Constitutive Expression of SALL4B in Mice Confers Properties of Leukemic Stem Cells to Committed Hematopoietic Progenitors.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 240-240 ◽  
Author(s):  
Li Chai ◽  
Wei Cui ◽  
Yupo Ma ◽  
Jianchang Yang ◽  
Nikki Kong ◽  
...  
Keyword(s):  
Stem Cell ◽  
Transgenic Mice ◽  
Myeloid Leukemia ◽  
Es Cells ◽  
Constitutive Expression ◽  
The Self ◽  
Beta Catenin ◽  
Hematopoietic Progenitors ◽  
Self Renewal ◽  
Leukemia Stem Cell

Abstract The homeotic gene SALL4 is a transcription factor involved in multi-organ developments. Recently, our group and others have shown that murine Sall4 plays an important role in maintaining the properties of embryonic stem (ES) cells by interacting with Oct4 and Nonag. Knock down of Sall4 expression in mouse ES cells led to their spontaneous differentiation and loss of self-renewal ability. Parallel to these findings, using quantitative RT-PCR approach, we found that human SALL4 was preferentially expressed in the CD34+CD38− hematopoietic stem cell (HSC) population, and was dramatically down regulated in the CD34+CD38+ progenitor (HPC) population, suggesting that SALL4 functions similarly at the HSC level as it does in ES cells. In contrast to normal hematopoiesis, we found that SALL4 was constitutively expressed in the leukemia initiation stem cell (CD34+CD38− and CD34+CD38+), and its expression in human acute myeloid leukemia (AML) was deregulated. To investigate the effect of constitutive expression of SALL4, we have generated a transgenic mouse model with constitutive expression of SALL4B, one of the SALL4 isoforms. The SALL4B transgenic mice developed myelodysplastic syndrome (MDS)-like features and subsequent AML that was transplantable. Further analysis on the pre-leukemic and leukemic SALL4B transgenic mice showed constitutive expression of SALL4B in the HSC and HPC subpopulations (GMP), which resulted in the expansion of the GMP population and eventually led to myeloid leukemia development. Activation of Wnt/beta catenin pathway and up-regulation of Bmi-1, the two key elements that are known to be involved in the self-renewal of HSC and leukemia stem cell (LSC), were found in the SALL4B transgenic mice. Taken together, our data suggests that constitutive expression of SALL4B contributes to leukemogenesis by conferring leukemia stem cell properties to committed murine hematopoietic progenitors. To our best knowledge, SALL4 is one the few genes, if not the only gene, that play an important role in the self-renewal properties of ES cell, HSC and LSC.

A Highly Selective Anti-ROR1 Monoclonal Antibody Inhibits Human Acute Myeloid Leukemia CD34+ Cell Survival and Self-Renewal.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2560-2560
Author(s):  
Larissa Balaian ◽  
Anil Sadarangani ◽  
George F. Widhopf ◽  
Rui-kun Zhong ◽  
Charles Prussak ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Myeloid Leukemia ◽  
The Self ◽  
Self Renewal ◽  
Leukemia Stem Cell ◽  
Cd34 Cells ◽  
Selective Expression ◽  
Dose Dependent ◽  
Acute Myeloid

