Regulation of Chronic Myelogenous Leukemia Stem Cells by Leukemia Oncogene Evi1

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 964-964
Author(s):  
Tomohiko Sato ◽  
Susumu Goyama ◽  
Keisuke Kataoka ◽  
Takako Tsuruta ◽  
Masahiro Nakagawa ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Chronic Myelogenous Leukemia ◽  
Targeted Therapies ◽  
Expression Profiles ◽  
Myelogenous Leukemia ◽  
Leukemic Cells ◽  
Hematopoietic Stem ◽  
Myeloid Progenitor

Abstract Abstract 964 Chronic myelogenous leukemia (CML) is a hematopoietic stem cell (HSC) disease caused by BCR-ABL oncogene, and now newly targeted therapies are warrented for CML due to the minimal effect of BCR-ABL-targeted tyrosine kinase inhibitors toward CML stem cells. As CML stem cells are known to show some similarity with HSC, utilizing HSC-specific factors as a guide for analyzing CML stem cells is of great significance. Since Evi1 is a transcription factor which is highly expressed within normal HSC compartment and it is frequently activated in myeloid malignancies including a blastic phase (blast crisis) of CML (CML-BC), it is supposed that CML stem cells could have a close relation with Evi1. Here in this study, with Evi1-GFP knock-in mice, which we have recently generated, we developed murine models of CML in a chronic phase (CML-CP) and CML-BC for uncovering new properties of CML stem cells. In Evi1-GFP knock-in CML-CP model, we found that Evi1 positive CML cells account for about 0.1–0.5% of total bone marrow (BM) cells and that almost all of them showed no lineage markers. Furthermore, Evi1 is predominantly expressed in the CML stem cell fraction (Lin- Sca-1+ c-kit+ (LSK)), but its expression is sharply downregulated even in myeloid progenitor (Lin- Sca1- c-kit+ (MP)) cells and in more differentiated cells. Even within CML LSK cells, Evi1 expression widely varies and Evi1-high LSK cells show an enhanced colony-forming capacity compared with Evi1-low LSK cells. As for cell cycle status, Evi1-high CML LSK cells are mostly in G0/G1 phase although Evi1-low CML LSK cells or CML myeloid progenitor are more in S/G2/M phase. When CML LSK cells are cocultured with OP-9 stromal cells, only Evi1-high LSK cells could made cobblestone areas. Comparison of Evi1-high cells with Evi1-low cells in normal and CML LSK compartments by gene expression profiles showed that a more quiescent feature and a less differentiated feature in Evi-high CML LSK cells than in Evi1-low CML LSK cells. Moreover, Evi1-high CML LSK cells have a close correlation with TGF-beta signaling and calcium signaling. In addition, Evi1-high normal LSK cells had the most quiescent and the least differentiated profiles, which suggested that Evi1-high CML LSK cells could keep self-renewal capacity with high proliferation capacity. In concert with our data of Evi1-trafficking CML mouse, in CML patients, we have also recently found that CD34+ 38- CML stem cells showed higher EVI1 expression than CD34+ 38+ CML progenitor cells or total CML cells, which implies that EVI1 could mark CML stem cells as well as normal HSCs. These data indicate that in our Evi1 trafficking CML model high Evi1 expression could enrich CML stem cells and that Evi1 could have a crucial role in CML pathogenesis. In Evi1-GFP knock-in CML-BC model, which more differentiated myeloid progenitors are likely to have a high leukemia initiating potential, a sizable fraction of MP leukemic cells show distinct Evi1 expression. Remarkably, in vivo transplantation assay revealed CML-BC stem cells that can recapitulate the disease are exclusively enriched in Evi1-high MP fraction. Evi1-high MP cells showed a replating capacity in colony assay while Evi1-low MP cells could not. Moreover, Evi1-high MP cells are more actively cycling than Evi1-low MP cells. Our data revealed a limited fraction with high Evi1 expression within stem/progenitor cells possesses enhanced proliferative and leukemia-initiating capacities in CML. As opposed to these CML models noted above, in Evi1-GFP knock-in AML model by MLL-ENL, Evi1-high leukemic cells showed no advantage in leukemia initiating potential. Additionally, other Evi1-GFP knock-in AML models by MOZ-TIF2 and TEL-PDGFRb/AML1-ETO never showed Evi1-high fraction both in BM and spleen, which might suggest the high affinity of Evi1 with stem cell disease as CML. The current study provides us with a new tool for dissecting pathogenesis and exploiting novel targeted therapies to eradicate CML stem cells. An establishment of Evi1-related therapy for CML stem cells, which could be applied to EVI1-high malignancies, is currently being explored. Disclosures: No relevant conflicts of interest to declare.

