MiR-150 Suppresses MLL-AF9 Associated Leukemia Through Simultaneously Targeting Multiple Oncogenes,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3461-3461
Author(s):  
Beiyan Zhou
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Expression Patterns ◽  
Gene Profiling ◽  
Fusion Genes ◽  
Leukemic Stem Cells ◽  
Hematopoietic Stem ◽  
Mll Gene

Abstract Abstract 3461 The mixed lineage leukemia (MLL) gene codes for an evolutionarily conserved histone methyltransferase that is crucial for early hematopoiesis. As a result of a chromosomal translocation involving locus 11q23 results in formation of chimeras composed of the 5' part of the MLL gene fused with more than 60 partner genes lead to disruption of normal function of MLL as a histone methytransferase and acquisition of transcriptional properties conferred by the partner genes. MLL fusion genes (MLL-FG) are often the causal mutations for aggressive acute myeloid and lymphoid leukemias (AML and ALL) that correlated with poor prognosis. In order to treat or even eliminate MLL-associated leukemias, extensive studies on the regulatory mechanism underlying MLL associated transformation and progression have been carried out. Leukemic stem cells (LSC) can derive from either hematopoietic stem or progenitor cells with the recruitment of MLL-fusion genes (MLL-FG) and wild type MLL protein. We report that miR-150, a key hematopoietic regulatory microRNA (miRNA) and one of the most downregulated miRNAs in MLL-associated leukemias, acts as a tumor suppressor to block the leukemogenic potency of leukemic stem cells. When expression of miR-150 was restored, a significantly suppressed leukemic stem cell potency of MLL-AF9 cells was observed both in vivo and in vitro. Gene profiling analysis demonstrated that elevated miR-150 altered various aspects of gene expression patterns in MLL-AF9 cells, including stem cell signatures, cancer pathways, and cell survival. By screening more than 30 predicted target genes, we identified multiple leukemia-associated oncogenes as bona fide miR-150 targets, and knockdown of these genes by shRNAs recapitulated the tumor suppressive effects observed after ectopically expression of miR-150 in MLL-AF9 cells. Disclosures: No relevant conflicts of interest to declare.

MiR-150 Inhibits MLL-AF9 Associated Leukemia By Suppressing Leukemic Stem Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3764-3764
Author(s):  
Patali S Cheruku ◽  
Marina Bousquet ◽  
Guoqing Zhang ◽  
Guangtao Ge ◽  
Wei Ying ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Colony Formation ◽  
Conflicts Of Interest ◽  
Expression Patterns ◽  
Gene Profiling ◽  
Leukemic Stem Cells ◽  
Hematopoietic Stem ◽  
Signature Genes

Abstract Leukemic stem cells (LSCs) are derived from hematopoietic stem or progenitor cells and often share gene expression patterns and specific pathways. Characterization and mechanistic studies of LSCs are critical as they are responsible for the initiation and potential relapse of leukemias, however the overall framework, including epigenetic regulation, is not yet clear. We previously identified microRNA-150 (miR-150) as a critical regulator of mixed lineage leukemia (MLL) -associated leukemias by targeting oncogenes. Our additional results suggest that miR-150 can inhibit LSC survival and disease initiating capacity by suppressing more than 30% of “stem cell signature genes,” hence altering multiple cancer pathways and/or stem cell identities. MLL-AF9 cells derived from miR-150 deficient hematopoietic stem/progenitor cells displayed significant proliferating advantage and enhanced leukemic colony formation. Whereas, with ectopic miR-150 expression, the MLL-AF9 associated LSC population (defined as Lin-ckit+sca1- cells) was significantly decreased in culture. This is further confirmed by decreased blast leukemic colony formation in vitro. Furthermore, restoration of miR-150 levels in transformed MLL-AF9 cells, which often display loss of miR-150 expression in AML patients with MLL-fusion protein expressing, completely blocked the myeloid leukemia development in a transplantation mouse model. Gene profiling analysis demonstrated that an increased level of miR-150 expression down regulates 30 of 114 stem cell signature genes by more than 1.5 fold, partially mediated by the suppressive effects of miR-150 on CBL, c-Myb and Egr2 oncogenes. In conclusion, our results suggest that miR-150 is a potent MLL-AF9 leukemic inhibitor that may act by suppressing the survival and leukemic initiating potency of MLL-AF9 LSCs. Disclosures: No relevant conflicts of interest to declare.