Abstract Abstract 2560 The mammalian orphan receptor tyrosine kinase-1 (ROR1) is expressed in a wide-variety of tissues during early embryonic development. By the late stages of embryogenesis the expression of this developmentally important protein is greatly diminished. Although not expressed in the tissues of post-partum animals, the ROR1 protein is expressed on neoplastic cells in chronic lymphocytic leukemia (CLL), some B-cell malignancies, and a variety of different carcinomas. We examined for expression of ROR1 in primary acute myeloid leukemia (AML) cells harvested from marrow aspirates and their normal counterparts by whole transcriptome paired-end RNA sequencing and by flow-cytometric analyses. These studies revealed selective expression of ROR1 in 62 (35%) of 179 AML samples examined. Many of these samples were found to have cells that co-expressed ROR1 and CD34, suggesting that ROR1 was present on the self-renewing leukemia stem-cell population, which resides in the marrow niche and potentially accounts for resistance to many cytotoxic drugs used in therapy. We investigated the activity of a chimeric anti-ROR1 mAb found effective in clearing CLL cells (UC99961) on AML expansion, growth, and renewal in a leukemia-stem-cell supportive niche assay. Mouse marrow cells lines SL/SL and M2–10B4 (transfected to produce hSCF,hIL3 and hIL3, hG-CSF respectively) were mixed 1:1 after mitomycin-C treatment, and used as a SLM2 stromal monolayer. CD34+ cells were selected from ROR1-positive (n=6) or negative (n=4) AML primary samples. As a normal control, CD34+ cells from cord blood (CB) were used (CB, n=3). In some experiments CD34+ cells were transfected with a GLP-lentivirus prior to co-culture. At the initiation of the co-culture, 10–50 μg/ml of the chimeric anti-ROR-1 mAb (UC99961) or control hIgG were added to the cultures. Two weeks after co-culture initiation, both stromal attached and floating cells were collected and their survival investigated by colony forming assay in methylcellulose. The UC99961 mAb was not cytotoxic to CB or ROR1-negative AML samples. In contrast, the UC99961 mAb provided a dose-dependent inhibition of colony formation for all ROR-1-positive AML samples examined. These results demonstrate the in vitro anti-leukemic specificity of this anti-ROR1 mAb in down-regulating AML stem and progenitor cell populations, without effecting normal CD34+ stem cells. To analyze the effect of ROR1 ligation on AML stem cell populations exclusively, AML self-renewal assays (2-ry colonies) were performed. In these studies, ROR1–positive AML samples were divided based on their response to mAb treatment. Half of the samples (n=3; 50%) demonstrated statistically significant (up to 90%) dose-dependent decreases in colony formation. However, another half was non-responsive and no correlation was found between ROR1 expression on leukemia CD34+ cells and response to anti-ROR1 mAb treatment in the self-renewal assays. Again UC99961 mAb treatment did not negatively impact CD34+ cells from CB or ROR1-negative AML, confirming the specificity and selective toxicity of the mAb for ROR1+ AML stem cells. These studies reveal selective expression of ROR1 on leukemia-stem-cells of large subset of AML patients. Furthermore, this study demonstrates that an anti-ROR1 mAb (UC99961) can inhibit survival and self-renewal in LSC supportive niche assays. Targeted ROR1 inhibition may represent a vital component of therapeutic strategies aimed at eradicating therapeutically recalcitrant malignant stem cells in AML and potentially other refractory cancer-stem-cell-driven malignancies. Disclosures: No relevant conflicts of interest to declare.

Inhibition of Chronic Myelogenous Leukemia Stem Cells with Novel Wnt Antagonists.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 238-238 ◽  
Author(s):  
Edward Kavalerchik ◽  
Jason Gotlib ◽  
Ifat Geron ◽  
Annelie Abrahamsson ◽  
Wolfgang Wrasidlo ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Chronic Myelogenous Leukemia ◽  
Reporter Gene ◽  
Myelogenous Leukemia ◽  
Beta Catenin ◽  
Self Renewal ◽  
Leukemia Stem Cell ◽  
Wnt Inhibitors

