Loss Of p53 Promotes Transformation Of Oncogenic Nras-Induced Chronic Myelomonocytic Leukemia To An Acute Phase

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3790-3790
Author(s):  
Jingfang Zhang ◽  
Yangang Liu ◽  
Guangyao Kong ◽  
Yuan-I Chang ◽  
Erik A. Ranheim ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Median Survival ◽  
Bone Marrow Cells ◽  
Chronic Myelomonocytic Leukemia ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Myelomonocytic Leukemia ◽  
Acute Myeloid ◽  
Marrow Cells ◽  
Myeloid Progenitors

Abstract Chronic myelomonocytic leukemia (CMML) primarily occurs in the elderly with the median age ranging from 65 to 75 years. As defined by WHO, CMML is characterized by persistent monocytosis in peripheral blood, hepatosplenomegaly, and the absence of BCR-ABL fusion gene. CMML is a devastating cancer for multiple reasons, one of which is that approximately 20% of CMML cases evolve into acute myeloid leukemia (AML) soon after their first diagnosis. However, little is known about the cellular and molecular mechanisms underlying this malignant transformation. Recently, our lab developed a CMML mouse model induced by oncogenic NrasG12D/+ expressed from its endogenous locus. Above 90% of recipient mice with NrasG12D/+ bone marrow cells developed CMML-like phenotypes with a median survival of ∼56 weeks. Interestingly, none of these mice spontaneously transform to AML. To identify the pathogenetic origins underlying CMML transformation to AML, we further deleted p53 expression in NrasG12D/+ bone marrow cells using p53fl/fl allele and Mx1-Cre because deletion of p53 is a common genetic event observed in oncogenic Ras-driven cancers. We found that ERK1/2 is significantly hyperactivated in NrasG12D/+; p53-/- hematopoietic stem/progenitor cells (enriched for myeloid progenitors) in the absence of cytokines or in the presence of low concentration of GM-CSF. Concomitantly, the mutant myeloid progenitors show significantly increased self-renewal in a serial replating assay in vitro. We transplanted NrasG12D/+, p53-/-, or NrasG12D/+; p53-/- bone marrow cells into lethally irradiated mice. Unlike recipients with p53-/- cells that died of a T-cell disease with 100% penetrance and a median survival of 24 weeks, ∼70% of recipients with NrasG12D/+; p53-/- cells died of AML or acute myeloid sarcoma with a median survival of 16 weeks. These malignant myeloid diseases are transplantable in secondary recipients. Interestingly, only mutant hematopoietic stem cells (HSCs) could initiate and maintain leukemia phenotypes in the NrasG12D/+ induced CMML model, whereas both NrasG12D/+; p53-/- HSCs and myeloid progenitors could initiate AML or acute myeloid sarcoma. Our results indicate that deletion of p53 cooperates with NrasG12D/+ mutation to transform CMML into an acute phase. This malignant transformation is initiated by mutant myeloid progenitors, which show increased self-renewal and potentially serve as leukemia initiating cells. Disclosures: No relevant conflicts of interest to declare.

Hmgb3 Deficiency Inhibits Down-Regulation of c-kit in Hematopoietic Stem Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 372-372
Author(s):  
Michael J. Nemeth ◽  
Stacie M. Anderson ◽  
Lisa J. Garrett-Beal ◽  
David M. Bodine
Keyword(s):  
Bone Marrow ◽  
Hind Limb ◽  
Bone Marrow Cells ◽  
Es Cells ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Bone Marrow Cellularity ◽  
Marrow Cellularity ◽  
Marrow Cells

