Leukemic Stem Cells and Progenitors Demonstrate Impaired Interaction with the Hematopoietic Microenvironment in Vivo in An Inducible Murine Model of Chronic Myelogenous Leukemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 191-191
Author(s):  
Amitava Sengupta ◽  
Jose Cancelas
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Chronic Myelogenous Leukemia ◽  
Murine Model ◽  
Chronic Phase ◽  
Myelogenous Leukemia ◽  
Cell Surface Expression ◽  
Leukemic Stem Cells ◽  
Hematopoietic Microenvironment

Abstract Chronic myelogenous leukemia (CML) is a stem cell malignancy induced by p210 BCR-ABL and characterized by myeloproliferation in BM and egression of leukemic stem cells and progenitors (LSC/P) to extramedullary sites. Persistence of BCR-ABL+ HSC in patients under Imatinib suggests inhibition of ABL-kinase alone is not sufficient to eliminate the LSC/P. One of the major hallmarks of CML induced by signaling downstream BCR-ABL is the loss of control of the hematopoietic microenvironment on LSC/P. Expression of p210 BCR-ABL has been associated with loss of adhesion to the bone marrow, impaired migration in response to CXCL12 and decreased retention in the BM. In order to study the putative LSC/P niches in steady-state chronic-phase leukemia, we have analyzed the ability of LSC/P to proliferate and get retained in the bone marrow (BM) in an inducible model of CML. Binary transgenic SCL-tTA/TRE-BCR-ABL mice (Koschmieder S et al., Blood 2005) express p210 BCR-ABL in LSC/P upon doxycycline withdrawal (CML mice). Induced myeloproliferation was associated with activation of the downstream signaling effectors CrkL and p38-MAPK and expansion of circulating (Table 1) and splenic LSC/P but not in BM, suggesting massive LSC/P egression from the marrow (Table 2). Proliferation analysis showed that myeloid expansion in the spleen was secondary to increased cycling of Lin−Sca1+c-Kit+ (LSK) cells (3.1-fold increase in S-phase cells, P<0.05), but not in Lin−/c-Kit+ (LK) population, compared with the control spleens. In agreement with the LSC/P BM content data, the frequency of BM-derived LSK and LK cells incorporating BrdU in CML and in control mice remained similar, suggesting a specific egression of LSC/P from the BM to extramedullary sites. To test whether this model truly represented a model of BM LSC/P egression, we compared the splenic and BM LSC/P compared with their controls regarding their adhesion molecule expression, interaction with the hematopoietic microenvironment (HM) and homing to the overall marrow cavity and endosteal space. Splenic, but not BM-derived, LSK and LSK CD34+ ST-HSCs had increased cell surface expression of CD44 compared to controls (1.35 fold, P=0.006 and 1.23 fold, P<0.05 respectively) and decreased expression of L-selectin (8.7 fold, P<0.05) while expression of CXCR4, α4β1 and α5β1 integrins remain similar in bone marrow and splenocytes from CML and control mice. CML BM progenitors also showed 18-fold reduced adhesion to fibronectin and 1.4-fold increased migration towards CXCL12 compared to control BM progenitors. Myeloproliferative disease was transplantable into non-transgenic littermates and homing of CML BM progenitors was increased (4.3 fold, P<0.005) in myeloablated littermate recipient BM. However, lineage-negative leukemic BM-derived cells which had increased homing in BM of recipient mice had an impaired ability to migrate to the BM endosteal space compared with their littermate controls (control: 31 ± 18% vs CML mice: 17.6 ± 17%), suggesting an specific impairment to lodge in specialized anatomically-defined hematopoietic “niches”. Altogether, this murine model may represent an adequate in vivo system to analyze the ability of p210 BCR-ABL-expressing LSC/P to interact with BM niches and study the control of the hematopoietic microenvironment on LSC/P survival, proliferation and retention. Table 1 Increase in circulating LSC/P in the CML mice after withdrawal of doxycyclin Peripheral Blood LSK (×103)Cells/mL Blood P<0.05 LT-HSC(×103)Cells/mL Blood P<0.05 CFU-GM+BFU-E/mL Blood P<0.05 Control 1.56 ± 0.25 0.459 ± 0.29 60.86 ± 51.09 CML mice 3.56 ± 1.52 2.159 ± 2.03 869.6 ± 628.4 Table 2. Immunophenotypic analysis of BM and splenocytes in control and CML mice Population BM (Cells ×104) (Control) BM (Cells x104) (CML) SP (Cells ×104) (Control) SP (Cells x104) (CML) C-Kit + Sca1 + 24.3 ± 9.9 21.3 ± 11 6.8 ± 4.5 30.1 ± 12.3 (P<0.05) Mac1 + Gr1 + 1779 ± 307 1583 ± 265 78.4 ± 32 608 ± 377 (P<0.05) CFU-C/10 5 Cells 342 ± 66 334 ± 99 63.3 ± 7.09 79 ± 6.54 (P<0.05)