Abstract Introduction A growing proportion of chronic myelogenous leukemia (CML) patients show evidence of disease progression. Recent research suggests that leukemia stem cells (LSC) that share phenotypic characteristics with granulocyte-macrophage progenitors (GMP) are involved in CML progression. These LSC have aberrantly gained self-renewal capacity as a result of enhanced Wnt/beta-catenin signaling. We assayed the capacity of novel Wnt/beta-catenin antagonists to inhibit CML LSC. Methods To assay the efficacy of a novel Wnt inhibitor, MC-001, HEK293 cells were transfected with a Wnt-dependent reporter gene and expression plasmid for Dsh. After 16h, the cells were treated for 24 h with MCC-001, a novel marine sponge derived inhibitor, at varying concentrations and the reporter gene activity was measured. All cells were also transfected with a b-gal reporter gene to control for transfection efficiency. To assess the effects of MCC-001 and other Wnt inhibitors on Wnt/beta-catenin induced self-renewal, hematopoietic stem cells (HSC), GMP and lineage positive cells from normal (n=8) and advanced phase CML (n=8) peripheral blood and marrow (n=8) were clone sorted with the aid of a FACS Aria into methocult media (Stem Cell Technologies) with or without Wnt inhibitors including recombinant Dkk1, lentiviral axin or MCC-001. On day 10, individual colonies were plucked and replated in new methylcellulose and the replating efficiency determined at day 10. To establish an in vivo CML LSC model, HSC, GMP and lineage positive cells were transduced with a lentiviral luciferase GFP for 48 hours and transplanted intrahepatically into newborn immunocompromised mice (RAG2−/−gamma−/−) mice that facilitate high levels of human hematopoietic progenitor engraftment. Results The HEK293 beta-catenin reporter assay revealed that the MC-001 IC50 was 2.1 microM. In comparative Wnt inhibitor replating assays (n=8), recombinant Dkk1 did not inhibit CML HSC (n=8) while lentiviral axin and MCC-001 (at 2 and 10 microM) inhibited both CML HSC and CML GMP at doses that spared normal HSC replating (Figure 1). Transplantation of CML HSC, GMP and lineage positive cells into RAG2−/−gamma−/− mice demonstrated that only CML GMP provided serial transplantation potential and thus, were enriched for the LSC population (Figure 2). Conclusions Selective Wnt/beta-catenin inhibition with a marine sponge derived beta-catenin antagonist, MCC-001, blocks in vitro replating capacity of CML LSC at doses that spare normal HSC. Current experiments focus on in vivo inhibition of LSC self-renewal with novel Wnt inhibitors in a robust CML LSC bioluminescent imaging model (Figure 2). Figure 1. Chronic Myelogenous Leukemia Stem Cell Inhibition with MCC-001: A novel β-catenin Inhibitor Figure 1. Chronic Myelogenous Leukemia Stem Cell Inhibition with MCC-001: A novel β-catenin Inhibitor Figure 2. Bioluminescent Chronic Myelogenous Leukemia Stem Cell Transplantation Model. Figure 2. Bioluminescent Chronic Myelogenous Leukemia Stem Cell Transplantation Model.

scholarly journals Inflammatory Cytokine Responsive Enzymatic Mutagenesis Fuels Myeloproliferative Neoplasm Pre-Leukemia Stem Cell Evolution

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3780-3780
Author(s):  
Catriona Jamieson ◽  
Qingfei Jiang ◽  
Frida Holm ◽  
Jane Isquith ◽  
Adam Mark ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cancer Progression ◽  
Inflammatory Cytokine ◽  
Myeloproliferative Neoplasm ◽  
Cytokine Signaling ◽  
Beta Catenin ◽  
Self Renewal ◽  
Leukemia Stem Cell ◽  
Hematopoietic Stem

Innate immune anti-viral adenosine to inosine (A-to-I) base editing enzymes (editases) promote hematopoietic stem cell (HSC) self-renewal and protect the human genome from retroviral integration in response to inflammatory cytokine signaling. However, hyper-editing has been linked to therapeutic resistance and cancer progression. Because myeloproliferative neoplasm (MPN) progression is typified by increased JAK2/STAT-mediated cytokine signaling, we investigated the cell type and context specific role of adenosine deaminase acting on RNA1 (ADAR1) editaseactivity in MPN pre-leukemia stem cell (pre-LSC) evolution into acute myeloid leukemia stem cells (LSCs). Here we show by whole transcriptome sequencing (RNA-seq) of 113 FACS-purified hematopoietic stem cells and progenitors from 78 individuals, including 54 MPN and AML patients and 24healthy young and aged individuals, that anti-viral signaling pathway activation and splice isoform switching from ADAR1p110 to JAK2/STAT-inducible ADAR1p150 RNA editase activation contributes to MPN progression. Pre-LSC evolution to LSC was characterized by ADAR1p150 upregulation, distinctive RNA editome patterns, STAT3 hyper-editing, increased replating as a measure of self-renewal. Moreover, LSC generation was typified by beta-catenin self-renewal pathway upregulation, which was recapitulated by lentiviral ADAR1p150 overexpression and reversed by lentiviral ADAR1p150 shRNA knockdown. Our studyunderscores the importance of inflammatory-cytokine fueled enzymatic mutagenesis in human MPN pre-LSC evolution to LSC. Thus, this study sets the stage for developing predictive RNA editome biomarkers of LSC generation to guidetherapeutic strategies aimed at preventing progression of hematopoietic malignancies. Disclosures Crews: Ionis Pharmaceuticals: Research Funding.

