absence the disease process accelerates. Recently, there has also been speculation that P-selectin may play a role in the aging hematopoietic stem cells (HSCs), as its expression in upregulated as a mouse ages. In this study, we show that the loss of P-selectin function dysregulates the balance of stem cells and progenitors and that these differences become more pronounced with age. We compared the percentages of HSCs, long-term (LT)-HSCs, short-term (ST)-HSCs, multipotent progenitors (MPPs), CMPs, GMPs and MEPs in bone marrow by flow cytometry between wild type (WT) and Selp-/- mice. An age-dependent LT-HSC expansion was observed in WT mice. However, this expansion was prevented by the loss of Selp as observed in Selp-/-mice. Further, we demonstrate that with age LT-HSCs in particular express more elevated levels of P-selectin. LT-HSCs and ST-HSC/MPPs were isolated from the bone marrow of young (2 months old) and old (15 months old) WT mice and examined P-selectin expression by FACS. A significant increase in P-selectin expression was observed in LT-HSCs of old mice, and this increase was not observed in the ST-HSC+MPP subpopulations. We also show that the loss of P-selectin gene has profound effects of stem cell function, altering the capacity of these cells to home. Despite impaired homing capacity, stem cells lacking P-selectin possess a competitive advantage over their wild type counterparts. Using a stem cell competition assay, HSCs derived from Selp-/- mice (CD45.2+) and WT control mice (CD45.2+GFP+) were mixed in 1:1 ratio and transplanted into irradiated WT recipients (CD45.1). The initial findings were potentially indicative of the ability of cells derived from GFP mice to more efficiently home and engraft. Despite this initial advantage, cells derived from Selp-/- eventually exhibited a competitive and statistically significant advantage over the cells derived from GFP mice. At 30 days post-transplant, 49.9±1.4% of the CD45.2 subpopulation was GFP+. At 86 days post-transplant, 25.7±3.3 % of the CD45.2 cells derived from the peripheral blood were GFP+. Similarly, 23.0±3.7% of the CD45.2 cells derived from the bone marrow of these mice were GFP+. Indeed, we demonstrate that recipients of P-selectin deficient bone marrow cells more efficiently repopulate the bone marrow than controls and that this advantage extends and expands in the long-term. Finally, we demonstrate that recipients of leukemic cells lacking P-selectin develop a more accelerated form of leukemia accompanied by significant increases in stem and progenitor cells. Bone marrow cells from donor WT and Selp-/- mice were infected with retrovirus expressing BCR-ABL-GFP, and irradiated WT recipients were transplanted with 2×105 of these transduced donor cells. At 14 days post-transplant, recipient mice from each of the groups were sacrificed, and bone marrow cells were harvested and analyzed by flow cytometry. Recipients of leukemic Selp-/- cells possessed 3.5-fold more LSCs than recipients of wild-type cells. There were 3.1-fold more LT-LSCs and 3.8-fold more ST-LSCs and MPPs in recipients of Selp-/- cells than WT cells. In addition, recipients of leukemic Selp-/- cells possessed significantly more CMP (16.9-fold) and MEP (4.5-fold) cells. Because P-selectin expression increases with age on LT-HSCs, we sought to determine the role that age plays in CML development and progression. Bone marrow cells derived from 15-month-old donor Selp-/- and WT mice were transduced with BCR-ABL, respectively, followed by transplantation of the transduced cells into recipient mice. All recipients of BCR-ABL transduced Selp-/- cells died by 23 days after induction of CML and had a median survival of 19 days, whereas recipients of the transduced WT cells survived significantly longer. This pro-leukemic role for cells lacking P-selectin expression is leukemic stem cell-specific rather than stromal cell-specific and supports an essential role for P-selectin on leukemic stem cells. Disclosures: No relevant conflicts of interest to declare.

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Dnmt3a is Required For Aberrant Self-Renewal Driven by PML-RARA

Blood ◽

10.1182/blood.v122.21.477.477 ◽

2013 ◽

Vol 122 (21) ◽

pp. 477-477

Author(s):

Christopher B Cole ◽

Angela M. Verdoni ◽

David H Spencer ◽

Timothy J. Ley

Keyword(s):