scholarly journals Ubiquitin-mediated interaction of p210 BCR/ABL with β-catenin supports disease progression in a murine model for chronic myelogenous leukemia

Blood ◽  
2013 ◽  
Vol 122 (12) ◽  
pp. 2114-2124 ◽  
Author(s):  
Ru Chen ◽  
Tinghui Hu ◽  
Gwendolyn M. Mahon ◽  
Ilona Tala ◽  
Nicole L. Pannucci ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Transplantation ◽  
Chronic Myelogenous Leukemia ◽  
Murine Model ◽  
Marrow Transplantation ◽  
Chronic Phase ◽  
Myelogenous Leukemia ◽  
Disease Phenotype ◽  
Key Points ◽  
Transplantation Model

Key Points p210 BCR/ABL interacts with β-catenin in the bone marrow transplantation model for chronic myelogenous leukemia. Loss of the interaction results in an altered disease phenotype, suggesting a role for β-catenin in chronic phase disease.

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.

Parathyroid Hormone-Induced Modulation of the Bone Marrow Microenvironment Reduces Leukemic Stem Cells in Murine Chronic Myelogenous-Leukemia-Like Disease Via a TGFbeta-Dependent Pathway

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1670-1670
Author(s):  
Daniela S. Krause ◽  
Keertik Fulzele ◽  
Andre Catic ◽  
Michael Hurley ◽  
Sanon Lezeau ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Parathyroid Hormone ◽  
Cell Line ◽  
Chronic Myelogenous Leukemia ◽  
Bone Marrow Microenvironment ◽  
Myelogenous Leukemia ◽  
Osteoblastic Cells ◽  
Leukemic Stem Cells ◽  
Antibody Treatment

Abstract Abstract 1670 It is known that osteoblastic cells regulate the normal hematopoietic stem cell (HSC) niche and control its size. Parathyroid hormone (PTH) is an important regulator of osteoblasts and osteoclasts maintaining calcium homeostasis and, additionally, increasing HSC number in transplant recipients and protecting HSCs from repeated exposure to cytotoxic chemotherapy. We, therefore, hypothesized, that PTH-treatment may allow normal HSCs to outcompete leukemic stem cells (LSCs) in a murine model of chronic myelogenous leukemia. Mice with osteoblastic cell-specific constitutive activation of the receptor for PTH and PTH-related protein (Col1-caPPR mice) are characterized by activation of osteoblastic cells and increases in osteoclast and osteoblast number, trabecular bone, bone turnover and cortical porosity. Col1-caPPR mice have significantly prolonged survival and reduced leukemic mortality compared to wildtype (wt) littermates in a murine retroviral transduction/transplantation model of BCR-ABL1-induced CML-like disease (p=0.002) and B-cell acute lymphoblastic leukemia (B-ALL) (p=0.0004). However, a leukemogenic fusion transcription factor, MLL-AF9, known to cause acute myeloid leukemia in this model, led to more rapid death in the Col1-caPPR recipients compared with their wt counterparts (p<0.0001), indicating that the increased survival of Col1-caPPR recipients is specific for BCR-ABL1-induced leukemia. Continuous infusion of human PTH(1–34) into wt mice with BCR-ABL1-induced CML led to a statistically significant decrease in spleen weights and decreased bone marrow infiltration by BCR-ABL+ cells. Limiting dilution secondary transplantation of BM cells from saline- or PTH-treated primary animals with fully established CML into wt recipients revealed a 15-fold reduction of LSCs in a PTH-treated environment. Secondary mice who received BM from saline-treated donors had an overall survival that was 1/4 that of recipients of marrow from a PTH-treated BM microenvironment. Transforming growth factor beta-1 (TGFβ-1), whose largest and most concentrated tissue source is bone, was increased in the bones of Col1-caPPR mice. TGFβ-1 significantly decreased the in-vitro growth of the BCR-ABL+ cell line K562, but not the MLL-AF9+ cell line THP-1 suggesting that TGFβ-1, increased in the bone marrow microenvironment of Col1-caPPR mice, may be actively suppressing the growth of the BCR-ABL+ diseases, but not of MLL-AF9+ AML. Conversely, blockade of TGFβ-1, -2, and -3 by anti- TGFβ antibody treatment increased the incidence of CML in Col1-caPPR mice from 50% to 75%. Knockdown of TGF Receptor I in transplanted BCR-ABL+ BM in the CML model increased the percentage of BCR-ABL+ myeloid cells in peripheral blood in wt and, more strikingly, in Col1-caPPR recipient mice and increased the overall incidence of CML in Col1-caPPR mice. These results argue that reduction in TGFβ-1 signaling may rescue the CML phenotype in Col1-caPPR mice. In conclusion, these studies suggest that modulation of the bone marrow microenvironment by PTH reduces the frequency of LSCs in CML, possibly by suppression of LSCs via TGFβ-1. Consequently, a clinical trial on the combined use of imatinib and PTH in patients with CML has been initiated at our institution. Disclosures: No relevant conflicts of interest to declare.