JMJD3 Plays Essential Roles in the Maintenance of Hematopoietic Stem Cells and Leukemic Stem Cells through the Regulation of p16INK4a

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2653-2653 ◽  
Author(s):  
Yuichiro Nakata ◽  
Norimasa Yamasaki ◽  
Takeshi Ueda ◽  
Kenichiro Ikeda ◽  
Akiko Nagamachi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Epigenetic Mark ◽  
M2 Macrophage ◽  
Leukemic Stem Cells ◽  
Cell Potential ◽  
Hematopoietic Stem

Abstract Hematopoiesis is a complex process that involves the interplay between lineage-specific transcription and epigenetic regulation, including histone modifications. Tri-methylation of histone H3 at Lys27 (H3K27me3) is an epigenetic mark for transcriptional repression. Jumonji domain-containing 3 (JMJD3) acts as a histone demethylase for H3K27 and contributes to various cellular processes including senescence and differentiation through transcriptional regulation. In the hematopoietic system, JMJD3 has been reported to be required for M2 macrophage development and terminal thymocyte differentiation. However, the roles of JMJD3 in normal hematopoiesis and leukemogenesis are still largely elusive. To address this issue, we generated pIpC-inducible Jmjd3 conditional KO (cKO) mice. Jmjd3-deficient (Jmjd3Δ/Δ) mice grew healthy and did not show obvious hematopoietic abnormalities, except a slight decrease of myeloid cells. To investigate the role of JMJD3in hematopoietic stem cell (HSC) function, a competitive repopulation assay was performed using control and Jmjd3Δ/Δ HSCs. The results showed that the chimerism of Jmjd3Δ/Δ cells was significantly decreased compared with that of control cells in all the hematopoietic lineages, indicating that JMJD3 is essential for long-term repopulating ability of HSCs. To further investigate the effect of Jmjd3 deletion in leukemogenesis, c-kit+ bone marrow (BM) cells from control and Jmjd3 cKO mice were transduced with MLL-AF9 fusion protein that rapidly induces acute leukemia. L-GMPs (the fraction containing leukemic stem cells (LSCs)) were sorted from MLL-AF9-transduced BM cells and subjected to colony replating and bone marrow transplantation (BMT) assays. In contrast control L-GMPs that continued to form colonies after multiple rounds of replating, Jmjd3Δ/Δ L-GMPs ceased to proliferate after third rounds of replating. In addition, recipients transplanted with Jmjd3Δ/Δ L-GMPs exhibited a significant delay in the onset of leukemia compared with those transplanted with controlL-GMPs. These results indicate that JMJD3 plays essential roles in maintaining stem cell properties not only in normal HSCs but also in LSCs. We next investigated underlying molecular mechanisms. Previous studies demonstrated the INK4a/ARF locus, a key executor of cellular senescence, is regulated by JMJD3. Thus, we examined whether JMJD3 regulates INK4a/ARF locus in hematopoietic cells under proliferative and oncogenic stresses. We found that enforced expression of Jmjd3 in in vitro-cultured and cytokine-stimulated hematopoietic stem-progenitor cells (HSPCs) significantly upregulated the expression of p16INK4a compared with control cells. In addition, transformation of HSPCs by MLL-AF9 induced expression of Jmjd3, but not other H3K27me3-related genes, such as Utx and EZH2, which was accompanied by the upregulation of p16INK4a. In contrast, no obvious expressional change was observed in p19ARF in both cases. In Jmjd3Δ/Δ HSPCs, no upregulation of p16INK4a was detected in HSPCs by cytokine-induced proliferation or MLL-AF9-induced transformation, where H3K27me3 was tightly associated with promoter region of p16INK4a locus. These results strongly suggest that proliferative and oncogenic stresses induces the expression of Jmjd3 in HSPCs, which subsequently upregulates p16INK4a through demethylating H3K27me3 on the p16INK4a promoter and consequently maintains stem cell potential by inhibiting excessive entry into cell cycle. Deficiency of Jmjd3 fails upregulation of p16INK4a, which induces continuous and excessive cell proliferation and finally causes exhaustion of stem cell pool. In conclusion, we propose the idea that JMJD3-p16INK4a axis plays essential roles in maintaining HSC and LSC pool size under proliferative and oncogenic stresses. Disclosures No relevant conflicts of interest to declare.

