The Regulation of β-arrestin1 in Leukemia Initiating Cells of Children B-Acute Lymphoblastic Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3538-3538
Author(s):  
Lin Zou ◽  
Shan Liu ◽  
Yi Shu ◽  
Ru Qin ◽  
Kang Li ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Mrna Expression ◽  
Promoter Region ◽  
Lymphoblastic Leukemia ◽  
The Self ◽  
Mrna Expression Level ◽  
Self Renewal ◽  
Nsg Mice

Abstract Background Leukemia is the most common malignant tumor in children under 15 years old. The main subtype of children leukemia is acute lymphoblastic leukemia (ALL), and B-lineage ALL (B-ALL) accounts for approximately 70%. The leukemia-initiating cells (LICs) are cancer stem cells with long-term repopulating potential and propagation ability, to maintain the leukemia cell phenotype, and possess leukemia-initiating activity. However, the regulation of LICs for the leukemia progression is poorly understood. The multifunctional scaffold proteins β-arrestins are proven to mediate H4 acetylation and gene expression. And β-arrestin2 is found to regulate the initiation and progression of chronic myeloid leukemia (CML). However, the role of β-arrestin1 in B-ALL is still unknown. Our preliminary data showed that both the high expression of β-arrestin1 and high proportion of CD34+CD38- cells are positively correlated with risk stratification and poor prognosis of childhood B-ALL. And β-arrestin1 binds with EZH2 to increase BCR/ABL H4 acetylation and thus promotes CML cell progression in vitro and in vivo. The aim of study is to investigate the essential function of β-arrestin1 in LICs from B-ALL. Materials and Methods The bone marrow (BM) and periphery blood (PB) of children B-ALL patients were collected, isolated and identified LICs by Magnetic-activated cell sorting (MACS) and flow cytometry. The total RNA and protein were purified for gene and protein expression by real-time RT-PCR and Western blot. The leukemia cells (LICs, Raji, and Reh) of β-arrestin1 depletion were constructed by transient or stable screening si-β-arrestin1 (siβ1) lentivirus vector. The serial cell colony formation and NSG mice survival analysis was measured the LICs self-renewal ability. The CCK8 and MTS assays were used to detect the cell proliferation, and annexin V-FITC and PI staining for cell apoptosis. The DNA methylation of gene promoter region was detected by methylation-specific PCR and the methltransferase activity by ELISA. The telomere length was indicated by Southern blot and FISH, and telomerase activity by TRAP. Electrophoretic mobility shift assay (EMSA) and dual-luciferase reporter assay were applied to explain gene transcription. Student’s t test and Log-Rank test were used in the corresponding statistical significance and P<0.05 were considered significant. All the statistical analysis was performed using the GraphPad Prism (Version 5.0) software packages and SPSS 17.0. Results The expression of β-arrestin1 was elevated in LICs from B-ALL patients, and the high level of β-arrestin1 was negatively correlated with the survival of these patients. Further study showed that the loss of β-arrestin1 in B-ALL LICs attenuates their self-renewal capacity and promotes their senescence in vitro and in vivo. The mRNA expression level of β-arrestin1 is negatively correlated with that of PTEN in LICs. Moreover, the DNA methylation of the PTEN promoter region, the activity and the expression of DNMTs were enhanced in the LICs. The inhibition of DNMT1 activity impaired the self-renewal and increased the expression of PTEN of LICs. In addition, depletion of β-arrestin1 significantly decreased DNMT1 activity and PTEN methylation, and consistently increased PTEN expression in LICs. For B-ALL cell senescence, the mRNA expression level of β-arrestin1 is negatively related with the length of telomere, positively related with the activity of telomerase and the mRNA expression of hTERT in B-ALL LICs and engrafted NSG mice. Moreover, the weakened effect of β-arrestin1 on telomere, telomerase and the gene of hTERT were observed by injected the inhibitor of telomerase in leukemic mice. In addition, depletion of β-arrestin1 significantly decreased the binding of SP1 to the promoter of hTERT and thus reduced the transcription of hTERT in B-ALL Raji and Reh cells. Furthermore, β-arrestin1 interacted with P300 to bind with SP1 in the -104bp to -113bp of hTERT core promoter region in B-ALL cells. Conclusions β-arrestin1 could regulate the self-renewal and senescence of LICs from B-ALL, by partially mediating DNMT1 activity and hTERT transcription respectively, indicating that β-arrestin1 is a potential therapeutic target for B-ALL. Disclosures No relevant conflicts of interest to declare.