Identification of human chronic myelogenous leukemia progenitor cells with hemangioblastic characteristics

Blood ◽  
2005 ◽  
Vol 105 (7) ◽  
pp. 2733-2740 ◽  
Author(s):  
Baijun Fang ◽  
Chunmei Zheng ◽  
Lianming Liao ◽  
Qin Han ◽  
Zhao Sun ◽  
...  
Keyword(s):  
Stem Cells ◽  
Endothelial Cells ◽  
Progenitor Cells ◽  
Chronic Myelogenous Leukemia ◽  
Blood Cells ◽  
Fusion Gene ◽  
Fetal Liver ◽  
Philadelphia Chromosome ◽  
Myelogenous Leukemia ◽  
Leukemic Cells

AbstractOverwhelming evidence from leukemia research has shown that the clonal population of neoplastic cells exhibits marked heterogeneity with respect to proliferation and differentiation. There are rare stem cells within the leukemic population that possess extensive proliferation and self-renewal capacity not found in the majority of the leukemic cells. These leukemic stem cells are necessary and sufficient to maintain the leukemia. Interestingly, the BCR/ABL fusion gene, which is present in chronic myelogenous leukemia (CML), was also detected in the endothelial cells of patients with CML, suggesting that CML might originate from hemangioblastic progenitor cells that can give rise to both blood cells and endothelial cells. Here we isolated fetal liver kinase-1–positive (Flk1+) cells carrying the BCR/ABL fusion gene from the bone marrow of 17 Philadelphia chromosome–positive (Ph+) patients with CML and found that these cells could differentiate into malignant blood cells and phenotypically defined endothelial cells at the single-cell level. These findings provide direct evidence for the first time that rearrangement of the BCR/ABL gene might happen at or even before the level of hemangioblastic progenitor cells, thus resulting in detection of the BCR/ABL fusion gene in both blood and endothelial cells.

Junb Regulates Hematopoietic Stem Cell Numbers in Normal and Leukemic Mice.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 560-560
Author(s):  
Emmanuelle Passegue ◽  
Erwin F. Wagner ◽  
Irving L. Weissman
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Blast Crisis ◽  
Myelogenous Leukemia ◽  
Hematopoietic Stem ◽  
Conditional Deletion ◽  
Stage Of Development ◽  
Myeloid Progenitor ◽  
P16 Ink4a

Abstract JunB is expressed in hematopoietic stem cells (HSC) as a partner for Fos in the composition of the AP-1 transcription factor. Previously, we have shown that junB inactivation in postnatal mice results in the development of a myeloproliferative disorder (MPD) resembling early human chronic myelogenous leukemia (CML) (Passegue et al., 2001, Cell, 104, 21-32). Here, we demonstrate that JunB is a critical transcriptional regulator of HSC numbers both in normal and leukemic mice. Overexpression of junB in long-term HSC (LT-HSC) dramatically decreases the frequency of LT-HSC, while inactivation of junB specifically expands the numbers of LT-HSC, and of granulocyte/macrophage progenitors (GMP), resulting in the development of a chronic MPD with many features of human CML, including progression to blast crisis, and death. JunB effects are mediated, at least in part, via the regulation of effectors genes such as the cell cycle inhibitor p16/INK4a, which is a direct junB-target gene and a key regulator of stem cell proliferation/senescence, as well as the anti-apoptotic proteins bcl2 and bcl-xl, two critical regulators of stem cell death. Using several models of conditional deletion of junB in hematopoietic cells, we demonstrate that junB inactivation must take place in LT-HSC, and not at later stages of myelopoiesis, to induce MPD. Most importantly, we show that only junB-deficient LT-HSC, and no other myeloid progenitor populations, are capable of transplanting the MPD to recipient mice. These results indicate a stem cell-specific role for JunB in normal and leukemic hematopoiesis, and provide an experimental demonstration that leukemia stem cells (LSC) can reside at the LT-HSC stage of development in a mouse model of chronic MPD.

