scholarly journals Setbp1 promotes the self-renewal of murine myeloid progenitors via activation of Hoxa9 and Hoxa10

Blood ◽  
2012 ◽  
Vol 119 (25) ◽  
pp. 6099-6108 ◽  
Author(s):  
Kevin Oakley ◽  
Yufen Han ◽  
Bandana A. Vishwakarma ◽  
Su Chu ◽  
Ravi Bhatia ◽  
...  
Keyword(s):  
Myeloid Leukemia ◽  
Blast Crisis ◽  
Myelogenous Leukemia ◽  
Mrna Levels ◽  
Self Renewal ◽  
Advanced Phase ◽  
Primary Mouse ◽  
Immortalized Cells ◽  
Leukemia Development ◽  
Myeloid Progenitors

Abstract Acquisition of self-renewal capability by myeloid progenitors to become leukemic stem cells during myeloid leukemia development is poorly understood. Here, we show that Setbp1 overexpression efficiently confers self-renewal capability to myeloid progenitors in vitro, causing their immortalization in the presence of stem cell factor and IL-3. Self-renewal after immortalization requires continuous Setbp1 expression. We also found that Hoxa9 and Hoxa10 mRNA are present at dramatically higher levels in Setbp1-immortalized cells compared with other immortalized cells, and are induced shortly after Setbp1 expression in primary myeloid progenitors. Suppression of either gene in Setbp1-immortalized cells drastically reduces their colony-forming capability. Interestingly, Setbp1 protein associates with Hoxa9 and Hoxa10 promoters in chromatin immunoprecipitation assays in these cells, suggesting that both are direct transcriptional targets of Setbp1. Setbp1 also promotes self-renewal of myeloid progenitors in vivo as its coexpression with BCR/ABL transforms primary mouse myeloid progenitors, generating aggressive leukemias in recipient mice resembling chronic myelogenous leukemia (CML) myeloid blast crisis. Increased SETBP1 mRNA levels were also detected in a subset of CML advanced phase/blast crisis patients with high levels of HOXA9 and HOXA10 expression. Thus, Setbp1 activation represents a novel mechanism conferring self-renewal capability to myeloid progenitors in myeloid leukemia development.

scholarly journals microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors, biased myeloid development, and acute myeloid leukemia

2010 ◽  
Vol 207 (3) ◽  
pp. 475-489 ◽  
Author(s):  
Yoon-Chi Han ◽  
Christopher Y. Park ◽  
Govind Bhagat ◽  
Jinping Zhang ◽  
Yulei Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Ectopic Expression ◽  
Hematopoietic Progenitors ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Myeloid Development ◽  
Acute Myeloid ◽  
Myeloid Progenitors

The function of microRNAs (miRNAs) in hematopoietic stem cells (HSCs), committed progenitors, and leukemia stem cells (LSCs) is poorly understood. We show that miR-29a is highly expressed in HSC and down-regulated in hematopoietic progenitors. Ectopic expression of miR-29a in mouse HSC/progenitors results in acquisition of self-renewal capacity by myeloid progenitors, biased myeloid differentiation, and the development of a myeloproliferative disorder that progresses to acute myeloid leukemia (AML). miR-29a promotes progenitor proliferation by expediting G1 to S/G2 cell cycle transitions. miR-29a is overexpressed in human AML and, like human LSC, miR-29a-expressing myeloid progenitors serially transplant AML. Our data indicate that miR-29a regulates early hematopoiesis and suggest that miR-29a initiates AML by converting myeloid progenitors into self-renewing LSC.

scholarly journals Expression of axl, a transforming receptor tyrosine kinase, in normal and malignant hematopoiesis