MLL-AF9 and FLT3 Cooperation during Myeloid Leukemogenesis: Development of a Model for Rapid Testing of New Therapeutics.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1605-1605
Author(s):  
Matthew C. Stubbs ◽  
Andrei Krivtsov ◽  
Yong-mi Kim ◽  
Renee D. Wright ◽  
Zhaohui Feng ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Bone Marrow Transplant ◽  
Acute Myelogenous Leukemia ◽  
High Frequency ◽  
Marrow Transplant ◽  
Myelogenous Leukemia ◽  
Leukemic Stem Cells ◽  
Acute Myelogenous

Abstract Human leukemias harboring chromosomal translocations involving the Mixed lineage leukemia (MLL, HRX, ALL-1) gene possess high-level expression, and frequent activating mutations of the receptor tyrosine kinase FLT3. We used a murine bone marrow transplant model to assess cooperation between MLL translocation and FLT3 activation, and demonstrate that MLL-AF9 expression induces leukemia in approximately 70 days whereas the combination of MLL-AF9 and FLT3-ITD does so in less than 30 days. In both cases, the resulting disease is a highly aggressive, clonal, acute myelogenous leukemia. Detailed immunophenotypic analysis demonstrates expansion of an IL-7Rα− Lin− Sca-1− c-Kit+ CD34int. granulocyte macrophage progenitor (GMP)-like population in both the MLL-AF9 and MLL-AF9/FLT3-ITD induced leukemias. Other progenitor populations such as IL-7Rα− Lin− Sca-1− c-Kit+ CD34+ FcγRII/IIIlo common myeloid progenitors (CMPs) and IL-7Rα− Lin− Sca-1− c-Kit+ CD34− FcγRII/III− megakaryocyte erythroid progenitors (MEPs) are absent in the bone marrow from leukemic mice. Secondary transplantation of splenic cells from diseased mice established that leukemic stem cells are present at a very high frequency of approximately 1:100 in both diseases. Cooperation between MLL-AF9 and FLT3-ITD was further verified by real-time assessment of leukemogenesis using non-invasive bioluminescence imaging. We developed a transgenic mouse model that expresses luciferase under control of the ubiquitin promoter to provide luminescent bone marrow cells that can be used as donor cells in bone marrow transplant assays. In these experiments, the development of leukemia was followed in secondary recipients via bioluminescent imaging. We used this model to confirm cooperation of MLL-AF9 and FLT3-ITD, and to demonstrate that MLL-AF9/FLT3-ITD induced leukemias are sensitive to FLT3 inhibition in a 2–3 week in vivo assay. These data (1) show that MLL-AF9 induces acute myelogenous leukemia with a high frequency of leukemic stem cells and expansion of a GMP-like progenitor population, (2) demonstrate that activated FLT3 cooperates with MLL-AF9 to accelerate leukemogenesis, and (3) provide a new genetically-defined model system that should prove useful for rapid assessment of potential therapeutics in vivo.