A New Flowcytometry-Based Method to Discriminate Malignant From Normal Stem Cells in CML.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3245-3245
Author(s):  
Jeroen J.W.M. Janssen ◽  
Wendy Deenik ◽  
Karlijn G.M. Smolders ◽  
Monique Terwijn ◽  
Angele Kelder ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Risk Scores ◽  
Fish Analysis ◽  
Leukemic Stem Cells ◽  
Aberrant Expression ◽  
Hematopoietic Stem ◽  
Culture Techniques

Abstract Abstract 3245 Poster Board III-182 Tyrosine kinase inhibitor (TKI) insensitivity of CML hematopoietic stem cells prevents eradication of the disease by these drugs and is presumably implicated in development of TKI resistance. Probably, improvement of treatment results will involve leukemic stem cell directed therapy. Therefore, more knowledge of stem cell specific targets would be instrumental. Previously, leukemic stem cells could only be identified indirectly by using culture techniques. We developed a new flowcytometric approach that enables to directly distinguish CML stem cells from their normal counterparts within single patient samples. In 24 newly diagnosed CML patients CML CD34+CD38- stem cells could be discriminated from normal stem cells by higher CD34 and CD45 expression and different forward/sideward light scatter properties, reflecting differences in size and granularity. In addition, aberrant expression of CD7, CD11b and CD56 was demonstrated on malignant stem cells, allowing clear discrimination from benign stem cells, that were always negative for these markers. Above all, in all tested CML patients we were able to demonstrate that high CD90 expression is a specific feature of CML stem cells, while CD90 expression is low on their normal counterparts. FISH analysis on FACS sorted cells proved that populations were BCR-ABL positive (in case of high CD34 and CD45 expression and high CD90 expression) or negative (in case of low CD34 and CD45 expression and low CD90 expression), while long term liquid culture assays with subsequent CFU assays and FISH analysis proved their malignant/normal stem cell character. Patients with a large proportion of non-leukemic stem cells had significantly lower clinical risk scores (Sokal, Euro) than patients with few remaining normal stem cells. This new technique will expand our possibilities to identify new CML stem cell specific targets and may improve efficacy assessment of CML treatment as well. Disclosures No relevant conflicts of interest to declare.

Bone Marrow Derived Mesenchymal Stem Cells and Radiation Response: a Kinetic Study of Gene Response Using Microarray Analysis.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4575-4575
Author(s):  
Philippe Garrigou ◽  
Jean-Francois Mayol ◽  
Catherine Mouret ◽  
Christophe Delaunay ◽  
Michel Drouet ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Short Term ◽  
Hematopoietic Stem ◽  
Pai 1