Blood ◽  
1994 ◽  
Vol 84 (6) ◽  
pp. 1931-1941 ◽  
Author(s):  
A Neubauer ◽  
A Fiebeler ◽  
DK Graham ◽  
JP O'Bryan ◽  
CA Schmidt ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Receptor Tyrosine Kinase ◽  
Intracellular Signaling ◽  
Myeloid Leukemia ◽  
Blast Crisis ◽  
Chronic Phase ◽  
Lymphocytic Leukemia ◽  
Myelogenous Leukemia ◽  
Hematopoietic Tissue ◽  
Blood Leukocytes

Abstract We previously reported the cloning, and characterization of a receptor tyrosine kinase, axl, from two patients with chronic myelogenous leukemia. Herein, we describe the expression pattern of axl in normal and malignant hematopoietic tissue axl message is detected in normal human bone marrow but not significantly in normal blood leukocytes. Cell separation experiments showed that axl is expressed in hematopoietic CD34+ progenitor and marrow stromal cells, at low levels in peripheral monocytes, but not in lymphocytes or granulocytes. Consistent with the normal pattern of axl expression, axl RNA was found predominantly in diseases of the myeloid lineage: 39 of 66 (59%) patients with myeloproliferative disorders (acute myeloid leukemia, chronic myeloid leukemia (CML) in chronic phase, CML in myeloid blast crisis, and myelodysplasia) showed significant axl transcription, as compared with 1 of 45 (2%) lymphoid leukemias (chronic lymphocytic leukemia, acute lymphocytic leukemia, and CML in lymphoid blast crisis). Treatment of K562 cells with the phorbol ester, 12-O- tetradecanoylphorbol-13-acetate (TPA), administration of interferon alpha (IFN alpha) to normal monocytes, and treatment of U937 cells with TPA and IFN tau significantly induced axl expression, supporting a role for this kinase in the intracellular signaling of myeloid cells through a variety of biochemical pathways. These results suggest that the axl kinase may be operative in normal and malignant myeloid biology.

Requirement of the E2F3 Transcription Factor for BCR/ABL Leukemogenesis.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 33-33
Author(s):  
Anna M. Eiring ◽  
Paolo Neviani ◽  
Ramasamy Santhanam ◽  
Joshua J. Oaks ◽  
Ji Suk Chang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Transcription Factor ◽  
Blast Crisis ◽  
Scid Mice ◽  
Myelogenous Leukemia ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Hnrnp A1 ◽  
Protein Levels ◽  
Abl Kinase