Abstract Hmgb3 is an X-linked member of a family of sequence-independent chromatin-binding proteins that is expressed in HSC-enriched lin−, c-kitHI, Sca-1HI, IL-7Rα− (KSIL) cells and Ter119+ erythroid cells. To define Hmgb3 function, we generated hemizygous mice (Hmgb3−/Y) using 129/SvJ ES cells. Hmgb3−/Y mice contain normal numbers of KSIL cells that are capable of normal repopulation and self-renewal. However, these mice have 1.6-fold fewer common lymphoid progenitors (CLP) and 3-fold fewer common myeloid progenitors (CMP) (p < 0.05). We hypothesized that the role of Hmgb3 in early hematopoiesis involves c-kit regulation. We observed that the level of c-kit mRNA in Hmgb3−/Y HSCs increased 30% compared to wild-type (WT) (p = 0.05). We used 5-fluorouracil (5-FU), which has been shown to down-regulate c-kit on HSCs, to characterize the interaction between Hmgb3 and c-kit. We monitored Hmgb3 expression in KSIL and lin−, Sca-1+, c-kit− cells before and after 5-FU treatment (150 mg/kg) using phenotypically normal transgenic mice containing an IRES-GFP cassette knocked into the 3′ UTR of Hmgb3. Prior to 5-FU treatment, 27% of KSIL cells were GFP+ (these cells were absent 4 days post-injection {p.i.}). In contrast, 1.8% of lin−, c-kit−, Sca-1+ cells were GFP+ before 5-FU treatment whereas 26% of lin−, c-kit−, Sca-1+ cells were GFP+ 4 days p.i. The increased proportion of GFP+ lin-, c-kit−, Sca-1+ cells after 5-FU treatment is consistent with previous findings that repopulating activity resides within the c-kit−/LO population in 5-FU treated bone marrow and our finding that Hmgb3 serves as a marker for long-term repopulating activity. To determine the time course of c-kit regulation, we compared bone marrow from 5-FU injected Hmgb3−/Y and WT mice for analysis at 2, 4, and 6 days p.i. Two days p.i., both WT and Hmgb3−/Y mice contained similar numbers of bone marrow cells (7 x 106 cells/hind limb) and the KSIL population was absent. By four days p.i., the bone marrow cellularity of WT mice declined to 5.5 ± 0.9 x 106 cells/hind limb and KSIL cells were still absent. However, in Hmgb3−/Y mice 4 days p.i., bone marrow cellularity stabilized at 7.9 ± 0.8 x 106 cells/hind limb, an increase of 43% compared to WT (p < 0.01), along with the re-emergence of the KSIL population. To determine whether the Hmgb3−/Y lin−, c-kit−, Sca-1+ population contains repopulating HSCs after 4 days of 5-FU treatment similar to WT mice, we performed repopulation assays using KSIL and lin−, c-kit−, Sca-1+ cells sorted from 4 day p.i. 5-FU treated Hmgb3−/Y mice. Recipients received either 2 x 104 KSIL or 2 x 105 lin−, c-kit−, Sca-1+ cells (Ly 5.2) from 5-FU treated Hmgb3−/Y mice along with a radioprotective dose of 3 x 105 congenic (Ly 5.1) bone marrow cells. FACS analysis performed on control recipients transplanted with congenic marrow exhibited < 1% Ly 5.2 cells in the bone marrow 16 weeks after transplant. Pre-5-FU treatment, 88% of bone marrow cells were donor derived in recipients of Hmgb3−/Y KSIL cells. There was no detectable engraftment of Hmgb3−Y lin−, c-kit−, Sca-1+ cells. In contrast to WT mice, both KSIL and lin−, c-kit−, Sca-1+ cells from 5-FU treated Hmgb3−/Y mice were capable of long-term repopulation (62–82% donor derived cells). We conclude that Hmgb3 deficiency facilitates the reemergence of c-kitHI HSCs following 5-FU treatment. Mechanisms involving either enhanced HSC self-renewal or delayed differentiation into CLPs and CMPs are both consistent with our results.

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

Blood ◽  
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&lt;0.05) which persisted at 3 months (22.8 vs 1.5, p&lt;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.

Non-Canonical NF-Kb Signaling Regulates Hematopoietic Stem Cell Self-Renewal and Microenvironment Interactions

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 859-859 ◽  
Author(s):  
Chen Zhao ◽  
Yan Xiu ◽  
John M Ashton ◽  
Lianping Xing ◽  
Yoshikazu Morita ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Wild Type ◽  
Double Knockout ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Physiological Processes ◽  
Marrow Cells