Dna Methylation ◽

Bone Marrow ◽

Progenitor Cells ◽

Bone Marrow Cells ◽

Ex Vivo ◽

Myeloid Cells ◽

Cd11b Expression ◽

Self Renewal ◽

Hematopoietic Stem ◽

Marrow Cells

We previously identified recurrent mutations in the DNA methyltransferase DNMT3A in patients with acute myeloid leukemia (AML). DNMT3A and the highly homologous gene DNMT3B encode the two methyltransferases that are primarily responsible for mediating de novo methylation of specific CpG residues during differentiation. Loss of Dnmt3a in hematopoietic stem cells impairs their ability to differentiate into committed progenitors (Challen et al Nat Gen 44:23, 2011). Importantly, DNMT3A mutations are mutually exclusive of the favorable prognosis AML-initiating translocations, including the t(15;17) translocation (which creates the PML-RARA fusion gene), and translocations involving MLL. PML-RARA has been shown to interact with DNMT3A in vitro (Di Croce et al Science 295:1079,2002), and to require DNMT3A to induce methylation and transcriptional silencing of a subset of specific target genes. These findings, and the lack of DNMT3A mutations in APL patients, suggest that PML-RARA may require functional DNMT3A to initiate leukemia. To investigate this possibility, we utilized a well-characterized transgenic mouse model (in a pure B6 background) in which expression of PML-RARA is driven in hematopoietic stem/progenitor cells by the mouse Cathepsin G locus (Ctsg-PML-RARA+/- mice). These mice spontaneously develop acute promyelocytic leukemia (APL) with high penetrance and long latency, and also exhibit a preleukemic phenotype marked by the accumulation of myeloid cells in bone marrow and spleen. In addition, myeloid progenitor cells derived from these mice have the ability to serially replate in methylcellulose cultures, demonstrating aberrant self-renewal. We generated Ctsg-PML-RARA+/- mice lacking Dnmt3a (PML-RARA+/- x Dnmt3a-/-) as well as mice in which conditional ablation of Dnmt3b in hematopoietic cells is driven by Vav-Cre (PML-RARA+/- x Dnmt3b fl/fl x Vav-Cre+). Loss of Dnmt3a completely abrogated the ex vivo replating ability of PML-RARA bone marrow (Figure 1). Although colonies from both PML-RARA+/- and PML-RARA+/- x Dnmt3a-/- mice appeared similar in morphology and number on the first plating, PML-RARA+/- x Dnmt3a-/- marrow ceased to form colonies with subsequent replating (see Figure), and cultured cells lost the expression of the myeloid marker CD11b. The same phenotype was also observed using bone marrow from both genotypes that was secondarily transplanted into wild type recipients, indicating that it is intrinsic to transplantable hematopoietic progenitors. Reintroduction of DNMT3A into bone marrow cells derived from PML-RARA+/- x Dnmt3a-/- mice with retroviral transduction restored replating ability and CD11b expression. Competitive repopulation experiments with PML-RARA+/- x Dnmt3a-/- marrow revealed a decreased contribution to peripheral lymphoid and myeloid cells at 4 weeks, relative to PML-RARA+/- or WT control animals. Finally, 12 weeks after transplantation, recipients of PML-RARA+/- x Dnmt3a-/- bone marrow did not display an accumulation of myeloid cells in the bone marrow and spleen. Importantly, bone marrow from PML-RARA+/- x Dnmt3b fl/fl x Vav-Cre+/- mice displayed no replating deficit or loss of CD11b expression ex vivo, indicating different functions for Dnmt3a versus Dnmt3b in this model. Finally, we interrogated the effect of Dnmt3a loss on bone marrow DNA methylation patterns using a liquid phase DNA capture technique that sampled ∼1.9 million mouse CpGs at >10x coverage. Loss of Dnmt3a caused a widespread loss of DNA methylation in whole bone marrow cells, with 36,000 CpGs that were highly methylated (methylation value >0.7) in the PML-RARA+/- and WT mice, but hypomethylated (methylation value <0.4) in Dnmt3a-/- and PML-RARA+/- x Dnmt3a-/- mice. Characterization of the effect of Dnmt3a loss on leukemia latency, penetrance, and phenotype in PML-RARA+/- mice is currently being defined in a tumor watch. In summary, we have demonstrated that PML-RARA requires functional Dnmt3a (but not Dnmt3b) to drive aberrant self-renewal of myeloid progenitors ex vivo, and that loss of Dnmt3a leads to widespread DNA hypomethylation in bone marrow cells, and abrogates preleukemic changes in mice expressing PML-RARA. This data may explain why DNMT3A mutations are not found in patients with APL initiated by PML-RARA. Disclosures: No relevant conflicts of interest to declare.

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Demonstration of Thy-1 antigen on pluripotent hemopoietic stem cells in the rat.