Abstract Several RNA binding proteins (RBPs) have been implicated in the progression of chronic myelogenous leukemia (CML) from the indolent chronic phase to the aggressively fatal blast crisis. In the latter phase, expression and function of specific RBPs are altered at transcriptional or post-translational levels by the increased constitutive kinase activity of the BCR/ABL oncoprotein, resulting in enhanced resistance to apoptotic stimuli, growth advantage and differentiation arrest of CD34+ CML blast crisis (CML-BC) progenitors. In the current study, we identified by RIP (RNA immunoprecipitation)-mediated microarray analysis that mRNA encoding the E2F3 transcription factor associates to the BCR/ABL-regulated RBP hnRNP A1. Moreover, RNA electrophoretic mobility shift and UV-crosslinking assays revealed that hnRNP A1 interacts with E2F3 mRNA through a binding site located in the 3’UTR of both human and mouse E2F3 mRNA. Accordingly, E2F3 protein levels were upregulated in BCR/ABL-transformed myeloid precursor cell lines compared to parental cells in a BCR/ABL-kinase- and hnRNP A1 shuttling-dependent manner. In fact, treatment of BCR/ABL-expressing myeloid precursors with the kinase inhibitor Imatinib (2mM, 24 hr) or introduction of a dominant-negative shuttling-deficient hnRNP A1 protein (NLS-A1) markedly reduced E2F3 protein and mRNA levels. Similarly, upregulation of BCR/ABL expression/activity in the doxycycline inducible TonB2.10 cell line resulted in increased E2F3 protein expression. BCR/ABL kinase-dependent induction of E2F3 protein levels was also detected in CML-BCCD34+ compared to CML-CPCD34+ progenitors from paired patient samples and to normal CD34+ bone marrow samples. Importantly, the in vitro clonogenic potential of primary mouse BCR/ABL+ lineage negative (Lin−) progenitors was markedly impaired in BCR/ABL+ E2F3−/− compared to BCR/ABL-transduced E2F3+/+ myeloid progenitors and upon shRNA-mediated downregulation of E2F3 expression (90% inhibition, P<0.001). Furthermore, subcutaneous injection of shE2F3-expressing BCR/ABL+ cells into SCID mice markedly impaired in vivo tumorigenesis (>80% reduction in tumor burden, P<0.01). Accordingly, BCR/ABL leukemogenesis was strongly inhibited in SCID mice intravenously injected with E2F3 shRNA-expressing 32D-BCR/ABL cells and in mice transplanted with BCR/ABL-transduced Lin− bone marrow cells from E2F3−/− mice. Specifically, we demonstrate that reduced or absent levels of E2F3 resulted in dramatically decreased numbers of circulating BCR/ABL+ cells as determined by nested RT-PCR at 4 weeks post-injection (P=0.0001), normal splenic architecture and bone marrow cellularity and the absence of infiltrating myeloid blasts into non-hematopoietic compartments (i.e. liver). By contrast, SCID mice transplanted with vector-transduced 32D-BCR/ABL cells or BCR/ABL+ E2F3+/+ Lin− BM progenitors showed signs of an overt acute leukemia-like process with blast infiltration of hematopoietic and non-hematopoietic organs. Altogether, these data outline the importance of E2F3 expression for BCR/ABL leukemogenesis and characterize a new potential therapeutic target for the treatment of patients with advanced phase CML.

Expression of Biomarkers That Predict Progression in Chronic Myeloid Leukemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4241-4241
Author(s):  
Mariana Selena Gonzalez ◽  
Patricia Martha Gargallo ◽  
Beatriz Moiraghi ◽  
Irene Larripa
Keyword(s):  
Myeloid Leukemia ◽  
Philadelphia Chromosome ◽  
Chronic Phase ◽  
Apoptotic Index ◽  
Myelogenous Leukemia ◽  
Control Group ◽  
Qrt Pcr ◽  
Advanced Phase ◽  
Bmi1 Expression ◽  
Healthy Donors