Hypoxia-Adapted Myeloma Cells Possess Stem Cell Character

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3088-3088
Author(s):  
Eishi Ashihara ◽  
Yoko Nakagawa ◽  
Hisayuki Yao ◽  
Asumi Yokota ◽  
Yasuo Miura ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Conflicts Of Interest ◽  
Flow Cytometric Analysis ◽  
Scid Mice ◽  
Myelogenous Leukemia ◽  
Leukemic Cells ◽  
Hematopoietic Stem ◽  
Hypoxic Conditions ◽  
Arterial Blood

Abstract The prognosis of patients with multiple myeloma (MM) has been improved by the emergence of new molecular targeting agents including proteasome inhibitors and immunomodurating agents. Nevertheless, MM remains incurable at present because it is likely that MM stem cells are resistant to these targeting agents. Thus, it is important to further investigate the biology of MM stem cells to cure the MM patients. We have demonstrated that b-catenin is a novel and attractive target against MM (Ashihara et al. Clin Cancer Res, 2009; Yao et al. Blood Cancer J, 2011). We next investigate novel targets focused on the hypoxic bone marrow (BM) environment. BM is known to have low levels of oxygen, particularly at the epiphysis, which is distant form the BM arterial blood supply. Normal hematopoietic stem cells (HSCs) reside in this hypoxic epiphyseal region “niche”, and HSCs are protected from DNA damage induced by reactive oxygen species. We have previously found that chronic myelogenous leukemia (CML) cells engrafted in the BM survived and proliferated in the severely hypoxic environment and that these hypoxia-adapted (HA) leukemic cells acquired stem cell-like characters (Takeuchi et al. Cell Death Differ, 2010). In this study, we investigated the characteristics of hypoxia-adapted MM (HA-MM) cells. We first confirmed oxygen status in the BM of the MM cell-engrafted mice. Irradiated NOD/SCID mice were inoculated with 2 x 106 AMO-1 cells. After 2 or 4 weeks transplantation, we sacrificed mice and confirmed engraftment. The inoculated MM cells engrafted in the epiphysis in recipient mice after 2 weeks transplantation, and populated endosteum of epiphysis after 4 weeks. These MM cells were positive for pimonidazole, which specifically accumulated in hypoxic cells (< 1.3% O2 concentration). These observations suggested that MM cells resided in the BM are hypoxic. We then established AMO-1, OPM-2, and IM-9 HA-MM cells cultured under hypoxic conditions (O2 1%). These HA cells can continue to proliferate in hypoxic conditions for more than six months. In flow cytometric analysis, the G0 fraction cells as well as side population fraction cells significantly increased in HA-MM cells compared with those in the parental MM cells. We next transplanted parental or HA-MM AMO-1 cells with same cell numbers into irradiated NOD/SCID mice. The survival durations of mice transplanted with HA-AMO-1 cells were significantly shorter than that of mice transplanted with parental cells. Moreover, in serial transplantation experiments, all 5 HA-MM cell-transplanted mice died of MM whereas 1 out of 5 parental MM cell-transplanted mice (Figure 1). Quantitative RT-PCR analysis demonstrated that Sox2, Oct3, and Nanog mRNA transcripts increased in the HA-MM AMO-1 cells (Figure 2). We next investigated the signaling pathway activated in HA-MM cells. Interestingly, phosphorylated Smad2 expression was increased in HA-AMO-1 cells. These findings suggest that HA-MM cells possess stem cell-like character, and these cells may provide a useful model to investigate the mechanism of MM stem cells (myeloma-initiating cells) resistant to molecular target agents. Disclosures: No relevant conflicts of interest to declare.