Abstract Abstract 4575 Mesenchymal Stem cells (MSC) are an important radiosensitive component of the so called hematopoietic stem cell niche. Importantly this supportive microenvironment influences the stem cell repopulation capacity as well as the quiescent/non proliferative state of hematopoietic cells. Senescence is considered as a major process in MSC response following irradiation. However, other studies have reported in mice the reduction of the pool of bone marrow mesenchymal stem/progenitor cells following TBI independently of senescence. An altered osteoblastic differentiation was pointed out in these studies. Furthermore, MSC have been shown to be involved in the repair of ionizing radiation damage of distant epithelial sites which requires adherence genes mitigation. The aim of this study was to clarify some of these points using an in vitro model of irradiation and short term culture. Briefly, confluent human BM-MSC were irradiated at the dose of 2.5 Gy (dose rate: 95 cGy.min-1) and immediately put into culture (Minimum essential medium supplemented with 10% FCS and 10 μg/ml of ciprofloxacin, penicillin and streptomycin). Six, 12, 24, 48 and 72 hours after irradiation, cells were harvested and lysed. Total RNAs were purified using the automatic Qiacube system (Qiagen,Courtaboeuf, France) and processed on DNA microarray scanner (Agilent technologies Inc.) according to supplier's recommendations. Data were analyzed with GeneSpring GX Expresion Analysis software version 10.0 (Agilent) in order to identify the transduction pathways involved. No apoptosis was observed during this short term incubation. Among other genes we identified plasminogen activator inhibitor 1 (PAI-1) as a factor highly upregulated after irradiation, in addition to CD151. This is in accordance with MSC response to nutrient-poor, hypoxic stress environment (Copland et al, Stem cells 2009). As MSC are radiosensitive cells, this may indicate that PAI impacts MSC survival through the mitigation of their adhesiveness to surrounding matrices. As PAI-1 is an important factor involved in the balance of blood coagulation and fibrinolysis as well as in the regulation of angiogenesis, one may speculate the consequences of PAI-1 release from MSC on blood homeostasis. Work is going on to describe the main response target genes. This could allow us to identify therapeutic strategies based on ex-vivo or in vivo manipulation of MSC in a purpose of tissue remodelling. 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 Twist-1 Increases Hematopoietic Stem Cell Self-Renewal and Causes Myeloid Skewing

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4320-4320
Author(s):  
Xiaotong Ma ◽  
Chengya Dong ◽  
Xiaoyan Liu ◽  
Nan Wang ◽  
Lina Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Conflicts Of Interest ◽  
Expression Patterns ◽  
Mouse Bone Marrow ◽  
Self Renewal ◽  
Myeloid Lineage ◽  
Hematopoietic Stem ◽  
Altered Expression ◽  
Twist 1

Abstract Twist-1 protein belongs to the large family of basic Helix-Loop-Helix transcription factors with diverse physiological functions in mesoderm-derived tissues. Growing evidences now link Twist-1 to the acquisition of stem-cell-like properties. However, there is little information available regarding its expression pattern and functional role in the hematopoietic system. We analyzed Twist-1 expression patterns in different hematopoietic cell populations from normal mouse bone marrow, and found that Twist-1 was most highly expressed in long-term hematopoietic stem cells (LT-HSCs) but showed a low abundance in more differentiated descendants. To investigate Twist-1 gene function, retroviral-mediated overexpression or removal experiments were performed. Competitive repopulation studies demonstrated that enforced expression of Twist-1 in HSC-enriched Lin−c-Kit+Sca-1+ (LKS) cells resulted in an increase in the size of the G0 population, and in their reconstitution ability after the first and a second transplantation. Conversely, removal of Twist-1 in LKS cells impaired their ability to repopulate. In addition, increased Twist-1 expression caused a shift toward production of myeloid cells. Twist-1 transduction in LKS cells activated myeloid lineage-determining factors PU.1 and GATA-1 and down-regulated lymphoid factor GATA-3 in vitro, suggesting that Twist-1-mediated myeloid skewing occurs in hematopoietic stem and progenitor cells (HSPCs). These findings indicate that Twist-1 is not only involved in the maintenance of HSC dormancy and self-renewal capacity but also implicated in the myeloid lineage fate choice of HSPCs. Exploration of the underlying mechanisms revealed that Twist-1 overexpression lead to altered expression of Runx1/c-Mpl/Tie2 regulatory pathway, and quiescence-associated N-cadherin and Hes1 in LKS cells, which may contribute to the phenotypes observed in Twist-1-overexpressing mice. These studies shed additional light on the mechanisms involved in the maintenance of normal HSC and myeloid lineage differentiation, and may also provide clues to the mechanisms controlling pathogenesis and preservation of leukemic stem cells. Disclosures No relevant conflicts of interest to declare.