Abstract Chronic Myelogenous Leukemia (CML) is associated with a chromosomal translocation, t(9;22)(q34;q11.2), that produces the Philadelphia chromosome (Ph). The molecular consequence of this translocation is the generation of the BCR/ABL oncogene, which encodes a chimeric protein of 210 kDa (p210Bcr/Abl) with elevated tyrosine kinase activity. BCR/ABL exerts its oncogenic effect in CML cells essentially by stimulating cell proliferation, inhibiting apoptosis and altering cell adhesion to bone marrow stroma. Despite of this consistent molecular abnormality, a marked heterogeneity in prognosis and response to treatment has been reported. Different molecular markers have been studied, such as: BMI1, ELA2, PR3, E2F1 and apoptotic genes (BCL-2, BCL-XL, BAX, BAD, BAK) in order to predict progression and overall survival in myeloid leukemia. The polycomb group gene BMI1 plays an essential role in regulating the proliferative activity in leukemic stem cell. The expression of this gene is related to a higher degree of malignancy. On the other hand, BCL-2 family genes involved in the mitochondrial-apoptotic pathway are related with clinical response and treatment failure. Enhanced expression of the apoptotic inhibitor BCL-2 or its homolog BCL-XL lead to tumor cells having a decreased susceptibility to cell death. Other BCL-2 family members such as BAX are able to induced apoptosis, so that the ratio of expression of proapoptotic and anti-apoptotic members might determine the apoptotic potencial of cancer cells. In this study we evaluated the expression of BMI1 and BAX/BCL-XL ratio (apoptotic index) to determine whether these genes could behave as biomarkers to predict disease aggressiveness and progression from chronic phase to more advanced phases. Total RNA was extracted from leucocytes of peripheral blood. using Trizol method. cDNA was synthesized with random hexamer primers and reverse transcriptase. The expression was assessed by quantitative real time (QRT-PCR) using the LightCycler 2.0 instrument (Roche), based on the Syber-Green method. All QRT-PCR reactions were performed in 20ul volume. The β-actin expression was used as the endogenous cDNA quality control. Groups of patients were compared using the Mann-Whitney test. The study was performed in 31 patients: 16 in chronic phase (CP), 15 in advanced phases (accelerated and blast crisis) and 10 healthy donors (control group). BMI1 expression levels were significantly lower in CP (mean ± SEM: 0.54±0.15) than in more advanced stages of CML (mean ± SEM: 4.54±1.4) (P&lt;0.0005). In peripherical blood of healthy donors, the expression of this gene was similar to CML-CP patients (0.4±0.13). The relationship of BAX/BCL-XL values were higher in CP (mean ± SEM: 13.81± 1.85) and lower in advanced phase (mean ± SEM: 0.88±0.17) than in the control group (mean ± SEM: 4.82 ± 0.49) (P&lt;0.0044 and P&lt; 0.0002, respectively). The CP patients showed a low BMI1 expression level and a high apoptotic index, this inverse correlation is associated with a benign stage of the disease and good treatment response. On the contrary, cases in more advance stage displayed overexpression of BMI1 gene and low BAX/BCL-XL ratio suggesting an aggressive stage and poor response. The identification of a genetic hostile profile in CP phase could predict an impending disease progression. Our results show that the simultaneous use of two biomarkers: BMI1 and the ratio BAX/BCL-XL represent sensitive indicators of clinical outcome in CML-CP. Therefore, the prospective screening of these biomarkers would help to refine CML disease staging and would be useful prognostic indicators for optimizing therapeutic strategies.

The Biology of CML Blast Crisis

Hematology ◽  
2007 ◽  
Vol 2007 (1) ◽  
pp. 384-391 ◽  
Author(s):  
Jerald P. Radich
Keyword(s):  
Tyrosine Kinase ◽  
Myeloid Leukemia ◽  
Blast Crisis ◽  
Chronic Phase ◽  
Allogeneic Transplantation ◽  
Cancer Evolution ◽  
Advanced Phase ◽  
History Of ◽  
Clinical Dilemma ◽  
Predictive Assays

Abstract The natural history of chronic myeloid leukemia (CML) progresses from a relatively benign chronic phase into a fatal blast crisis, which resembles acute leukemia, but is incurable by chemotherapy. Fortunately, the progression can usually be blocked by tyrosine kinase therapy or allogeneic transplantation. The seemingly stereotypical march of progression involves changes in genetic instability and DNA repair, proliferation, differentiation, and apoptosis, and thus may serve as a unique model of cancer evolution and progression. Given that all treatments work much better in chronic-phase than advanced-phase disease, the clinical dilemma is predicting and detecting patients bound to evolve into advanced disease. This is especially important in the age of tyrosine kinase inhibition (TKI) therapy. The purpose of this review is to address the biology of blast crisis in the age of tyrosine kinase therapy, with an emphasis on what genes or pathways may be future targets of predictive assays or treatments of progression.

scholarly journals Orlistatand and Rosuvastatin Suppressed Proliferation of K562 Human Myelogenous Leukemia Cell Line through AMPK/Akt/c-Myc Signaling

2020 ◽  
Author(s):  
Behnam Mojjarad ◽  
Yaghub Pazhang
Keyword(s):  
Cell Cycle ◽  
Chronic Myeloid Leukemia ◽  
Cell Line ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
K562 Cells ◽  
Myelogenous Leukemia ◽  
Leukemia Cell Line ◽  
Mrna Levels ◽  
Hsp 70