Deletion of Rac2 inhibits Proliferation of Chronic Myelogenous Leukemia (CML) Stems Cells and Progenitors (HSC/P) In Vivo and Promotes Survival of Scl/p210-BCR-ABL Mice.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3253-3253
Author(s):  
Amitava Sengupta ◽  
Jorden Arnett ◽  
Susan Dunn ◽  
Jose Cancelas
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Chronic Myelogenous Leukemia ◽  
Research Funding ◽  
Myelogenous Leukemia ◽  
Leukemic Stem Cells ◽  
Hematopoietic Stem ◽  
Rac Gtpases ◽  
And Migration

Abstract Abstract 3253 Poster Board III-1 Chronic myelogenous leukemia (CML) is a hematopoietic stem cell (HSC) malignancy induced by p210-BCR-ABL and is characterized by myeloproliferation in the bone marrow (BM) and egress of leukemic stem cells and progenitors (LSC/P) to extramedullary sites. Persistence of BCR-ABL+ HSCs in patients under imatinib suggests that inhibition of ABL-kinase alone is not sufficient to completely eliminate the LSC/P population. Rac GTPases represent integrative molecular switches for p210-BCR-ABL-induced HSC transformation and combined pharmacological and genetic attenuation of Rac GTPases significantly prolong survival in vivo, as reported in a retroviral transduction/transplantation model (Thomas EK & Cancelas JA et al, Cancer Cell 2008). Here, we analyzed the role of Rac2 GTPase in the leukemic maintenance and in the interaction of LSC/P with the leukemic microenvironment in vivo. We used a stem cell leukemia (Scl) promoter-driven, tet-off, Scl-tTA x TRE-BCR-ABL (Scl/p210-BCR-ABL) binary transgenic mouse model (Koschmieder S et al., Blood 2005), where expression of BCR-ABL is restricted to the HSC/P compartment, allowing the study of the intrinsic molecular changes in LSC/P during leukemogenesis. In these mice, Scl-driven expression of BCR-ABL is active in HSC (Lin-/Sca1+/c-kit+; LSK) and progenitors (Lin-/c-kit+/Sca-1-; LK), and CML development is associated with the activation of downstream signaling effectors CrkL, p38-MAPK and JNK. Additionally, Scl/p210-BCR-ABL mice had increased cycling of LSK cells and expansion of circulating and splenic, but not BM, LSC/P, suggesting egress of LSC/Ps from the marrow. These mice share all the characteristics of HSC/P transformation in CML, including increased HSC/P proliferation and survival, severely reduced adhesion to fibronectin, increased migration towards CXCL12, increased cell surface expression of CD44 and decreased expression of L-selectin. Myeloproliferative disease (MPD) in these mice is transplantable into recipient mice, and CML splenocytes have a 10-fold increase in homing to the spleen than towards BM (P<0.05). Leukemic splenocytes are also enriched in endosteal lodging progenitors, compared to the BM-derived progenitors (1.9-fold, P≤0.05). In order to determine the contribution of Rac2 GTPase in the transformation phenotype of leukemic stem cells and progenitors, Scl/p210 mice were intercrossed with Rac2-/- mice. Interestingly loss of Rac2 GTPase alone significantly prolongs survival of the leukemic mice (P≤0.001). Prolonged survival, as observed in Scl/p210 x Rac2-/-, is associated with significantly reduced proliferation of leukemic LK (3-fold, P<0.05) and LSK (6-fold P<0.005) cells, both in BM as well as in spleen, in vivo. Scl/p210 x Rac2-/- mice are also characterized by increased apoptosis (1.7-fold) and lower frequency of LSK cells (2-fold) compared to the Scl/p210 mice in vivo. However, deletion of Rac2 does not significantly reverse the adhesion and migration transformation phenotype of LSC/P. In summary, Rac2 deficiency induces a significant survival of CML mice in a HSC-initiated model of disease through decrease proliferation and survival but does not reverse the transformation phenotype affecting adhesion and migration. This murine model may represent an adequate in vivo system to dissect out the specific signaling pathways involved in p210-BCR-ABL-induced stem cell transformation. Disclosures: Cancelas: CERUS CO: Research Funding; CARIDIAN BCT: Research Funding; HEMERUS INC: Research Funding.