Abstract Abstract 859 RelB and NF-kB2 are the main effectors of NF-kB non-canonical signaling and play critical roles in many physiological processes. However, their role in hematopoietic stem/progenitor cell (HSPC) maintenance has not been characterized. To investigate this, we generated RelB/NF-kB2 double-knockout (dKO) mice and found that dKO HSPCs have profoundly impaired engraftment and self-renewal activity after transplantation into wild-type recipients. Transplantation of wild-type bone marrow cells into dKO mice to assess the role of the dKO microenvironment showed that wild-type HSPCs cycled more rapidly, were more abundant, and had developmental aberrancies: increased myeloid and decreased lymphoid lineages, similar to dKO HSPCs. Notably, when these wild-type cells were returned to normal hosts, these phenotypic changes were reversed, indicating a potent but transient phenotype conferred by the dKO microenvironment. However, dKO bone marrow stromal cell numbers were reduced, and bone-lining niche cells supported less HSPC expansion than controls. Further, increased dKO HSPC proliferation was associated with impaired expression of niche adhesion molecules by bone-lining cells and increased inflammatory cytokine expression by bone marrow cells. Thus, RelB/NF-kB2 signaling positively and intrinsically regulates HSPC self-renewal and maintains stromal/osteoblastic niches and negatively and extrinsically regulates HSPC expansion and lineage commitment through the marrow microenvironment. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽  
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.

MOZ-TIF2, but Not BCR-ABL, Confers Properties of Leukemic Stem Cells to Committed Murine Hematopoietic Progenitors.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 62-62
Author(s):  
Brian J.P. Huntly ◽  
Hirokazu Shigematzu ◽  
Kenji Deguchi ◽  
Benjamin Lee ◽  
Shinichi Mizuno ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mononuclear Cells ◽  
Hierarchical Organization ◽  
Leukemic Stem Cells ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Acute Myeloid ◽  
Myeloid Progenitors

Abstract The existence of leukemia stem cells has been demonstrated in acute myeloid and lymphoblastic leukemias (AML and ALL). The origins of these cells are unknown, but it has been suggested that they result from the transformation of adult hematopoietic stem cells (HSC). To challenge this hypothesis we tested the ability of representative leukemia oncogenes to transform committed myeloid progenitor cells that lack the capacity for self-renewal. Flow-sorted populations of common myeloid progenitors (CMP), and granulocyte-monocyte progenitors (GMP) were transduced with the fusion oncogenes MOZ-TIF2 and BCR-ABL, respectively and their self-renewal and leukemogenic potential were tested in in vitro and in vivo assays. Utilizing the same experimental design we were also able to address the poorly understood question of the contribution of the cell of transformation to the eventual leukaemia phenotype. In contrast to CMP or GMP transduced with BCR-ABL or non-transduced control cells, CMP or GMP that were retrovirally transduced with MOZ-TIF2 could be serially replated in methylcellulose cultures, and continuously propagated in liquid culture media containing IL-3. In further contrast, transplantation of CMP or GMP transduced with MOZ-TIF2 into recipient mice also resulted in an acute myeloid leukemia (AML). This leukaemia could be transplanted to secondary recipients, documenting the long-term self-renewal properties of the leukemic stem cells, yet in limiting dilution experiments did not cause disease below a transplanted cell dose of 1 x104 cells, suggesting that the probability of transferring leukemia to secondary recipients relates to the frequency of self-renewing leukemic stem cells within the total leukemic population. This in turn suggests that our retroviral bone marrow transduction and transplantation models have the same hierarchical organization of self-renewal as has been shown for human AML. The phenotype of the leukemias were virtually indistinguishable regardless of whether the initially transduced cell population was CMP, GMP or the control populations of whole bone marrow mononuclear cells or HSC, suggesting that MOZ-TIF2 may also have a dominant effect upon the eventual leukaemia phenotype. These observations indicate that MOZ-TIF2, but not BCR-ABL, can confer properties of leukemic stem cells to committed myeloid progenitors. Control experiments conducted with with MOZ-TIF2 point mutants that do not cause leukemia in the murine BMT system and with BCR-ABL, a fully active leukemogenic tyrosine kinase, were insufficient to cause in-vitro changes in self-renewal or leukaemia. Together, these data argue strongly that retroviral insertional mutagenesis alone cannot explain these results. However, we cannot exclude the possibility that an active MOZ-TIF2, but not BCR-ABL, can collaborate with mutations induced by retroviral mutagenesis to confer properties of leukemic stem cells to committed progenitors. These findings have important implications regarding the origin of leukemic stem cells, and provide tools for understanding the transcriptional programs that confer properties of self-renewal in malignant and non-malignant cells.