Abstract Background: Chronic myeloid leukemia is a myeloproliferative cancer with worldwide incidence, has become as a clinical concern due to chemoresistance in the patients received chemotherapy. Here, we investigated the effect of Orlistat and Rosuvastatin on K562 human myelogenous leukemia cell line in vitro and attempted to illuminate their possible underlying mechanisms. Methods: Cells were exposed to Orlistat and Rosuvastatin, the inhibitors of lipogenesis, then survival and apoptosis rate of K562 cells were examined by MTT assay and flow cytometric analysis respectively. The real time-PCR analysis was used to quantify mRNA levels of Bax, Bcl-2, and Hsp-70 genes. Cell cycle analysis was performed using flow cytometry, whereas the subcellular distribution of c-Myc was measured via immunofluorescence imaging technique. Additionally, the protein level of AMPK, p-AMPK Akt-1, and p-Akt-1 were studied by western blotting. Results: The results showed Orlistat and Rosuvastatin had synergistic anticancer effects on cells and in comparison with the control group, viability and apoptosis rate decreased and increased in treated cells respectively in a dose/time-dependent manner (P<0.05). The mRNA levels of Bax increased while expression of Hsp-70 decreased (P< 0.05). K562 cells treated with Orlistat and Rosuvastatin showed a cell cycle arrest in sub-G1 phase and a decreased level of c-Myc positive cells. Upon outlining the mechanism, it was revealed that AMPK/p-AMPK and p-Akt-1/Akt-1 ratio decreased in treated cells (P< 0.05). Conclusions: Data suggest Orlistat and Rosuvastatin could synergically suppress proliferation of K562 cells through AMPK/Akt/c-Myc axis, proposing a theoretical basis for upcoming application in the treatment of chronic myeloid leukemia

scholarly journals Characteristics of the TCR Vbeta Repertoire and Identical Clonally Expanded T Cells in Chronic Myeloid Leukemia Patients in Advanced Phase with ABL Mutations

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5136-5136
Author(s):  
Ling Xu ◽  
Yuhong Lu ◽  
Jing Lai ◽  
Wei Yu ◽  
Zhenyi Jin ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Chronic Myeloid Leukemia ◽  
Natural Science ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Blast Crisis ◽  
Chronic Phase ◽  
Cytotoxic Lymphocyte ◽  
Advanced Phase

Abstract Abstract Tumor specific or related antigen cytotoxic lymphocyte (CTL) have been identified in chronic myeloid leukemia patients, however, whether they are constituted by specific type of T cell receptor chains has not been illustrated so far. Previous studies have reported abnormal TCR repertoires and clonally expanded TCR Vβ T cells in chronic myeloid leukemia in chronic phase (CP-CML). In this study, we investigated the distribution and clonality of the TCR Vβ repertoire in 5 CML patients in blast crisis (BC-CML) and one in acceleration phase (AP-CML) with ABL kinase domain mutations (KDMs) including T315I, E255K, F317L+S417Y, Y-253F and L387M+T-315A. Examination of TCR Vβ expression and clonality was performed by reverse transcription-polymerase chain reaction (RT-PCR) combined with GeneScan analysis. Significantly skewed TCR Vβ repertoires were observed in those patients, and 4 to 8 oligoclonally expanded TCR Vβ subfamilies could be identified in each sample, which distributed in 15/24 different subfamilies (TCR Vβ4, Vβ5, Vβ6, β8, Vβ9, Vβ10, Vβ15, Vβ16, Vβ17, Vβ18, Vβ19, Vβ21, Vβ22, Vβ23, Vβ24). Intriguingly, a relatively highly expanded Vβ9 clone with the same length as CDR3 (139 bp) was found in all three CML patients in lymphoid blast crisis (LBC-CML) who had different KDMs, but the clone was not detected in the other two CML patient in myeloid blast crisis (MBC-CML) or the one CML patients in accelerated phase. In conclusion, restricted TCR Vβ repertoire expression and decreased clone complexity was a general phenomenon in the BC-CML patients with different KDMs, indicating the T-cell immunodeficiency status of these patients, and clonally expanded Vβ9 T cell clones may represent a specific immune response to leukemia-associated antigens in LBC-CML patients. Disclosures Li: The Foundation for High-level Talents in Higher Education of Guangdong, China ([2013]246-54),and the Guangzhou Science and Technology Project Foundation (201510010211): Research Funding; National Natural Science Foundation of China (81270604, U1301226, and 81400109), the Guangdong Natural Science Foundation (S2013040016151 and S2013020012863): Research Funding.