Cytokine-STATs Signalings Upregulate C/EBPβ In BCR-ABL+ Leukemic Cells Independently From BCR-ABL/JAK-STAT Pathway

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1468-1468
Author(s):  
Asumi Yokota ◽  
Hideyo Hirai ◽  
Yoshihiro Hayashi ◽  
Akihiro Tamura ◽  
Atsushi Sato ◽  
...  
Keyword(s):  
Stem Cells ◽  
Progenitor Cells ◽  
Therapeutic Strategy ◽  
Myelogenous Leukemia ◽  
Leukemic Cells ◽  
Bone Marrow Niche ◽  
Hematopoietic Stem ◽  
Complete Cure ◽  
Novel Therapeutic ◽  
Stat Pathway

Abstract Since residual chronic myelogenous leukemia (CML) stem cells may be a cause of relapse after Imatinib (IM) cessation, targeting these IM-resistant stem cells is mandatory for complete cure of this disease. CCAAT/enhancer binding protein β (C/EBPβ), a leucine zipper transcription factor, promotes both cell cycle progression and differentiation toward granulocytes in hematopoietic stem/progenitor cells under stress conditions such as infection. We have recently reported that C/EBPβ was upregulated in leukemic stem/progenitor cells derived from patients in chronic phase of CML (CP-CML) through STAT5, a major downstream target of BCR-ABL. In CML mouse model, C/EBPβ enhanced exhaustion of CML stem cells through promoting their differentiation (Hayashi Y et al, Leukemia 2013). In spite of the upregulation of C/EBPβ by BCR-ABL, CML stem cells will not be exhausted spontaneously in patients with CP-CMP. Therefore, we hypothesized that quiescent CML stem cells maintain their immature status in the bone marrow niche by suppressing C/EBPβ expression or function induced by BCR-ABL and that induction of C/EBPβ expression in CML stem cells through BCR-ABL-independent pathways may be a novel therapeutic strategy targeting CML stem cells. The aim of this study is to propose a novel therapeutic strategy that can stimulate quiescent CML stem cells with cytokine-STATs signalings and induce their exhaustion through C/EBPβ-mediated differentiation. STATs are family members of molecules which convey signals from various kinds of cytokine receptors to nucleus. In order to investigate whether STAT molecules can induce C/EBPβ expression, we first examined the effects of constitutive active (CA) form of STAT1, STAT3 and STAT5 on C/EBPβ expression in a murine hematopoietic stem cell line, EML cells. Retroviral transduction of CA-STAT5 significantly upregulated C/EBPβ mRNA and protein in EML cells. EML cells begun to differentiate toward CD11b+ myeloid lineage upon introduction of CA-STAT5. CA-STAT1 and CA-STAT3 also upregulated C/EBPβ mRNA when they were retrovirally transduced into EML cells (Figure 1). These results suggest that signaling mediated by various kinds of STATs can upregulate C/EBPβ. Consensus binding sites for STATs were not found in the proximal (∼ 4 kb) promoter region of C/EBPβ and we are currently identifying the cis-regulatory elements responsible for the STATs-dependent activation of C/EBPβ.Figure 1Figure 1. Interferon-α (IFNα) exerts STATs-mediated signaling in hematopoietic stem cells and has been used for therapy of CML. Therefore we investigated the possible involvement of C/EBPβ in efficacy of IFNα in CML treatments. Stimulation of EML cells with 500 U/ml IFNα upregulated C/EBPβ mRNA (Figure 2). Higher levels of phosphorylation of STAT1 than those of STAT3 or STAT5 were observed after stimulation with IFNα, suggesting that STAT1 mediated activation of C/EBPβ was induced by IFNα. As previously reported, C/EBPβ was upregulated in EML cells transduced with BCR-ABL (EMLBCR-ABL). Treatment of EMLBCR-ABL cells with IFNα augmented this effect significantly. IM effectively inhibited phosphorylation of STAT5 and blunted the upregulation of C/EBPβ in EMLBCR-ABL cells. Simultaneous treatment of EMLBCR-ABL cells with IFNα and IM resulted in maintained upregulation of C/EBPβ with increased phosphorylation of STAT1 and decreased phosphorylation of STAT5. These data suggested that IFNα treatment can upregulate C/EBPβ independently of signals mediated by BCR-ABL.Figure 2Figure 2. In conclusion, cytokine-STATs signalings can induce C/EBPβ expression in BCR-ABL+ leukemic cells independently from BCR-ABL/JAK-STAT pathway. Stimulations of dormant CML stem cells with cytokines might be a novel treatment strategy to eliminate these populations, leading to complete cure of CML. We are currently evaluating the in vivo effects of IFNα treatment on CML stem cells in mice models. Disclosures: No relevant conflicts of interest to declare.