Wnt5a Inhibits Wnt3a-Mediated HSC Differentiation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2271-2271
Author(s):  
Michael Nemeth ◽  
David Bodine
Keyword(s):  
Bone Marrow ◽  
Formation Control ◽  
Bone Marrow Cells ◽  
Wnt Signaling Pathway ◽  
Retroviral Vectors ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Canonical Wnt ◽  
Cells Cultured ◽  
Marrow Cells

Abstract Activation of the canonical Wnt signaling pathway by Wnt3a has been implicated in hematopoietic stem cell (HSC) self-renewal (Reya et al., Nature, 2003). Wnt5a has been observed to inhibit Wnt3a signaling (Topol et al., J Cell Biol, 2004). We hypothesized that Wnt3a and 5a act as antagonists on HSC function. 1 x 106 lineage negative cells (lin−) were cultured for 4 days in the presence of 50 ng/ml SCF and Flt3L (control) plus 100 ng/ml rmWnt3a and/or 500 ng/ml rmWnt5a (all factors added on day 0 and day 2). Control lin− cell numbers expanded more than lin− cells cultured with Wnt3a, 5a, or both (control 8.3 ± 0.3-fold; Wnt3a 6.9 ± 0.2-fold (p &lt; .01); Wnt5a 4.8 ± 0.2-fold (p &lt; .001); Wnt3a and 5a 2.6 ± 0.6-fold (p &lt; .001); n = 3). After 4 days, cells were analyzed for myeloid colony formation. Control cells and cells cultured in Wnt3a had similar numbers of CFU-GM/5000 lin− cells (control 13.1 ± 11.1; Wnt3a 21.8 ± 15.3; p = .21; n = 8), while cells cultured in Wnt5a and Wnt3a and 5a had 2-fold and 5.9-fold more CFU-GM/5000 lin− cells than control (Wnt 5a 26.8 ± 13.3 (p = .04); Wnt3a and 5a 77.9 ± 48.3 (p &lt; .01); n = 8). To analyze repopulating ability, 4 x 105 lin− Ly5.1 cells, cultured under the same conditions, were transplanted with 2 x 106 Ly5.2 bone marrow cells into lethally-irradiated Ly5.2 recipients. 16 weeks after transplant, repopulation by control lin− cells increased 2-fold compared to lin− cells cultured in Wnt3a or Wnt5a (control 7.3 ± 3.8%; Wnt3a 3.37 ± 1.2% (p &lt; .01); Wnt5a 3.6 ± 1.1% (p &lt; .01); n = 9-10). However, lin− cells cultured in Wnt3a and 5a showed normal repopulating activity (n = 10; 8.7 ± 5.3%; p = .52). 1 x 104 HSCs (lin−, c-kitHI, Sca-1HI, IL-7Rα −) were cultured for 6 days with SCF, Flt3L, Wnt3a and 5a (factors added on day 0 and day 3) as described above. Control HSC numbers expanded more than HSCs cultured with Wnt3a, Wnt 5a, or both (control 20.7 ± 10.4-fold; Wnt 3a 7.0 ± 4.1-fold (p = .05); Wnt5a 1.7 ± 1.7-fold (p = .01); Wnt3a and 5a 1.2 ± 1.0-fold (p &lt; .01); n = 4). Similar numbers of control HSCs and HSCs cultured with Wnt3a or 5a were lin+ (control 21.7 ± 0.2%; Wnt 3a 15.4 ± 5.3% (p = .10); Wnt5a 14.4 ± 5.2% (p = .07); n = 3). However, culturing HSCs with Wnt3a and 5a resulted in a 50% decrease in the number of lin+ cells compared to control (12.3 ± 2.0% (p = .001)). Cultured Ly5.1 HSCs were transplanted with Ly5.2 bone marrow cells at a 1:100 ratio. There was no difference in repopulation between control HSCs and HSCs cultured with Wnt3a (control 5.8 ± 6.1%; Wnt3a 3.6 ± 0.4%; p = .43; n = 5). To examine the effects of enforced expression of Wnt ligands in HSCs, 5-FU treated bone marrow was transduced with Wnt3a-IRES-GFP, Wnt5a-IRES-dsRED, or IRES-GFP retroviral vectors. Sorted IRES-GFP+, Wnt3a-GFP+ and Wnt5a-dsRed+ cells (Ly5.1) were transplanted with equal numbers of mock-transduced cells and 3 x 105 Sca-1− bone marrow cells (Ly5.2) into lethally-irradiated Ly5.2 mice. 16 weeks later, recipients of IRES-GFP+ and Wnt5a-dsRed+ cells contained a similar number of engrafted cells expressing the vector (3.4 ± 1.8% GFP+ Ly5.1 and 3.5 ± 0.4% dsRed+ Ly5.1 respectively; n = 8). In contrast, no GFP+ Ly5.1 cells were detected in Wnt3a-GFP+ recipients (n = 8). 33.4 ± 3.7% of bone marrow cells were Ly5.1+ indicating successful engraftment and retroviral DNA was detected by PCR, suggesting that transduction had occurred but that only cells in which the vector was silenced survived. We conclude that activation of the canonical Wnt pathway in HSCs promotes differentiation of primitive hematopoietic cells and that other signals, such as Wnt5a, are required to maintain the balance between HSC differentiation and self-renewal.