scholarly journals MLL-AF9– and HOXA9-mediated acute myeloid leukemia stem cell self-renewal requires JMJD1C

2016 ◽  
Vol 126 (3) ◽  
pp. 997-1011 ◽  
Author(s):  
Nan Zhu ◽  
Mo Chen ◽  
Rowena Eng ◽  
Joshua DeJong ◽  
Amit U. Sinha ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Stem Cell ◽  
Myeloid Leukemia ◽  
Self Renewal ◽  
Leukemia Stem Cell ◽  
Acute Myeloid

Hematopoietic Stem Cells and Primitive Progenitors Express Leukemia-Associated Antigens That May Be Targets for Graft-Versus-Leukemia Effect or for Vaccine-Based Immunotherapy in Chronic Myeloid Leukemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2125-2125
Author(s):  
Agnes S.M. Yong ◽  
Keyvan Keyvanfar ◽  
Rhoda Eniafe ◽  
Bipin N. Savani ◽  
Elaine M. Sloand ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Healthy Individuals ◽  
Hematopoietic Progenitors ◽  
Leukemia Stem Cell ◽  
Hematopoietic Stem ◽  
Myeloid Progenitor ◽  
Graft Versus Leukemia

Abstract The cure of chronic myeloid leukemia (CML) following allogeneic stem cell transplantation (SCT) is attributed to a graft-versus-leukemia (GvL) effect which eliminates residual leukemia cells that might otherwise have the capacity to regenerate the leukemia. Whether the GvL effect is directed against the most primitive leukemia stem cell or only against a more mature leukemia progenitor cell is unknown. In contrast, although tyrosine kinase inhibitors (TKI), notably imatinib, effectively suppresses at least 2 logs of CML hematopoiesis in most patients, they appear not to affect the leukemia stem cell or the most primitive progenitor and so fail to eradicate the leukemia. To characterize further the GvL reaction and to identify targets that might be exploited for vaccine therapy outside the context of a stem cell transplant, we studied expression of selected proteins in CML progenitors at various levels of commitment. CD34+ progenitor subpopulations were purified by fluorescence activated cell sorting from 10 CML patients (5 chronic phase, 3 accelerated phase and 2 blastic phase) and from 8 healthy individuals. We used real-time quantitative polymerase chain reaction (RQ-PCR) for BCR-ABL, WT1, proteinase 3 (PR3), β-catenin and BMI-1. Hematopoietic stem cells, CD34+CD38-Lin-CD90+ and primitive progenitor subpopulations CD34+CD38- Lin- CD7+, CD34+CD38+ Lin-CD7+, and more differentiated CD34+CD38+ Lin + CD7- and CD34+CD38+ Lin+ CD7+ populations were collected. In addition, CD34+CD38+Lin- cells were sorted into common myeloid progenitor (CMP) CD34+CD38+Lin- IL3Rα+CD45RA- and granulocyte-macrophage progenitor (GMP) CD34+CD38+Lin- IL3Rα+CD45RA+ populations and their CD7+ and CD7- subpopulations collected. Mature myeloid progenitor subpopulations CD34+CD38+Lin-CD33+ were also separated into CD7+ and CD7- groups. All primitive leukemic CD34+ subpopulations expressed similar levels of BCR-ABL mRNA and contained greater than 80% BCR-ABL positive cells by fluorescence in situ hybridization. By RQ-PCR we detected higher expression of the leukemia-associated antigen WT1 in the most primitive CD34+CD38-CD90+Lin- subset of hematopoietic progenitors in CML compared to normal. In more mature CMP and GMP populations, the expression of both WT1 and PR3 was higher in CML than normal. The CD7+ subpopulation of GMPs in CML, which are implicated in disease progression, also have higher expression of leukemia-associated antigens than normal. The expression of β-catenin and BMI-1 was lower in CML CD34+ progenitors in comparison to those from healthy individuals in all subpopulations except in CD7+ GMPs, indicating low self-renewal activity. These results suggest that the most primitive leukemic hematopoietic progenitors, as well as their more differentiated progeny, express WT1 and PR3 at relatively high levels and may thus be targets for peptide-based vaccines. The mechanism that protects primitive leukemic progenitors from the effects of TKI remains undefined but may be related to their quiescent status or to their high levels of BCR-ABL expression.