Transforming Growth-Interacting Factor (TGIF) Modulates Hematopoietic Stem Cell Functions

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1279-1279
Author(s):  
Ling Yan ◽  
Bethany Womack ◽  
Stephen J. Brandt ◽  
Rizwan Hamid
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Flow Cytometric Analysis ◽  
Myelogenous Leukemia ◽  
Leukemic Cells ◽  
Term Survival ◽  
Hematopoietic Stem ◽  
Cell Functions

Abstract Abstract 1279 We have shown that quantitative expression of the stem cell expressed Transforming-growth-interfering factor (TGIF) is a predictor of patient survival in acute myelogenous leukemia (AML). By Kaplan-Meier analysis, patients whose leukemic cells expressed decreased levels of TGIF RNA had a mean survival of 12 months, while patients whose leukemic cells expressed TGIF at a higher level had a mean survival of 60 months (p=0.00001). TGIF is a homeobox transcriptional repressor and although it has been implicated in holoprosencephaly (HPE), whether it has a direct role in hematopoiesis is not known. It is known, however, that TGIF is expressed in hematopoietic stem cells (HSCs) and that it can down-regulate both TGF-b and retinoic acid (RA) signaling, and there is incontrovertible evidence that both of these pathways play an important role in hematopoiesis. To understand the biological basis for our clinical finding, we hypothesized a role for TGIF in hematopoiesis and in particular HSC function. We then proceeded to test this hypothesis in a Tgif knockout mouse model. Flow cytometric analysis of non-lineage depleted bone marrow (BM) cells showed that Tgif-null mice had statistically increased numbers of long-term HSCs (LT-HSCs) as defined by CD34-Flk2-Lin-ckit+Sca1+ status or by CD150+CD48-Lin-cKit+Sca+ status and decreased numbers of multipotent progenitors (MPP) as defined by CD34+FlK+Lin-ckit+Sca1+ status. The short-term (ST) HSC (defined by CD34+Flk2-lin-ckit+Sca1+ or by CD150-CD48-lin-ckit+Sca+) populations were not statistically different between the null and the WT-type mice. Tgif-null BM cells produced statistically significant higher total colony number in methylcellulose colony forming unit (CFU) compared to the WT-mice though the percentages of specific colonies types remained the same. We then compared the Tgif-null BM cells with WT-BM cells in competitive repopulation assays (CRA). These experiments showed that Tgif-null BM cells were more competitive and showed higher engraftment compared to the WT-BM cells. In conclusion, our data suggest that Tgif has an important role in hematopoietic stem cell function and may play a role in determining the balance between quiescence and self-renewal. One hypothesis to explain these data is that HSCs with no or low Tgif are more quiescent and if so this provides one biological explanation of how might TGIF affect AML prognosis; increased quiescence of HSCs or the Leukemic stem cells derived from these HSCs may make them more resistant to myelotoxic injury following chemotherapy, increasing the likelihood of relapse and/or poor long-term survival. These hypotheses continue to be an active area or research in our laboratory. Disclosures: No relevant conflicts of interest to declare.

Bmi1 Is Essential for Leukemic Reprogramming of Myeloid Progenitor Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1423-1423
Author(s):  
Jin Yuan ◽  
Masahiro Takeuchi ◽  
Hideyuki Oguro ◽  
Masamitsu Negishi ◽  
Hitoshi Ichikawa ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Target Genes ◽  
Expression Profiles ◽  
Myelogenous Leukemia ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Myeloid Progenitor ◽  
Immortalized Cells