Mutant p53 Drives the Development of Pre-Leukemic Hematopoietic Stem Cells through Modulating Epigenetic Regulators

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 307-307
Author(s):  
Sarah C Nabinger ◽  
Michihiro Kobayashi ◽  
Rui Gao ◽  
Sisi Chen ◽  
Chonghua Yao ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Leukemia Stem Cells ◽  
Mutant P53 ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Tp53 Mutations ◽  
Marrow Cells

Abstract AML is thought to arise from leukemia stem cells (LSCs); however, recent evidence suggests that the transforming events may initially give rise to pre-leukemic hematopoietic stem cells (pre-leukemic HSCs), preceding the formation of fully transformed LSCs. Pre-leukemic HSCs have been shown to contribute to normal blood development and harbor a selective growth advantage compared to normal HSCs. Pre-leukemic HSCs can acquire subsequent mutations, and once differentiation capacity is impaired, leukemia emerges. Recently, acquired somatic TP53 mutations, including p53R248W and p53R273H, were identified in healthy individuals as well as AML patients, suggesting that TP53 mutations may be early events in the pathogenesis of AML. We found that p53R248W HSCs showed a multi-lineage repopulation advantage over WT HSCs in transplantation experiments, demonstrating that mutant p53 confers a pre-leukemic phenotype in murine HSCs. Although TP53 mutations are limited in AML, TP53 mutations do co-exist with mutations of epigenetic regulator, ASXL-1, or receptor tyrosine kinase, FLT3, in AML. Mutations in Asxl-1 are present in ~10-30% of patients with myeloid malignancies and confer poor prognosis. Loss of Asxl-1 in the hematopoietic compartment leads to a myelodysplastic-like syndrome in mice and reduced stem cell self-renewal. Internal tandem duplications in Flt3 (Flt3-ITD) occur in ~30% of AML patients and are associated with adverse clinical outcome. Flt3-ITD-positive mice develop a myeloproliferative neoplasm (MPN) and HSCs expressing Flt3-ITD have decreased self-renewal capabilities. We hypothesize that mutant p53 drives the development of pre-leukemic HSCs with enhanced self-renewal capability, allowing clonal expansion and subsequent acquisition of Asxl-1 or Flt3 mutations leading to the formation of fully transformed leukemia stem cells. To define the role of mutant p53 in Asxl-1+/- HSCs, we generated p53R248W/+ Asxl-1+/- mice and performed in vitro serial replating assays as well as in vivo competitivebone marrow transplantation experiments. We found that p53R248W significantly enhanced the serial replating ability of Asxl-1-deficient bone marrow cells. Interestingly, while bone marrow from Asxl-1+/- mice had very poor engraftment compared to wild type bone marrow cells 16 weeks post-transplantation, the expression of p53R248W in Asxl-1+/- bone marrow rescued the defect. To examine the role of mutant p53 in Flt3-ITD-positive HSCs, we generated p53R248W/+ Flt3ITD/+ mice. We found that p53R248W enhanced the replating ability of Flt3ITD/+ bone marrow cells. Despite the fact that Flt3ITD/+ bone marrow cells displayed decreased repopulating ability compared to wild type cells 16 weeks post-transplant, expression of p53R248W in Flt3ITD/+ cells rescued the defect. We are monitoring leukemia development in primary and secondary transplant recipients as well as in de novo p53R248W/+ Asxl-1+/- and p53R248W/+ Flt3ITD/+ animals and predict that mutant p53 may cooperate with Asxl-1 deficiency or Flt3-ITD in the formation of LSCs to accelerate leukemia development in Asxl-1 deficient or Flt-ITD-positive neoplasms. Mechanistically, dysregulated epigenetic control underlies the pathogenesis of AML and we discovered that mutant p53 regulates epigenetic regulators, including Ezh1, Ezh2, Kdm2a, and Setd2, in HSCs. H3K27me3 is catalyzed by EZH1 or EZH2 of the Polycomb repressing complex 2 (PRC2). Both Ezh1 and Ezh2 are important for HSC self-renewal. SETD2 is a histone H3K36 methyltransferase and mutations in SETD2 have been identified in 6% of patients with AML. SETD2 deficiency resulted in a global loss of H3K36me3 and increased self-renewal capability of leukemia stem cells. We found that there were increased levels of H3K27me3 and decreased levels of H3K36me3 in p53R248W/+ HSCs compared to that of the WT HSCs. In ChIP experiments, we found that p53R248W, but not WT p53, was associated with the promoter region of Ezh2 in mouse myeloid progenitor cells, suggesting that p53R248W may directly activate Ezh2 expression in hematopoietic cells. Given that Asxl-1 has been shown to regulate H3K27me3 in HSCs, the synergy between mutant p53 and Asxl-1 deficiency on LSC self-renewal could be due to changes in histone modifications. Overall, we demonstrate that mutant p53 promotes the development of pre-leukemic HSCs by a novel mechanism involving dysregulation of the epigenetic pathways. Disclosures No relevant conflicts of interest to declare.