scholarly journals Ing4-Deficiency Enhances Hematopoietic Stem Cell Quiescence and Confers Resistance to Inflammatory Stress

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1095-1095
Author(s):  
Zanshé Thompson ◽  
Georgina A Anderson ◽  
Seth Gabriel ◽  
Melanie Rodriguez ◽  
Vera Binder ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Target Genes ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Loss Of Function ◽  
Wild Type ◽  
Hematopoietic Stem

Abstract In a screen for epigenetic regulators of hematopoiesis in zebrafish, we identified a requirement of the tumor suppressor protein, Ing4, in hematopoietic stem and progenitor cell (HSPC) specification. Though the Ing4 mechanism of action remains poorly characterized, loss of Ing4 has been shown to promote stem cell-like characteristics in malignant cells and it is a frequent target of inactivation in various types of cancer. Mutations in Ing4 cause deregulation of both NF-kB and c-Myc target gene expression. We have also identified a requirement for Ing4 in murine hematopoiesis. Ing4-/- mice have aberrant hematopoiesis and elevated cytokine expression in bone marrow cells. Using RNA-sequencing, we found that Ing4-deficient HSPCs express high levels of c-Myc target genes and genes associated with oxidative phosphorylation and ribosomal biogenesis. Yet, Ing4 deficiency induces G 0 arrest in HSPCs and they have low levels of reactive oxygen species. This places Ing4-deficient HSPCs in a poised state, where they are quiescent, but express elevated levels of genes associated with differentiation. Under stress hematopoiesis following low-dose irradiation, Ing4-deficient long-term hematopoietic stem cells (LT-HSCs) do not expand, but short-term hematopoietic stem cells (ST-HSCs) function comparably to wild-type. Similarly, under transplantation stress, LT-HSCs fail to contribute to multilineage chimerism, while ST-HSCs contribute at levels equal to wild-type cells. These results are striking, particularly when compared to other models of enhanced NF-kB activity, where HSPCs cannot contribute to multilineage chimerism in transplantation. We sought to target the misregulated pathways in Ing4-deficient HSCs to rescue to effects of Ing4 deficiency. To this end, we chose to target the c-Myc pathway for several reasons: c-Myc target genes are over-represented in our RNA-seq data, c-Myc lies upstream of several of the misregulated pathways observed in Ing4-/- HSCs, and Ing4 has previously been reported to negatively regulate c-Myc activity directly. When treated with the c-Myc inhibitor, 10058-F4, both LT-HSCs and ST-HSCs are pushed into cycling, but this treatment also resulted in fewer cells overall. These results suggest that dampening of the c-Myc pathway can partially rescue Ing4 loss of function. Overall, our findings suggest that Ing4 plays a crucial role in the regulation of hematopoiesis and provides key tools for further identification and characterization of Ing4 pathways and functions. Given the role of Ing4 in both normal hematopoiesis and cancer, this gene likely has a critical role in regulation of stem cell self-renewal and maintenance. Disclosures No relevant conflicts of interest to declare.

scholarly journals The quiescent fraction of chronic myeloid leukemic stem cells depends on BMPR1B, Stat3 and BMP4-niche signals to persist in patients in remission

Haematologica ◽  
2020 ◽  
Vol 106 (1) ◽  
pp. 111-122 ◽  
Author(s):  
Sandrine Jeanpierre ◽  
Kawtar Arizkane ◽  
Supat Thongjuea ◽  
Elodie Grockowiak ◽  
Kevin Geistlich ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Tyrosine Kinase ◽  
Stromal Cells ◽  
Kinase Inhibitor ◽  
Bmp Signaling ◽  
Myelogenous Leukemia ◽  
Leukemic Stem Cells ◽  
Hematopoietic Stem