Abstract Abstract 1423 Poster Board I-446 The polycomb group (PcG) protein Bmi1 plays an essential role in the maintenance of self-renewing hematopoietic stem cells (HSCs). Derepressed p16Ink4a and p19Arf are tightly associated with a loss of self-renewing capacity of HSCs in Bmi1-deficient mice. Deletion of both Ink4a and Arf genes substantially restores the self-renewal capacity of Bmi1−/− HSCs. Thus, Bmi1 maintains HSCs by acting as a critical failsafe against the p16Ink4a- and p19Arf-dependent senescence pathway. Meanwhile, Bmi1 was originally identified as a collaborating oncogene in the induction of lymphoma and was subsequently reported to be overexpressed in various human cancers including leukemia. Recent studies have demonstrated that PcG proteins bind to multiple regions of the genome and regulate a bunch of target genes. Therefore, we asked whether Bmi1 is essential for leukemic stem cells (LSCs) and tried to identify critical target genes for Bmi1 other than Ink4a and Arf in leukemia. We expressed the MLL-AF9 leukemic fusion gene in purified Lin−Sca-1−c-Kit+CD34+FcγRII/ IIIhi granulocyte/macrophage progenitors (GMPs) from wild-type, Bmi1−/−, Ink4a-Arf−/−, and Bmi1−/−Ink4a-Arf−/− mice and performed in vitro myeloid progenitor replating assay. GMPs from 4 different genetic backgrounds were all immortalized in vitro, although Bmi1-deficient cells showed a slightly decreased replating efficiency. We then infused the immortalized cells into lethally irradiated recipient mice. Mice infused with wild-type and Ink4a-Arf−/− cells developed acute myelogenous leukemia (AML) at 30 to 60 days after infusion. Mice infused with Bmi1−/− cells did not develop leukemia at all. While a significant portion of mice infused with Bmi1−/−Ink4a-Arf−/− cells developed AML, although they took much longer time compared to those mice infused with wild-type and Ink4a-Arf−/− cells. These results indicate that as in HSCs, the Ink4a /Arf locus is one of the major targets for Bmi1 in leukemogenesis. In order to find unknown targets of Bmi1 in LSCs, we compared gene expression profiles of purified c-KithiFcRγII/IIIhiCD34+ cells from Ink4a-Arf−/− and Bmi1−/−Ink4a-Arf−/− immortalized cells. We found that the loss of Bmi1 did not affect the induction of MLL-AF9 target gene expression. By contrast, a number of genes were derepressed in the absence of Bmi1. Among these, Tbx15, a transcriptional co-repressor gene, appeared to be regulated by Bmi1 and a potential tumor suppressor gene in the development of leukemia. Of interest, the majority of derepressed target genes in transformed Bmi1−/−Ink4a-Arf−/− cells, including Tbx15, remained unchanged by re-expression of Bmi1. Correspondingly, re-introduction of Bmi1 to transformed Bmi1−/−Ink4a-Arf−/− cells failed to rescue their compromised leukemogenic activity in vivo. Our findings suggest that Bmi1 is required for faithful epigenetic reprogramming of myeloid progenitors into LSCs by leukemic fusions and contributes to establish LSC-specific transcriptional profiles to confer full leukemogenic activity on LSCs. Disclosures: No relevant conflicts of interest to declare.

Identification Of Leukemia Suppressive Genes By Inducible Overexpression Of The DNA Methyltransferase DNMT3B During Leukemogenesis In Mice

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2493-2493
Author(s):  
Isabell Schulze ◽  
Petra Tschanter ◽  
Christian Rohde ◽  
Annika Krause ◽  
Heinz Linhart ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Stem Cells ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Expression Patterns ◽  
Leukemic Cells ◽  
Leukemic Stem Cells ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Serial Transplantation