Effects of the Homeodomain Gene Pitx2 on Self-Renewal of Hematopoietic Stem Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 95-95 ◽  
Author(s):  
Hui Z. Zhang ◽  
Svetlana Rogulina ◽  
Wendy Chen ◽  
Barbara A. Degar ◽  
Bernard G. Forget
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Leukemic Cells ◽  
Wild Type ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Colony Forming Units ◽  
Marrow Cells

Abstract Pitx2, a homeodomain gene preferentially expressed in murine hematopoietic stem/progenitor cells, is also a downstream target of genes important for hematopoiesis such as MLL and Wnt/Dvl/β-Catenin. We have previously reported that Pitx2 null hematopoietic stem cells (HSCs) can contribute to multi-lineage hematopoiesis under physiologic conditions. We have now carried out serial bone marrow transplantation experiments and demonstrated that after the 3rd round of serial transplantation, Pitx2 null cells reconstituted only 28.6% of the recipient hematopoietic cells as compared to 60% in the case of wild type cells (P<0.001). There were no Pitx2 null donor-derived cells in recipient mice after the 4th round of transplantation, whereas donor-derived chimerism was 57% with wild type cells (P<0.001), and 26% with Pitx2 +/− cells (P<0.001). Therefore, Pitx2 null HSCs have decreased self renewal capacity. To further study the function of Pitx2 in HSC, we constitutively overexpressed the Pitx2 gene in murine bone marrow cells following transduction using a MSCV/IRES/GFP retroviral vector, and analyzed the effects on hematopoiesis in vitro and in vivo. Bone marrow cells overexpressing Pitx2 were isolated on the basis of their GFP expression and analyzed for their colony forming ability in vitro. Retrovirally transduced bone marrow cells were also transplanted into lethally irradiated mice, and the transplanted mice were observed for long-term reconstitution. Colony-forming unit assays showed that Pitx2 overexpressing bone marrow cells, compared to control cells transduced with vector only, had increased numbers of GM colony forming units and reduced numbers of megakaryocytic colony forming units. Pitx2-overexpressing cells continued to form GM colonies after more than eight serial replatings. When these cells were cultured in liquid medium containing SCF, IL-3 and IL-6, they gave rise to cells that stained positively either for alpha naphthyl butyrate, indicating monocytic differentiation, or for peroxidase, indicating neutrophilic differentiation. The ability of these GM-colony forming cells to cause leukemia is currently under investigation. Long-term reconstitution of hematopoiesis in mice by Pitx2 over-expressing HSCs was demonstrated by identifying GFP positive multi-lineage peripheral blood cells four months following transplantation. One of these mice manifested leukemia at this time, as evidenced by a markedly elevated WBC count and other hematologic abnormalities. The leukemic WBCs had very high levels of GFP and Pitx2 expression and were shown to contain two retroviral integration sites, neither of which involved a known oncogene or overexpression of the gene at the integration site. Immunophenotyping by flow cytometry demonstrated that the majority of the leukemic cells were c-kit positive and expressed the megakaryocytic marker CD41, as well as the common myeloid progenitor marker, CD16/32. Some of the cells expressed the erythroid marker Ter119. The leukemic cells did not express any lymphoid markers, including CD3ε, B220, CD19, and IL7R3. This Pitx2-overexpression-associated leukemia was transplantable. Experiments are under way to characterize the leukemia initiating cells. Taken together, our results provide evidence that the homeodomain gene Pitx2 plays a role in the self-renewal of hematopoietic stem/progenitor cells.