Journal of Experimental Medicine ◽

10.1084/jem.148.5.1351 ◽

1978 ◽

Vol 148 (5) ◽

pp. 1351-1366 ◽

Cited By ~ 66

Author(s):

I Goldschneider ◽

L K Gordon ◽

R J Morris

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Bone Marrow Cells ◽

Cell Activity ◽

Hemopoietic Stem Cell ◽

Hemopoietic Stem Cells ◽

Stem Cell Activity ◽

Series Of Experiments ◽

Marrow Cells

Three approaches were used to demonstrate the presence of Thy-1 antigen on the surface of pluripotent hemopoietic stem cells in the rat. In the first, stem cells from fetal liver, neonatal spleen, and adult bone marrow were prevented from forming hemopoietic colonies in the spleens of irradiated recipients spleen (colony-forming unit assay) by incubation with antibodies to Thy-1 antigen. Highly specific rabbit heteroantiserum to purified rat brain Thy-1 antigen and mouse alloantisera to Thy-1.1-positive thymocytes were equally effective. This inhibition was neutralized by purified Thy-1 antigen. In a second series of experiments, Thy-1-positive and Thy-1-negative populations of nucleated bone marrow cells were separated by the FACS. All of the hemopoietic stem cell activity was recovered in the Thy-1-positive population. The stem cells were among the most strongly positive for Thy-1 antigen, being in the upper 25th percentile for relative fluorescence intensity. The relationships of Thy-1 antigen to the rat bone marrow lymphocyte antigen (BMLA) was shown in a third series of experiments. Rabbit anti-BMLA serum, which is raised against a null population of lymphocyte-like bone marrow cells, has been shown to have anti-stem cell activity. Here we demonstrate by double immunofluorescence, cocapping, and differential absorption studies that Thy-1 and BMLA are parts of the same molecule.

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Retroviral gene transfer of human adenosine deaminase in murine hematopoietic cells: effect of selectable marker sequences on long-term expression

Blood ◽

10.1182/blood.v78.2.310.bloodjournal782310 ◽

1991 ◽

Vol 78 (2) ◽

pp. 310-317 ◽

Cited By ~ 1

Author(s):

JF Apperley ◽

BD Luskey ◽

DA Williams

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Gene Transfer ◽

Bone Marrow Cells ◽

Selectable Marker ◽

Retroviral Vector ◽

G418 Selection ◽

Marrow Cells

Retroviral-mediated gene transfer of human adenosine deaminase (hADA) provides a model system for the development of somatic gene therapy as a therapy for diseases of bone marrow-derived cells. We have previously demonstrated that hADA can be observed in all hematopoietic lineages in a minority of mice transplanted with bone marrow cells infected with a simplified retroviral vector, ZipPGK-ADA. Here we report a majority of mice (six of eight) demonstrate expression of hADA in the peripheral blood at least 6 months after transplantation with bone marrow infected with this simplified retroviral vector, which contains no selectable marker. The failure to express hADA in two of eight mice was associated with the absence of the recombinant retroviral provirus in DNA prepared from bone marrow cells of these mice apparently due to failure to efficiently infect the reconstituting hematopoietic stem cell. In an effort to preselect bone marrow stem cells containing proviral integrations, we incorporated the selectable marker neo phosphotransferase (NEO) into a retroviral vector encoding hADA, N2/ZipPGK-ADATKNEO, and used G418 selection of infected bone marrow cells before transplantation. In contrast to the simplified retroviral vector, hADA expression in these recipients was short lived (less than 8 weeks), despite the continued presence of intact provirus in DNA prepared from bone marrow of these mice. To determine whether the preselection of bone marrow using G418 was responsible for the lack of sustained hADA expression, we repeated the infection with the N2/ZipPGK- ADATKNEO vector but omitted the G418 selection step. Again, the majority of recipient mice failed to express hADA long term, although the continued presence of provirus in DNA prepared from peripheral blood cell mononuclear cells was clearly demonstrated. Finally, we demonstrate clonal fluctuation of infected stem cells, and observe a temporal correlation between cessation of expression of hADA and the emergence of a dominant stem cell clone between 14 and 20 weeks posttransplantation in one recipient. These data suggest that inclusion of a second transcriptional unit that includes neo phosphotransferase sequences in this simplified vector is associated with decreased expression of the nonselectable ADA sequences.

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The Tumor Suppressor Role of the Msr1 Gene in Cancer Stem Cells of Chronic Myeloid Leukemia.