Chronic myelogenous leukemia arises from the transformation of hematopoietic stem cells by the BCR-ABL oncogene. Though transformed cells are predominantly BCR-ABL-dependent and sensitive to tyrosine kinase inhibitor treatment, some BMPR1B+ leukemic stem cells are treatment-insensitive and rely, among others, on the bone morphogenetic protein (BMP) pathway for their survival via a BMP4 autocrine loop. Here, we further studied the involvement of BMP signaling in favoring residual leukemic stem cell persistence in the bone marrow of patients having achieved remission under treatment. We demonstrate by single-cell RNA-Seq analysis that a sub-fraction of surviving BMPR1B+ leukemic stem cells are co-enriched in BMP signaling, quiescence and stem cell signatures, without modulation of the canonical BMP target genes, but enrichment in actors of the Jak2/Stat3 signaling pathway. Indeed, based on a new model of persisting CD34+CD38- leukemic stem cells, we show that BMPR1B+ cells display co-activated Smad1/5/8 and Stat3 pathways. Interestingly, we reveal that only the BMPR1B+ cells adhering to stromal cells display a quiescent status. Surprisingly, this quiescence is induced by treatment, while non-adherent BMPR1B+ cells treated with tyrosine kinase inhibitors continued to proliferate. The subsequent targeting of BMPR1B and Jak2 pathways decreased quiescent leukemic stem cells by promoting their cell cycle re-entry and differentiation. Moreover, while Jak2-inhibitors alone increased BMP4 production by mesenchymal cells, the addition of the newly described BMPR1B inhibitor (E6201) impaired BMP4-mediated production by stromal cells. Altogether, our data demonstrate that targeting both BMPR1B and Jak2/Stat3 efficiently impacts persisting and dormant leukemic stem cells hidden in their bone marrow microenvironment.

Characterization of the Putative Stem Cells From Umbilical Cord Blood.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4231-4231
Author(s):  
Amos S. Gaikwad ◽  
Michael Cubbage ◽  
Tatiana Goltsova ◽  
Christopher Threeton ◽  
Maria Ty ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cord Blood ◽  
Cell Population ◽  
Cell Biology ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Side Population ◽  
Hematopoietic Stem ◽  
Cell Fractions

Abstract Abstract 4231 Cord blood (CB) is a rich source of hematopoietic stem cells (HSC) with long-term repopulating activity necessary for allogeneic stem cell transplantation. CD34+ stem cells are considered sufficient for transplantation, however recent progress in stem cell biology indicates that cells with other surface markers such as CD133 or cells expressing high aldehyde dehydrogenase activity with low side scatter (ALDHhigh/SSClow) or a rare side population (SP) of cells that exclude the Hoechst 33342 vital dye via multi drug transporters have been shown to possess stem cell properties. We characterized CD34+, CD133+, ALDH+ and SP in mononuclear cells (MNC) isolated from human CB. While the SP cell population is rare and detectable in few CB-MNC examined, we found abundant CD34+ and CD133+ cells (1.0+/-0.5 and 0.8+/-0.4 per 100 CD45+ MNC cells, respectively) following the ISHAGE protocol. A distinct ALDH+ cell population (median of 0.26%; range of 0.1 to 0.5%) was also present in all of the CB-MNC analyzed. Over 90% of the ALDH+ cells were also CD34+ and CD133+. The ability of CB-MNC to form colonies in methocult semi-solid media supplemented with cytokines yielded myeloid, lymphoid and erythroid colonies. The clonogenic potential of CB-MNC ranged from 16-48%. We are assessing the colony forming ability of purified stem cell fractions using flow cytometry. The clonogenic efficiency of these individual putative stem cells will be discussed. Disclosures: No relevant conflicts of interest to declare.