Establishment of a murine model for therapy-treated chronic myelogenous leukemia using the tyrosine kinase inhibitor STI571

Blood ◽  
2001 ◽  
Vol 98 (9) ◽  
pp. 2808-2816 ◽  
Author(s):  
Nicholas C. Wolff ◽  
Robert L. Ilaria
Keyword(s):  
Bone Marrow ◽  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitor ◽  
Chronic Myelogenous Leukemia ◽  
Murine Model ◽  
High Efficiency ◽  
Kinase Inhibitor ◽  
Chronic Phase ◽  
Myelogenous Leukemia ◽  
Leukemic Cells

Abstract The murine bone marrow retroviral transduction and transplantation model of chronic myelogenous leukemia (CML) imperfectly mimics human CML because the murine CML-like disease causes death of all animals from an overwhelming granulocytosis within 3 to 4 weeks. In this report, mice reconstituted with P210BCR/ABL-transduced bone marrow cells received posttransplantation therapy with either the tyrosine kinase inhibitor STI571 or placebo. Compared with the rapidly fatal leukemia of placebo-treated animals, 80% of the STI571-treated mice were alive on day 74, with marked improvement in peripheral white blood counts and splenomegaly. There was decreased tyrosine phosphorylation of STAT5, Shc, and Crk-L in leukemic cells from STI571-treated animals, consistent with STI571-mediated inhibition of the Bcr/Abl tyrosine kinase in vivo. In some STI571-treated animals Bcr/Abl messenger RNA and protein expression were markedly increased. In contrast to the polyclonal leukemia of placebo-treated mice, STI571-treated murine CML was generally oligoclonal, suggesting that STI571 eliminated or severely suppressed certain leukemic clones. None of the STI571-treated mice were cured of the CML-like myeloproliferative disorder, however, and STI571-treated murine CML was transplanted to secondary recipients with high efficiency. These results demonstrate the utility of this murine model of CML in the evaluation of novel therapeutic agents against Bcr/Abl-induced leukemias. This improved murine chronic-phase CML model may be a useful tool for the study of STI571 resistance, CML progression, and the anti-CML immune response.

scholarly journals Vcam1 Is a "Don't-Eat-Me" Signal on Healthy Hematopoietic and Leukemic Stem Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 565-565 ◽  
Author(s):  
Sandra Pinho ◽  
Wei Qiaozhi ◽  
Maria Maryanovich ◽  
Halley Pierce ◽  
Fumio Nakahara ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Steady State ◽  
Research Funding ◽  
Marked Reduction ◽  
Myelogenous Leukemia ◽  
Leukemic Stem Cells ◽  
Wild Type ◽  
Blocking Antibody