scholarly journals Spontaneous chronic subdural hematoma development in chronic myeloid leukemia cases at remission phase under maintenance therapy, management strategy - a series with literature review

2016 ◽  
Vol 30 (3) ◽  
pp. 451-460
Author(s):  
Amol Raheja ◽  
Guru Dutta Satyarthee
Keyword(s):  
Subdural Hematoma ◽  
Myeloid Leukemia ◽  
Surgical Intervention ◽  
Haematological Malignancy ◽  
Blast Crisis ◽  
Chronic Subdural Hematoma ◽  
Myelogenous Leukemia ◽  
Temporal Region ◽  
Current Article ◽  
Remission Phase

AbstractChronic subdural hematoma (CSDH) is common squeal of trauma and rarely associated with anticoagulant therapy, antiplatelet, chemotherapeutic drugs, arteriovenous malformation, aneurysms and post-craniotomy. However its occurrence is very unusual with systemic haematological malignancy and mostly reported with acute myeloid leukemia; however incidence of SDH occurrence in chronic myelogenous leukemia (CML) is very rare. CML is a haematological malignancy characterized by chromosomal alteration, pathologically represents increased proliferation of the granulocytic cell line without loss of capacity to differentiate. CML has three phases - remission phase, accelerated phase and blast crisis. About 85 % of patients present in remission phase of disease and carries a favorable prognosis. As intracranial, subdural hematoma usually occur in the accelerated phase or blast crisis phase or extremely uncommon during chronic remission phase, although only those affected, who are neglecting therapeutic medication or discontinued therapy or rarely as an adverse effect of medications. However, important role of neurosurgeon lies in early detection and correction of platelet count and associated hematological abnormality as quite sizeable proportion of cases may not need surgical intervention instead can be managed conservatively under regular supervision in association with oncologist colleague, but few cases may need urgent surgical intervention. So, selecting a subgroup of CML cases in the remission phase requiring surgical intervention, presenting with CSDH is not only challenging, as failure to make an informed and timely precise decision can lead to catastrophic worse outcome and even mortality. So, purpose of current article is to formulate the management therapeutic plan. Authors report three cases of CML in chronic remission phase, receiving treatment under guidance of Haemto-oncologist at our institute presented with spontaneous chronic SDH. The mean age was 36 years (range 29- 44 years), 66% were male, headache was presenting feature in all 100% (n=3), 66% cases were hemiplegic and 33% unconscious each, in 66% cases CSDH were located on right fronto-temporal region and 33% had small left sided thin CSDH. About were 66% cases (n=2) were managed surgically by burr hole placement and drainage drain placement while 33% case (n=1), who had thin CSDH was managed conservatively.Favorable outcome was observed in 100% cases (n=3) Outcome was favorable in all of our cases.