Blood ◽

10.1182/blood.v114.22.188.188 ◽

2009 ◽

Vol 114 (22) ◽

pp. 188-188

Author(s):

Yaoyu Chen ◽

Con Sullivan ◽

Shaoguang Li

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Chronic Myeloid Leukemia ◽

Tumor Suppressor ◽

Myeloid Leukemia ◽

Bone Marrow Cells ◽

Leukemia Cells ◽

Wild Type ◽

Marrow Cells

Abstract Abstract 188 We have previously shown that the arachidonate 5-lipoxygenase gene (Alox5) functions as a critical regulator of leukemia stem cells (LSCs) in BCR-ABL-induced chronic myeloid leukemia (CML) in mice (Chen Y, Hu Y, Zhang H, Peng C, Li S. Loss of the Alox5 gene impairs leukemia stem cells and prevents chronic myeloid leukemia. Nature Genetics 41:783-792, 2009). We believe that the Alox5 pathway represents a major molecular network in LSCs. Therefore, we decided to further dissect this pathway by comparing gene expression profiles between wild type and Alox5−/− LSCs from CML mice using the DNA microarray analysis. We identified a small group of candidate genes that were changed in expression in the absence of Alox5. Among these genes, we have identified the Msr1 gene and chosen to test the function of this gene in regulating LSC function, because this gene was up-regulated, indicating that it might play a tumor suppressor role in LSCs. In our CML mouse model, we observed that recipients of BCR-ABL transduced Msr1−/− bone marrow cells developed CML much rapidly than recipients of BCR-ABL transduced wide type bone marrow cells. To test whether this accelerated CML is related to abnormal function of LSCs, we carried out a serial transplantation assay by transferring bone marrow cells from primary recipients of BCR-ABL-transduced wild type or Msr1−/− donor bone marrow cells into secondary and next-generation of recipient mice to biologically assess the effect of Msr1 on LSCs. BCR-ABL-expressing wild type leukemia cells from bone marrow of CML mice were only able to transfer CML once, whereas BCR-ABL-expressing Msr1−/− leukemia cells were able to transfer lethal CML for five genrations. This observation indicates that BCR-ABL-expressing Msr1−/− LSCs have markedly increased stem cell function. To further compare the stem cell function, we performed the leukemia stem cell competition assay by 1:1 mixing wild type (CD45.1) and Msr1−/− (CD45.2) bone marrow cells from CML mice. At day 25 or 30 after transplantation, more than 60% and 95% of GFP+Gr-1+ cells in peripheral blood of the mice were CD45.2+Msr1−/− myeloid leukemia cells, and all these mice developed CML and died of CML derived from Msr1−/− LSCs. To confirm the tumor suppressor role of Msr1 in CML development, we co-expressed BCR-ABL and Msr1 in MSR1−/− bone marrow cells by retroviral transduction, followed by transplantation of these cells into recipient mice. The ectopically-expressed Msr1 in MSR1−/− bone marrow cells rescued the accelerated CML phenotype, and some recipient mice did not even develop the CML. Together, these results demonstrate that Msr1 plays a tumor suppressor role in LSCs. The Msr1 pathway is a novel molecular network in LSCs, and it will be important to fully study this pathway for developing curative therapeutic strategies for CML. Disclosures: No relevant conflicts of interest to declare.

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ATRA and the RARα Specific Agonist, NRX195183, Have Opposing Effects on the In Vitro Clonogenicity of Pre-Leukemic Murine AML1-ETO Bone Marrow Cells

Blood ◽

10.1182/blood.v116.21.999.999 ◽

2010 ◽

Vol 116 (21) ◽

pp. 999-999

Author(s):