scholarly journals Histone demethylase JMJD2D promotes the self-renewal of liver cancer stem-like cells by enhancing EpCAM and Sox9 expression

2020 ◽  
pp. jbc.RA120.015335
Author(s):  
Yuan Deng ◽  
Ming Li ◽  
Minghui Zhuo ◽  
Peng Guo ◽  
Qiang Chen ◽  
...  
Keyword(s):  
Liver Cancer ◽  
Cancer Progression ◽  
Liver Tumors ◽  
The Self ◽  
High Rate ◽  
Sox9 Expression ◽  
Self Renewal ◽  
Lysine Demethylase

Cancer stem-like cells (CSCs) contribute to the high rate of tumor heterogeneity, metastasis, therapeutic resistance, and recurrence. Histone lysine demethylase 4D (KDM4D or JMJD2D) is highly expressed in colon and liver tumors, where it promotes cancer progression; however, the role of JMJD2D in CSCs remains unclear. Here, we show that JMJD2D expression was increased in liver cancer stem-like cells (LCSCs); downregulation of JMJD2D inhibited the self-renewal of LCSCs in vitro and in vivo and inhibited the lung metastasis of LCSCs by reducing the survival and the early lung seeding of circulating LCSCs. Mechanistically, JMJD2D promoted LCSC self-renewal by enhancing the expression of CSC markers EpCAM and Sox9; JMJD2D reduced H3K9me3 levels on the promoters of EpCAM and Sox9 to enhance their transcription via interaction with β-catenin/TCF4 and Notch1 intracellular domain, respectively. Restoration of EpCAM and Sox9 expression in JMJD2D-knockdown liver cancer cells rescued the self-renewal of LCSCs. Pharmacological inhibition of JMJD2D using 5-c-8HQ reduced the self-renewal of LCSCs and liver cancer progression. Collectively, our findings suggest that JMJD2D promotes LCSC self-renewal by enhancing EpCAM and Sox9 expression via Wnt/β-catenin and Notch signaling pathways and is a potential therapeutic target for liver cancer.

In vivo monitoring of JAK/STAT and PI3K/mTOR signal transduction inhibition in pediatric CRLF2-rearranged acute lymphoblastic leukemia (ALL).

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. 9506-9506
Author(s):  
Sarah Kathleen Tasian ◽  
Shannon L. Maude ◽  
Junior Hall ◽  
Tiffaney Vincent ◽  
Charles Grenfell Mullighan ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Ex Vivo ◽  
Lymphoblastic Leukemia ◽  
Nsg Mice ◽  
Signal Transduction Inhibitors ◽  
Background Therapy ◽  
Pediatric All ◽  
Therapeutic Responses

9506 Background: Therapy intensification for children with B-precursor ALL with high-risk genetic lesions has improved relapse-free survival. CRLF2 rearrangements and JAK2 and IL7RA mutations occur in 10-15% of adult and pediatric ALL patients, most of whom relapse. We and others identified aberrant kinase signatures and perturbed JAK/STAT and PI3K/mTOR signal transduction via in vitro studies of CRLF2-rearranged (CRLF2r) ALLs, suggesting the therapeutic relevance of signal transduction inhibitors (STIs). Our creation of CRLF2r ALL xenograft models has enabled rapid preclinical testing of STIs and measurement of in vivo target inhibition. We hypothesized that inhibition of JAK/STAT and PI3K/mTOR phosphosignaling correlates with therapeutic responses in these models. Methods: NOD/SCID/γc-null (NSG) mice well-engrafted with pediatric ALL samples were treated with the JAK inhibitor ruxolitinib, the mTOR inhibitor sirolimus, or vehicle for 72 hours (for signaling response) or 4 weeks (for therapeutic response). Splenocytes were briefly stimulated ex vivo with thymic stromal lymphopoietin (ligand for CRLF2) and stained with human-specific surface and intracellular phosphoantibodies for multi-parameter phosphoflow cytometry analysis. Results: Ruxolitinib-induced inhibition of phospho (p)-JAK2 and pSTAT5 was most pronounced in non-CRLF2r ALLs with novel JAK2-activating BCR-JAK2 and IL7RA/LNK mutations. Sirolimus potently inhibited pS6 and other PI3K/mTOR pathway phosphoproteins in the CRLF2r r ALLs. PSTAT5 and pS6 inhibition correlated with longer-term ruxolitinib- and sirolimus-induced decreases in ALL cell burden, demonstrating therapeutic responses to STIs. Conclusions: Ruxolitinib inhibited JAK/STAT phosphosignaling and markedly decreased leukemic burden in the JAK2-activating BCR-JAK2 and IL7RA/LNK mutant ALL xenografts. Sirolimus potently inhibited PI3K/mTOR (as well as some JAK/STAT) phosphosignaling and had greater therapeutic efficacy than ruxolitinib in the CRLF2r ALLs. The safety of ruxolitinib and of temsirolimus with cytotoxic chemotherapy are currently being established in Children’s Oncology Group Phase I trials.