scholarly journals Stem cell factor, interleukin-3, and interleukin-6 promote retroviral- mediated gene transfer into murine hematopoietic stem cells

Blood ◽  
1992 ◽  
Vol 80 (2) ◽  
pp. 396-402 ◽  
Author(s):  
BD Luskey ◽  
M Rosenblatt ◽  
K Zsebo ◽  
DA Williams
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Gene Transfer ◽  
Hematopoietic Stem Cells ◽  
Stem Cell Factor ◽  
Bone Marrow Cells ◽  
Self Renewal ◽  
Retroviral Infection ◽  
Hematopoietic Stem ◽  
Marrow Cells

The efficiency of retroviral-mediated gene transfer into hematopoietic stem cells (HSC) is dependent on the survival and self-renewal of HSC in vitro during retroviral infection. We have examined the effect of prestimulation of bone marrow with various cytokines, including the product of the Steel gene, Steel factor or stem cell factor (SCF) (the ligand for the c-kit receptor) on the efficiency of retroviral transduction of the human adenosine deaminase (hADA) cDNA into murine HSC. Bone marrow cells were prestimulated for 48 hours with hematopoietic growth factors, then cocultivated with the packaging cell line producing the ZipPGK-ADA simplified retrovirus for an additional 48 hours with continued growth factor exposure. Nonadherant cells from these cocultures were injected into lethally irradiated recipients. The content of day 12 colony-forming unit-spleen (CFU-S12) in SCF/interleukin 6 (IL-6)-prestimulated and cocultured bone marrow was more than threefold greater than that of IL-3/IL-6-prestimulated bone marrow cells. All mice receiving bone marrow cells infected with the PGK-ADA virus after prestimulation with IL-3/IL-6 or SCF/IL-6 demonstrated hADA expression in the peripheral blood after full hematopoietic reconstitution. While all recipients of IL-3/IL-6- prestimulated bone marrow expressed hADA at 4 months posttransplant, in three independent experiments examining a total of 33 mice, in most recipients of SCF/IL-6-prestimulated and infected bone marrow cells, the expression of human enzyme was higher than IL-3/IL-6 mice. Southern blot analysis of DNA from hematopoietic tissues from these same mice prepared at least 4 months posttransplantation also demonstrated a higher infection efficiency of HSC as measured by proviral integration patterns and genome copy number analysis. These results suggest that the higher level of hADA expression seen in mice receiving marrow prestimulated with SCF/IL-6 before retroviral infection is due to more efficient infection of reconstituting HSC. Other growth factor combinations were also studied; however, prestimulation with SCF/IL-6 or IL-3/IL-6 appeared optimal. Using retroviral-mediated gene transfer and viral integration patterns, Steel factor (SCF) in combination with IL-6 appears to increase the survival and self-renewal of reconstituting hematopoietic stem cells and proves useful in effecting expression of foreign genes in transplant recipients. Such pretreatment may also be useful in the application of retroviral transfer methods to human cells.