Abstract DNA methyltransferases (DNMT) play an important role in regulation of DNA methylation and mutations of DNMT3A are frequently found in AML. In previous studies using a tetracycline-inducible DNMT3B mouse model, we could show that overexpression of DNMT3B affected leukemia initiation and maintenance upon retroviral transduction and serial transplantation of hematopoietic stem and progenitor cells with MSCV-MLL-AF9-GFP and MSCV-cmyc-bcl2-mcherry oncogenic vectors, respectively. Sublethally irradiated recipient mice of DNMT3B overexpressing MLL-AF9 and cmyc/bcl2 leukemic cells developed leukemia with a prolonged latency when compared to recipients of wildtype cells. We performed serial transplantation assays of MLL-AF9 leukemic stem cells, which were sorted for high expression of ckit. The life-prolonging effect of DNMT3B expression was stem cell-specific, as the potential to initiate leukemia was maintained upon serial retransplantation and recipients of DNMT3B overexpressing leukemic stem cells also died significantly later in secondary (p<0.001) and tertiary transplantations (p<0.001). Analysis of global DNA methylation levels in MLL-AF9 ckit+ leukemic stem cells and cmyc/bcl2 leukemic cells via Reduced Representation Bisulfite Sequencing (RRBS) revealed a strong hypermethylation in DNMT3B overexpressing cells, independent of the oncogene used for leukemia induction. Differentially methylated CpG sites were defined as CpGs with at least 20% methylation difference between wildtype and DNMT3B overexpressing samples. Hypermethylation in MLL-AF9 leukemic cells directly correlated with observed hypermethylation in cmyc/bcl2 leukemic cells and inversely correlated with hypomethylation in cmyc/bcl2 cells, indicating that in both leukemias, the same sites are prone to DNMT3B induced DNA methylation. To investigate, if these changes in DNA methylation resulted in different gene expression patterns, we performed microarray analysis of the same MLL-AF9 leukemic wildtype and DNMT3B expressing samples which were also used for DNA methylation analysis. In microarray analyses, we could identify several genes differentially expressed in DNMT3B overexpressing cells when compared to wildtype samples. Interestingly, changes in expression levels could not be attributed to differential DNA methylation in promoter regions. Instead, hypermethylation in exons and gene bodies resulted in downregulation of the respective genes, whereas genes with hypomethylated exons and gene bodies showed higher expression levels. Genes downregulated in DNMT3B overexpressing cells, were mainly cancer-associated genes, which are known to have functions in cellular growth and proliferation, as well as in the hematopoietic system development and maintenance. Gene Set Enrichment Analysis (GSEA) of wildtype cells revealed a strong enrichment of genes upregulated in different stages of hematopoietic stem and progenitor cells as well as in leukemic stem cells, whereas DNMT3B overexpressing samples were enriched in genes which have been shown to be downregulated in hematopoietic and leukemic stem cells and upregulated in mature hematopoietic cells. This strengthens our hypothesis that DNMT3B induced DNA methylation mainly influences the phenotype and function of hematopoietic stem cells and thereby, exerts its inhibitory function on leukemia initiation and maintenance. Taken together, these findings demonstrate that DNMT3B exerts its anti-leukemic effect mainly via induction of aberrant DNA methylation in hematopoietic and leukemic stem cells, thereby changing expression patterns of genes known to be important for stem cell function. The identification of differentially expressed DNMT3B target genes could help to find promising targets for new therapeutic strategies in AML. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Clonal analysis of bcr-abl rearrangement in T lymphocytes from patients with chronic myelogenous leukemia

Blood ◽  
1992 ◽  
Vol 79 (4) ◽  
pp. 1017-1023 ◽  
Author(s):  
D Jonas ◽  
M Lubbert ◽  
ES Kawasaki ◽  
M Henke ◽  
KJ Bross ◽  
...  
Keyword(s):  
Stem Cell ◽  
T Cell ◽  
T Lymphocytes ◽  
Chronic Myelogenous Leukemia ◽  
Philadelphia Chromosome ◽  
Precursor Cell ◽  
Myelogenous Leukemia ◽  
Hematopoietic Stem ◽  
T Cell Clones ◽  
Cell Clones

The cytogenetic hallmark of chronic myelogenous leukemia (CML) is the Philadelphia chromosome (Ph1), which reflects a chromosomal translocation t(9;22) and a rearrangement of the ABL and bcr genes. This marker is found in all cells arising from the same malignant precursor cell and can be detected in CML cells of the myeloid, monocytic, erythroid, and B-lymphocyte lineage. It is, however, controversial as to whether T lymphocytes of CML patients carry this gene rearrangement. An answer to this question would clarify whether the translocation in CML occurs in a pluripotent hematopoietic stem cell or in a precursor cell already committed to certain lineages, but not the T-cell lineage. To address this question, we established T-cell clones from peripheral venous blood cells of four patients with CML and screened these clones for bcr-abl fusion transcripts by means of polymerase chain reaction and Southern blot analysis. In four T-cell clones of three of these patients, the bcr-abl transcript could be detected. None of 12 T-cell clones of the fourth patient disclosed detectable bcr-abl amplification product. Both CD4+ as well as CD8+ clones displayed fused bcr-abl sequences. These data imply that in CML some but not all T lymphocytes may originate from the Ph1-positive stem cell.

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