Bioluminescent Imaging of Human Leukemic Stem Cell Engraftment.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 696-696
Author(s):  
Catriona Jamieson ◽  
Mobin Karimi ◽  
Remi Creusot ◽  
Robert Negrin ◽  
Jason Gotlib ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cord Blood ◽  
Imaging System ◽  
Blast Crisis ◽  
Bioluminescent Imaging ◽  
Beta Catenin ◽  
Self Renewal ◽  
Advanced Phase

Abstract Previously, we found that candidate leukemic stem cells (LSC) involved in chronic myelogenous leukemic (CML) progression to myeloid blast crisis (BC) shared phenotypic characteristics with granulocyte-macrophage progenitors (GMP). However, CML GMP had activated a key self-renewal gene - beta-catenin. Aberrant in vitro self-renewal capacity could be specifically inhibited with axin - a potent beta-catenin antagonist (Jamieson et al, New Engl J Med2004;351:657–67). In order to determine whether these candidate LSC had enhanced in vivo self-renewal potential, we FACS-sorted hematopoietic stem cells (HSC), common myeloid progenitors (CMP), GMP, megakaryocyte-erythroid progenitors (MEP), CD34+CD38+ cells and blasts (lineage-positive cells) from advanced phase CML versus normal bone marrow or cord blood and transplanted them intrahepatically into newborn T, B and NK cell deficient (RAG2−−-γ−/−) mice (Traggiai et al. Science2004;304:104–7). Engraftment of human (ge;1%) CD45, CD19, CD3, and CD14-positive cells in the hematopoietic organs including bone marrow, liver, spleen and thymus of recipient animals was analyzed by FACS and compared with non-transplanted controls. In seven transplantation experiments performed with normal cord blood or bone marrow (n=24 mice), populations enriched for HSC, showed evidence of long-term engraftment, while committed progenitors including GMP did not. Conversely, in six experiments with myeloid BC CML (n=28 mice), GMP gave rise to long-term engraftment (7 of 11 mice) more frequently than HSC (2 of 6 mice) and blasts seldom engrafted (2 of 7 mice). These results suggested that LSC were enriched within the GMP fraction of myeloid BC CML. Subsequently, bioluminescent imaging (IVIS 100) was employed in order to track the kinetics of normal versus LSC engraftment more precisely. In 7 experiments involving normal marrow or cord blood (n=28 mice) and 3 experiments with advanced phase CML (n=18 mice), HSC, progenitor and blast (Lin+) populations were transduced with a lentiviral luciferase GFP vector and transplanted intrahepatically into newborn RAG2−/−γ−/− mice. Engraftment was monitored by weekly bioluminescent imaging as well as by tail vein bleeds to detect GFP expression. When mice were sacrificed, human engraftment in the liver, spleen, bone marrow and thymus was assessed by FACS analysis and sorted human CD45+ cells were transplanted into secondary recipients (n=4 experiments). In primary bioluminescent transplantation studies, CML HSC, CMP and GMP engrafted. Normal HSC demonstrated serial engraftment potential while more committed normal progenitors such as CMP, GMP and MEP did not. In contrast, CML blast crisis GMP demonstrated serial (2o and 3o) engraftment potential suggesting that they had gained the capacity to self-renew in vivo and thus, behaved like LSC. Hence, bioluminescent imaging of LSC engraftment in a highly immunocompromised mouse model can be used to detect LSC and may be utilized for pre-clinical evaluation of the effects of molecularly targeted therapy on LSC. Figure 1. Bioluminescent imaging was performed with the aid of a Xenogen™. IVIS 100 imaging system at 9 weeks post-transplant. Upper: RAG2−/γ0−/− mouse transplanted with no cells. Lower: Bioluminescence of 2° human CD45+GFP+ cells derived from mice transplanted with CML blast crisis GMP (mouse 1) or normal HSC (mouse 2) were compared with mice transplanted with primary normal Figure 1. Bioluminescent imaging was performed with the aid of a Xenogen™. IVIS 100 imaging system at 9 weeks post-transplant. Upper: RAG2−/γ0−/− mouse transplanted with no cells. Lower: Bioluminescence of 2° human CD45+GFP+ cells derived from mice transplanted with CML blast crisis GMP (mouse 1) or normal HSC (mouse 2) were compared with mice transplanted with primary normal

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