Abstract Hematopoietic stem cells (HSCs) possess the ability to maintain the entire population of blood cells throughout life and to replenish the hematopoietic system after transplantation into marrow-ablated recipients. In mammalian adults, HSCs predominantly reside in the bone marrow to generate and maintain all blood cell types. HSCs, however, retain their ability to migrate to ectopic niches via the bloodstream, and traffic back to the bone marrow microenvironment via in part the interactions of alpha4 integrins with Vascular Cell Adhesion Molecule-1 (Vcam1, also known as CD106), constitutively expressed by endothelial and stromal cells. In the course of studies to investigate the role of Vcam1 in the macrophage erythroblastic niche, we have found using Vcam1-floxed mice crossed with a Csf1r-iCre (hereafter Vcam1Δ/Δ) that Vcam1 and Cre, using this transgenic line, were expressed on Lineage− (Lin−) c-Kit+ Sca1+ CD48− CD150+ HSCs. Although Vcam1Δ/Δ mice show no significant steady-state hematopoietic defect, transplantation of Vcam1Δ/Δ bone marrow, both in competitive and non-competitive settings, failed to engraft irradiated wild-type recipients. Indeed, ~80% of animals transplanted with Vcam1Δ/Δcells died, and the few that survived failed to successfully engraft secondary recipient mice. Vcam1Δ/Δ HSC/progenitors homed to bone marrow as well as wild-type counterparts and showed no detectable change in apoptosis in the bone marrow. However, syngeneic Vcam1Δ/Δ HSC/progenitors were selectively cleared in vivo by bone marrow macrophages over 4 days. The in vivo Vcam1Δ/Δ HSC/progenitor clearance did not require irradiation-induced damage since in steady-state parabiosis (Vcam1Δ/Δ and wild-type co-joined pairs), Vcam1Δ/Δ HSCs did not engraft the partner after G-CSF-induced mobilization whereas wild-type HSCs engrafted the parabiont partner. In addition, in vitro incubation of bone marrow-derived macrophages with Lin− cells revealed rapid phagocytosis by macrophages of Vcam1Δ/Δ, but not wild-type, cells and phagocytosis was also induced when the integrin alpha4 beta1 (also known as VLA-4) was blocked in wild-type Lin− cells. Thus, these results suggest that Vcam1 expressed on HSCs serves as a novel don't-eat-me signal, controlling the migration in the bone marrow by a macrophage-enabled checkpoint. Since we found that Vcam1 is expressed at higher levels on acute myelogenous leukemia (AML) cells than on healthy HSCs, and high VCAM1 expression correlates with poor prognosis in human AML patients (Ley et. al. NEJM 2013), we examined whether Vcam1 expression by AML cell could affect leukemogenesis in vivo. To test this idea, we generated AML-Vcam1Δ/Δand AML-Control cells by transduction of bone marrow Lin− c-Kit+ Sca1+ cells with the pMSCV-MLL-AF9-GFP oncogene. Strikingly, FACS analysis of primary AML recipient bone marrow revealed a marked reduction (>99%) in the number and percentage of phenotypic Lin− IL7Rα− Sca1− MLL-AF9 GFP+ c-Kithigh CD34low FcγRII/IIIhigh leukemic stem cells in AML-Vcam1Δ/Δmice compared to control. These results were confirmed by imaging analyses, which showed a marked reduction in leukemic infiltration in AML-Vcam1Δ/Δmice compared to control. Kaplan-Meier survival analysis of secondary recipient mice receiving 20,000 GFP+ leukemic cells revealed a significantly greater survival of mice harboring AML-Vcam1Δ/Δ cells relative to AML-Control (P<0.0001). To assess in a pre-clinical experimental setting whether Vcam1 inhibition can alter the course of AML, we established AML in immunocompetent C57BL/6 recipients and started therapy of moribund leukemic mice (>50% circulating AML-GFP+ cells) with a daily injection of saline, IgG control, anti-Vcam1 blocking antibody (100 μg/day), cytarabine (100 mg/kg), or a combination of anti-Vcam1/cytarabine for 5 days. Remarkably, this short treatment with anti-Vcam1 blocking antibody was able to preferentially and significantly reduce the frequency and absolute number of phenotypic bone marrow leukemic stem cells in vivo. In addition, our results suggest that using a blocking anti-Vcam1 antibody synergizes with cytarabine treatment to decrease leukemic burden in vivo. These studies lay the groundwork for the development of a new therapeutic strategy for eliminating leukemia stem cells by modulating the innate immune response. Disclosures Frenette: GSK: Research Funding; Pfizer: Consultancy; PHD Biosciences: Research Funding.

Imatinib-Resistant Activation of β-Catenin by BCR-ABL in a Murine Model of Chronic Myelogenous Leukemia Stem Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2126-2126
Author(s):  
Yosuke Minami ◽  
Scott Stuart ◽  
Tomokatsu Ikawa ◽  
Asoka Banno ◽  
Irina Hunton ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Chronic Myelogenous Leukemia ◽  
Murine Model ◽  
Expression Profiles ◽  
Blast Crisis ◽  
Myelogenous Leukemia ◽  
Hematopoietic Progenitor Cell ◽  
Cell Counts ◽  
Imatinib Resistant