Canonical and Non-Canonical Wnt Signaling Regulate the Balance between HSC Self-Renewal and Differentiation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 862-862
Author(s):  
Michael Nemeth ◽  
Yingzi Yang ◽  
David Bodine
Keyword(s):  
Bone Marrow ◽  
Wnt Signaling ◽  
Bone Marrow Cells ◽  
Retroviral Vectors ◽  
Canonical Wnt Signaling ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Serum Free ◽  
Canonical Wnt ◽  
Marrow Cells

Abstract We and others have implicated activation of canonical Wnt signaling by Wnt3a in promoting hematopoietic stem cell (HSC) self-renewal. Since Wnt5a can inhibit canonical Wnt signaling (Topol, et al. 2003), we hypothesized that Wnt3a and Wnt5a act as antagonists on HSC function. To examine the effect of Wnt3a and Wnt5a on canonical Wnt signaling in HSCs, we isolated HSCs (lin−, c-kitHI, Sca-1HI, IL-7Rα−) that contain a lacZ reporter gene that is induced by canonical signaling and cultured them in serum-free media in the presence of 30 ng/ml SCF and Flt3L and recombinant Wnt3a (3a; 100 ng/ml) with and without recombinant Wnt5a (5a; 500 ng/ml). We observed a significant 50% reduction (p &lt; .05) in lacZ mRNA in HSCs cultured with 3a + 5a compared to 3a alone, indicating that Wnt5a inhibits canonical signaling in HSCs. Treatment with 3a, 5a, and 3a + 5a caused the percentage of actively cycling HSCs to decrease 1.4 to 1.6-fold compared to control (SCF + Flt3L) (p &lt; .02). To examine the role of Wnt signaling in HSC self-renewal, wild-type CD45.1+ HSCs were cultured for 6 days before being transplanted into lethally irradiated recipients (1:100 ratio HSCs to CD45.2+ whole bone marrow cells; total number of CD45.1+ HSCs transplanted ranged from 3 to 5 × 103). There was no difference in long-term repopulation between control HSCs (6.7 ± 5.5% CD45.1+ bone marrow cells, n = 14) and HSCs cultured with 3a (4.1 ± 3.7%, n = 8, p = .24). However, HSCs cultured with 5a (24.1 ± 21.2%, n = 10) or 5a + 3a (38.5 ± 36.4%, n = 11) showed significant increases in repopulation compared to control (p &lt; .01; Mann-Whitney test). To determine if additional survival signals were necessary for 3a to induce HSC self-renewal, we transplanted cultured HSCs isolated from transgenic mice that overexpressed the anti-apoptotic gene Bcl2. We observed that control HSCs engrafted 7/7 mice (average repopulation: 17.8 ± 6.8% CD45.1+ bone marrow) whereas when 3a was added, only 1/7 mice showed engraftment (1.2%). Together, these data suggest that induction of canonical signaling by Wnt3a results in decreased HSC expansion and self-renewal and that Wnt5a positively regulates HSC self-renewal by non-canonical Wnt signaling pathways independent of its ability to inhibit canonical signaling. We examined the effects of Wnt3a and Wnt5a on Hoxb4, Notch1, and c-myc expression in HSCs. We observed that adding 3a to serum-free cultures caused no change in Hoxb4 mRNA, a 2-fold reduction in Notch1 mRNA (p &lt; .01) and a 2.4-fold increase in the canonical Wnt pathway target gene c-myc (p &lt; .05; n = 3) compared to control. No changes in Hoxb4, Notch1, or c-myc mRNA were observed in HSCs cultured with 5a or 5a + 3a. Since overexpression of c-myc has been linked to loss of HSC self-renewal, we hypothesized that overexpression of Wnt3a would inhibit HSC self-renewal and repopulation. We transduced CD45.1+ bone marrow with Wnt3a-IRES-GFP or IRES-GFP retroviral vectors and sorted GFP+ cells were transplanted with equal numbers of CD45.2+ mock-transduced cells. Four months post transplantation, 3.4 ± 1.8% of bone marrow cells in IRES-GFP recipients were GFP+ (n = 8). In contrast, no GFP+ cells were detected in Wnt3a-GFP recipients (n = 8). Wnt3a-GFP retroviral DNA was detected by PCR, suggesting that vector silencing was required for transduced cell survival. We propose that imbalanced canonical Wnt signaling in HSCs deregulates hematopoiesis by inducing pro-differentiation genes such as c-myc and that additional signals, e.g. Wnt5a, are required to maintain the balance between HSC differentiation and self-renewal.

Sign in / Sign up

Export Citation Format

Share Document