Lynette C.Y. Chee ◽

Jean Hendy ◽

Louise Purton ◽

Grant A. McArthur

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Bone Marrow Cells ◽

Myeloid Cells ◽

Myeloid Cell ◽

Sustained Remission ◽

Self Renewal ◽

Cells Cultured ◽

Marrow Cells

Abstract Abstract 999 All-trans retinoic acid (ATRA) is used successfully to treat acute promyelocytic leukemia (APML), however, to date it has not shown promise in treating other AML subtypes. ATRA has been shown to enhance hematopoietic stem cell (HSC) self-renewal (requiring RARγ activation) but promotes differentiation of myeloid progenitors likely through RARα activation. We hypothesized that (1) the lack of success of ATRA in treating other AML subtypes may be due to the potential ability of ATRA to enhance self-renewal of the leukemic stem cell and (2) the use of a specific RARα agonist may have more promise in enhancing AML differentiation. We therefore compared the effects of pharmacological levels (1μM) of ATRA and an RARα-specific agonist, NRX195183, on bone marrow cells harvested from a Cre-inducible conditional AML1-ETO (AE) knock-in murine model. AE cells cultured for 2 weeks with ATRA showed significant reductions in the proportions of mature myeloid cells (Gr1brightCD11b+) by fluorescence activated cell sorting (FACS) (DMSO: 14.2±4.3%, ATRA: 4.0±1.6%, p=0.04, n=4). By 4 weeks of culture, ATRA-treated AE cells had increased blast and reduced maturing myeloid cell proportions (Blasts %: DMSO 70.2 ± 3.0, ATRA 95.3 ± 1.2, p=0.08; Intermediate %: DMSO 14.3 ± 2.6, ATRA 3.8 ± 1.0, p=0.01; Neutrophils %: DMSO 2.3± 1.0, ATRA 0.3 ± 0.2, p=0.07, n=6). Furthermore, ATRA potentiated the clonogenicity of the AE cells after 5 weeks of treatment in vitro (Mean±SEM for colony #/ 5×104 cells: DMSO 505.8±337.0, ATRA 4394±388.9, p=0.001; n=6). In contrast, AE cells cultured for 2 weeks with NRX195183 showed significant increases in the proportions of mature myeloid cells by FACS (DMSO: 15.8±3.5%, NRX195183 26.7±3.0%, p=0.03; n=5). By 4 weeks of culture, NRX195183-treated AE cells had decreased blast and increased maturing myeloid cell proportions (Blasts %: DMSO 82.4±3.0, NRX195183 58.8±9.1, p=0.03; Intermediate %: DMSO 14.5±2.5, NRX195183 29.0±6.8, p=0.07; Neutrophils %: DMSO 1.6±0.8, NRX195183 8.2±4.7 p=ns; DMSO n=8, NRX195183 n=5). Moreover, NRX195183 reduced the clonogenicity of the AE cells after 5 weeks of treatment in vitro (Mean±SEM for colony #/ 5×104 cells DMSO 554.8±252.6, NRX195183 82.6±61.6, p=0.05; n=8). Short-term in vivo transplants of fetal liver cells overexpressing the truncated AE gene, AE9a, into sublethally irradiated recipients revealed similar findings in the NRX195183-treated mice with a decrease in blasts and an increase in mature neutrophils in the peripheral blood on morphological analysis after 4 weeks of treatment (Blasts x106/ml: DMSO 3.1±1.0, NRX195183 0.9±0.3, p=0.08; Neutrophils x106/ml: DMSO 0.5±0.1, NRX195183 0.8±0.1, p=0.04; DMSO n=16, NRX195183 n=11). Taken together, these findings support a model whereby ATRA promotes self-renewal of leukemic blasts whilst NRX195183 has the opposing effect. To understand the mechanism by which ATRA promotes self-renewal in AE cells, we performed genome-wide gene expression analyses on the ATRA- versus control-treated AE cells. This revealed 16 differentially upregulated genes after 24 hours of treatment. Using Ingenuity Pathway Analysis, the top scoring network in the ATRA-treated AE cells was cell-to-cell signalling and interaction (p=1.1E-7-2.4E-3), lipid metabolism (p=2.3E-7-2.0E-3) and small molecule biochemistry (p=2.3E-7-2.1E-3); SERPINE1 and BMP2 were the genes with the highest connectivity within the network interacting with molecules known for their roles in tumorigenesis, including AKT, NF-kβ complex and TGFβ1. SERPINE1 upregulation has been shown to be RARα-mediated whilst BMP2 has been shown to be a RARγ-regulated gene. Interestingly, the specific RARγ agonist, NRX204723, had no effect on the clonogenic potential of these AE progenitors thus raising the hypothesis that both RARα and RARγ activation are required to promote self-renewal of the AE progenitors. Further studies using both RARα/RARγ agonists are warranted to assess if the ATRA effects on AE cells are phenocopied. Collectively, these findings reveal the contrasting roles of specific RARα activation in promoting loss of self-renewal ability and enhancing differentiation in the AE cells whilst ATRA promotes clonogenicity of these cells. This has potential significant implications in AML treatment as specific RARα agonists may be beneficial in improving the efficacy of current treatment modalities to achieve sustained remission in other AML subtypes. Disclosures: No relevant conflicts of interest to declare.

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