Abstract [INTRODUCTION] Chronic myelogenous leukemia (CML) is effectively treated with imatinib, a small molecule inhibitor of the BCR-ABL tyrosine kinase that is expressed in the hematopoietic compartment including stem and progenitor cells in CML patients. While imatinib induces disease remission, it does not eradicate BCR-ABL-positive stem cells. Recently, granulocyte-macrophage progenitors (GMP) with nuclear β-catenin and an aberrant potential for self-renewal were detected in CML blast crisis (Jamieson et al, NEJM, 2004). We have established a murine model that generates BCR-ABL-positive GMP and a transplantable CML. In this model, BCR-ABL activates β-catenin through an imatinib-resistant pathway. [METHODS] Retroviral mediated gene transfer was employed to express BCR-ABL (p210) and/or GFP in a pluripotent murine hematopoietic progenitor cell line (mHPC) (Ikawa et al, Immunity, 2004). Infected cells were isolated using GFP-sorting and characterized ex vivo for leukemogenic phenotypes and gene expression profiles. The GFP-sorted cells were transplanted into lethally irradiated congenic mice. The distribution of GFP-positive cells in the hematopoietic compartments was determined by FACS. [RESULTS] BCR-ABL expression induced leukemogenic traits in mHPC, i.e., stroma- and cytokine-independent survival and proliferation. BCR-ABL also induced the expression of myeloid lineage markers, a process that was inhibited by imatinib. By contrast, imatinib did not abolish the stabilization of β-catenin in BCR-ABL transduced mHPC. Transplantation of BCR-ABL-transduced mHPC, but not GFP-transduced mHPC, induced in mice a CML-like disease presented with increased white blood cell counts and splenomegaly. The bone marrow cells from these mice reproducibly transferred CML phenotypes to secondary recipient mice. The diseased bone marrow contains an expanded population of GFP-positive GMP. The effect of imatinib on the leukemogenic potential of these BCR-ABL-positive GMP in vivo is under investigation. [CONCLUSIONS] Expression of BCR-ABL in the context of mHPC induces leukemogenic traits, including the stabilization of β-catenin, without requirement for additional genetic events. The BCR-ABL-transduced mHPC generates CML-like disease in mice accompanied by the expansion of a BCR-ABL-positive GMP population, similar to that observed in blast crisis CML patients. The stabilization of β-catenin by BCR-ABL is not inhibited by imatinib; consistent with the notion that imatinib cannot eradicate CML stem cells. This murine model can be used to identify therapeutics that targets the CML stem cells.

Do Microenvironmental Factors Play Important Role in Supporting the Maintenance of Leukemic Stem Cells of Chronic Myelogenous Leukemia?

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4219-4219
Author(s):  
Yeon Hee Cho ◽  
Dong Hwan Kim ◽  
Jun Ho Jang ◽  
Seok Jin Kim ◽  
Kihyun Kim ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stromal Cells ◽  
Bone Marrow Cells ◽  
Notch Pathway ◽  
Chronic Phase ◽  
Myelogenous Leukemia ◽  
Leukemic Stem Cells ◽  
Hematopoietic Stem ◽  
Marrow Cells

Abstract Background : In the era of tyrosine kinase inhibitor(TKI) for chronic myelogenou leukemia(CML), it is becoming more important to develop the way to get rid of the leukemic stem cells(LSC), as there is frequent relapse when TKI therapy is stopped even when molecular remission is achieved. Some researchers reported macrophages and Wnt signaling in bone marrow microenvironment might play significant role in the development and the progression of CML. Methods : We isolated CD34+CD38− LSC from bone marrow cells from 8 patients with CML chronic phase and also CD34+CD38− normal hematopoietic stem cell from 7 normal controls using magnetic cell separation system. CD34+CD38− cells were cultured on stromal cells for 5 weeks and transfered to methylcellulose media for long-term culture initiating cell(LTC-IC) assay. We checked whether there is a difference in LTC-IC capacity of LSC when cultured on leukemic or normal stromal feeder cells. CD45− stromal supporting cells were established by 3-week culture after 12Gy irradiation of CD34− bone marrow cells. We studied the expression of VCAM, ICAM, E-selectin in stromal cells and the presence of BCR-ABL transcript. Wnt and β-catenin, frizzled, Notch1 were analyzed in the LSCs. CD14+ cells were isolated and added in LTC-IC assay whether the presence of leukemic or normal CD14+ cells would make a change in the maintenance of LSCs. Results : The number of CD34+CD38− cells in CML was in the range of 1.3 × 103 to 9.2 × 104, that we could do only two sets of 2 × 2 LTC-IC assay that statistical analysis was impossible. LTC-IC frequency of LSCs was about 5-times higher compared to that of normal stem cells irrespective of the kind of stromal cells used. CD14+ cell co-culture did not changed significantly the LTC-IC frequency both in normal and LSCs. Wnt 2B, 5A and 10B were highly expressed in LSCs irrespective of the stromal cells. β-catenin was also strongly expressed in LSCs compared to normal stem cells. Notch1 was expressed only in CD34+CD38− LSCs but not in normal stem cells. The kind of stromal cells or the addition of CD14+ cells in LTC-IC assay did not affected the Notch1 expression in LSCs. Conclusion : CML CD34+CD38− LSCs seem to have more LTC-ICs and wnt and notch pathway may play important role in the maintenance of LSCs that could be therapeutic targets.

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