T(15;17)-PML/RAR-Induced Leukemogenesis: Long-Term Repopulating Hematopoietic Stem Cells as the Initial Target and More Mature Progenitors as the Potential Targets for Final Leukemic Transformation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3980-3980 ◽  
Author(s):  
Claudia Oancea ◽  
Brigitte Rüster ◽  
Jessica Roos ◽  
Afsar Ali Mian ◽  
Tatjana Micheilis ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Population ◽  
Conflicts Of Interest ◽  
Cell Model ◽  
Leukemic Cell ◽  
Hematopoietic Stem ◽  
Proliferation Potential

Abstract Abstract 3980 Poster Board III-916 Stem cells have been shown to play an important role in the pathogenesis and maintenance of a significant number of malignancies, including leukemias. Similar to normal hematopoiesis the AML cell population is thought to be hierarchically organized. According to this model, only a few stem cells (LSC) are able to initiate and maintain the disease. The inefficient targeting of the leukemic stem cells (LSC) is considered responsible for relapse after the induction of complete hematologic remission (CR) in AML. Acute promyelocytic leukemia (APL) is a subtype of AML characterized by the t(15;17) translocation and expression of the PML/RARα fusion protein. Treatment of APL with all-trans retinoic acid (t-RA) as monotherapy induces CR, but not molecular remission (CMR), followed by relapse within a few months. In contrast arsenic as monotherapy induces high rates of CR and CMR followed by a long relapse-free survival. We recently have shown that in contrast to t-RA, arsenic efficiently targets PML/RAR-positive stem cells, whereas t-RA increases their proliferation. For a better characterization of LSC in APL which has to be targeted for an efficient eradication of the disease we wanted to characterize the leukemia-initiating cell and the cell population able to maintain the disease in vivo. The model was based on a classical transduction/transplantation system of murine Sca1+/lin- HSC combined with a novel approach for the enrichment of transformed cells with long-term stem cell properties. We found that PML/RAR induced leukemia from the Sca1+/lin- HSC with a frequency of 40% and a long latency of 8-12 months independently of its capacity to increase dramatically replating efficiency and CFU-S12 potential as expression of the differentiation block and proliferation potential of derived committed progenitors. Based on the hypothesis that PML/RAR exerts its leukemogenic effects on only a small proportion of the Sca1+1/lin- population, we proceeded to select and to amplify rare PML/RAR-positive cells with the leukemia-initiating potential, by a negative selection of cell populations with proliferation potential without long term stem cell-capacity (LT). Therefore we expressed PML/RAR in Sca1+/lin- cells and enriched this population for LT- (lin-/Sca1+/c-Kit+/Flk2-) and ST-HSC (lin-/Sca1+/c-Kit+/Flk2+). After a passage first in semi-solid medium for 7 days and subsequent transplantation into lethally irradiated mice, cells from the ensuing CFU-S day12 were again transplanted into sublethally recipient mice. After 12 to 36 weeks, 6/6 mice developed acute myeloid leukemia without signs of differentiation in the group transplanted with the lin-/Sca1+/c-Kit+/Flk2- population but not from that transplanted with lin-/Sca1+/c-Kit+/Flk2+ cells. This leukemia was efficiently transplanted into secondary recipients. The primary leukemic cell population gave origin to 6 clearly distinct subpopulations defined by surface marker pattern as an expression of populations with distinct differentiation status, able - after sorting - to give leukemia in sublethally irradiated recipients: Sca1+/c-Kit+/CD34- (LT-HSC), Sca1+/c-Kit+/CD34+ (ST-HSC), Sca1-/c-Kit+, B220lo/GR1+/Mac1+, B220hi/GR1+/Mac1+, B220-/Gr1-/Mac1-. Interestingly, all leukemias from the different population presented an identical phenotype. These findings strongly suggest that there is a difference between a leukemia-initiating (L-IC) and leukemia-maintaining (L-MC) cell population in the murine PML/RAR leukemia model. In contrast to the L-IC, represented by a very rare subpopulation of primitive HSC, recalling a hierarchical stem cell model, the L-MC is represented by a larger cell population with a certain grade of phenotypical heterogeneity, but a high grade of functional homogeneity recalling a stochastic cancer induction model. Disclosures: No relevant conflicts of interest to declare.

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