scholarly journals Genome-scale expression and transcription factor binding profiles reveal therapeutic targets in transgenic ERG myeloid leukemia

Blood ◽  
2013 ◽  
Vol 122 (15) ◽  
pp. 2694-2703 ◽  
Author(s):  
Liat Goldberg ◽  
Marloes R. Tijssen ◽  
Yehudit Birger ◽  
Rebecca L. Hannah ◽  
Sarah J. Kinston ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Stem Cell ◽  
Transgenic Mice ◽  
Myeloid Leukemia ◽  
Therapeutic Targets ◽  
Leukemia Stem Cell ◽  
Factor Binding ◽  
Key Points ◽  
Program Characteristic ◽  
Genome Scale

Key Points ERG overexpression in transgenic mice induces a transcriptional leukemia stem cell program characteristic of human AML. PIM1 and RAS are relevant ERG therapeutic targets.

Modeling the functional heterogeneity of leukemia stem cells: role of STAT5 in leukemia stem cell self-renewal

Blood ◽  
2009 ◽  
Vol 114 (19) ◽  
pp. 3983-3993 ◽  
Author(s):  
Michael Heuser ◽  
Laura M. Sly ◽  
Bob Argiropoulos ◽  
Florian Kuchenbauer ◽  
Courteney Lai ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Constitutive Expression ◽  
Leukemia Stem Cells ◽  
Functional Heterogeneity ◽  
Self Renewal ◽  
Leukemia Stem Cell ◽  
Gm Csf ◽  
Stat Signaling

Abstract Although the cancer stem cell (CSC) concept implies that CSCs are rare, recent reports suggest that CSCs may be frequent in some cancers. We hypothesized that the proportion of leukemia stem cells would vary as a function of the number of dysregulated pathways. Constitutive expression of MN1 served as a 1-oncogene model, and coexpression of MN1 and a HOX gene served as a 2-oncogene model. Leukemia-initiating cell (LIC) number and in vitro expansion potential of LICs were functionally assessed by limiting dilution analyses. LIC expansion potential was 132-fold increased in the 2- compared with the 1-oncogene model, although phenotypically, both leukemias were similar. The 2-oncogene model was characterized by granulocyte-macrophage colony-stimulating factor (GM-CSF) hypersensitivity and activated STAT/ERK signaling. GM-CSF hypersensitivity of the 2-oncogene model (MN1/HOXA9) was lost in Stat5b−/− cells, and the LIC expansion potential was reduced by 86- and 28-fold in Stat5b−/− and Stat1−/− cells, respectively. Interestingly, in 201 acute myeloid leukemia (AML) patients, coexpression of MN1 and HOXA9 was restricted to patients with the poorest prognosis and was associated with highly active STAT signaling. Our data demonstrate the functional heterogeneity of LICs and show that STAT signaling is critical for leukemia stem cell self-renewal in MN1- and HOXA9-expressing leukemias.

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