NOTCH1 Signaling Defines a Leukemia Initiating Cell Population in T-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1507-1507
Author(s):  
Wenxue Ma ◽  
Kristen M. Smith ◽  
Alejandro Gutierrez ◽  
Heather S. Leu ◽  
Qingfei Jiang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Notch Signaling ◽  
Lymphoblastic Leukemia ◽  
Therapeutic Resistance ◽  
Self Renewal ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Notch1 Receptor

Abstract Abstract 1507 Leukemia initiating cells (LIC) contribute to therapeutic resistance as a result of their capacity to accumulate mutations in pathways, such as the NOTCH1 receptor signaling pathway, that promote self-renewal and survival within specific niches. Activating mutations in NOTCH1 occur commonly in T cell acute lymphoblastic leukemia (T-ALL) and have been implicated in driving therapeutic resistance. However, the role of NOTCH1 activation in human T-ALL LIC propagation and LIC sensitivity to selective NOTCH1 receptor inhibition has not been examined. The difficulties in maintaining primary cultures of leukemia cells have hampered investigations into the biology of T-ALL LIC and underscore the need for a direct transplantation model to characterize human LIC in vivo and as a paradigm for screening candidate drugs that inhibit self-renewal pathways active in T-ALL LIC. Pediatric T-ALL serially transplantable LIC were found to be enriched in the CD34+CD4− and CD34+CD7− fractions of newly diagnosed patient samples. More recently, a CD7+CD1a− glucocorticoid resistant LIC population, capable of engrafting leukemia in NOD/SCID IL2Rƒn gamma null (NSG) mice, was identified in primary adult T-ALL without an in vitro expansion. In this study, we identified and molecularly characterized potential LIC populations in pediatric T-ALL without preceding in vitro culture and examined the role of NOTCH1 activation in LIC propagation. To further define the T-ALL LIC, CD34+CD2+CD7+ or CD34+CD2+CD7− cells were isolated from T-ALL primary patients' blood by FACS sorting and transplanted into neonatal RAG2−/− gamma chain−/− mice to determine their leukemic engraftment potential. Limiting dilution experiments were performed with cells from six T-ALL patient samples. Mice transplanted with CD34+CD2+CD7+ or CD34+CD2+CD7− cells developed a T-ALL-like disease characterized by pale bone marrow and enlarged spleen, thymus and liver. Hematopoietic organs were analyzed by flow cytometry and showed engraftment of bone marrow, spleen, thymus and liver. Furthermore, the disease could be serially transplanted. LIC were uniquely susceptible to targeted inhibition in vivo with a therapeutic human NOTCH1 negative regulatory region selective monoclonal antibody (mAb) while normal human hematopoietic progenitors were spared thereby highlighting the cell type and context specific effects of NOTCH signaling. Both the NOTCH1 mAb treatment and lentiviral shRNA knockdown of NOTCH1 reduced NOTCH1, HES1 and c-MYC transcript levels, underscoring the selectivity of NOTCH1 mAb inhibition of NOTCH signaling. These results demonstrate that CD34+CD2+CD7+ and CD34+CD2+CD7− subpopulations are enriched for LIC activity in pediatric T-ALL. Moreover, inhibition of NOTCH signaling by either mAb or shRNA-mediated Notch1 knockdown might be another strategy to target the LIC in T-ALL. Disclosures: No relevant conflicts of interest to declare.

Self-Renewal and Differentiation in Hematopoietic Stem and Progenitor Cells Is Controlled By the APC/C Coactivator Cdh1

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2370-2370
Author(s):  
Daniel Ewerth ◽  
Stefanie Kreutmair ◽  
Birgit Kügelgen ◽  
Dagmar Wider ◽  
Julia Felthaus ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Research Funding ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Nsg Mice ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells

Abstract Introduction: Hematopoietic stem and progenitor cells (HSPCs) represent the lifelong source of all blood cells and continuously renew the hematopoietic system by differentiation into mature blood cells. The process of differentiation is predominantly initiated in G1 phase of the cell cycle when stem cells leave their quiescent state. During G1 the anaphase-promoting complex or cyclosome (APC/C) associated with the coactivator Cdh1 is highly active and marks proteins for proteasomal degradation to regulate proliferation. In addition, Cdh1 has been shown to control terminal differentiation in neurons, muscle cells or osteoblasts. Here we show that Cdh1 is also a critical regulator of human HSPC differentiation and self-renewal. Methods: Human CD34+ cells were collected from peripheral blood (PB) of G-CSF mobilized donors and cultured in the presence of different cytokine combinations. To analyze cell division and self-renewal versus differentiation, CFSE staining was used in combination with flow cytometric detection of CD34 expression. The knockdown and overexpression of Cdh1 was achieved by lentiviral delivery of suitable vectors into target cells. After cell sorting transduced (GFP+) CD34+ cells were used for in vitro differentiation in liquid culture or CFU assay. For in vivo experiments purified cells were transplanted into NSG mice. Results: G-CSF mobilized CD34+ cells showed effective differentiation into granulocytes (SCF, G-CSF), erythrocytes (SCF, EPO) or extended self-renewal (SCF, TPO, Flt3-L) when stimulated in vitro. The differentiation was characterized by a fast downregulation of Cdh1 on protein level, while Cdh1 remained expressed under self-renewal conditions. A detailed analysis of different subsets, both in vitro and in vivo, showed high Cdh1 level in CD34+ cells and low expression in myeloid cells. Analysis of proliferation revealed lowest division rates during self-renewal, accompanied by higher frequency of CD34+ cells. The fastest proliferation was found after induction of erythropoiesis. These experiments also showed a more rapid decrease of HSPCs' colony-forming ability and of CD34+ cells during granulopoiesis after 2-3 cell divisions in contrast to a moderate decline under self-renewal conditions. The depletion of Cdh1 (Cdh1-kd) had no effect on total cell numbers or proliferation detected by CFSE during differentiation and self-renewal, but showed an increase in S phase cells. These results were confirmed at the single cell level by measuring the cell cycle length of individual cells. Independent of cell cycle regulation, Cdh1-kd cells showed a significant maintenance of CD34+ cells under self-renewal conditions and during erythropoiesis with lower frequency of Glycophorin A+ cells. In CFU assays, the Cdh1-kd resulted in less primary colony formation, notably CFU-GM and BFU-E, but significantly more secondary colonies compared to control cells. These results suggest that the majority of cells reside in a more undifferentiated state due to Cdh1-kd. The overexpression of Cdh1 showed reversed results with less S phase cells and tendency to increased differentiation in liquid culture and CFU assays. To further validate our results in vivo, we have established a NSG xenotransplant mouse model. Human CD34+ cells depleted of Cdh1 engrafted to a much higher degree in the murine BM 8 and 12 weeks after injection as shown by higher frequencies of human CD45+ cells. Moreover, we also found an increased frequency of human CD19+ B cells after transplantation of CD34+ Cdh1-kd cells. These results suggest an enhanced in vivo repopulation capacity of human CD34+ HSCs in NSG mice when Cdh1 is depleted. Preliminary data in murine hematopoiesis support our hypothesis showing enhanced PB chimerism upon Cdh1-kd. Looking for a mediator of these effects, we found the Cdh1 target protein TRRAP, a cofactor of many HAT complexes, increased upon Cdh1-kd under self-renewal conditions. We use currently RT-qPCR to determine, if this is caused by a transcriptional or post-translational mechanism. Conclusions: Loss of the APC/C coactivator Cdh1 supports self-renewal of CD34+ cells, represses erythropoiesis in vitro and facilitates engraftment capacity and B cell development of human HSPCs in vivo. This work was supported by Josè Carreras Leukemia Foundation grant DCJLS R10/14 (to ME+RW) Disclosures Ewerth: Josè Carreras Leukemia Foundation: Research Funding. Wäsch:German Cancer Aid: Research Funding; Comprehensiv Cancer Center Freiburg: Research Funding; Janssen-Cilag: Research Funding; MSD: Research Funding.

scholarly journals Repression of kit Expression by Plzf in Germ Cells

2007 ◽  
Vol 27 (19) ◽  
pp. 6770-6781 ◽  
Author(s):  
Doria Filipponi ◽  
Robin M. Hobbs ◽  
Sergio Ottolenghi ◽  
Pellegrino Rossi ◽  
Emmanuele A. Jannini ◽  
...  
Keyword(s):  
Germ Cells ◽  
Transcriptional Repression ◽  
Promoter Region ◽  
Spermatogonial Stem Cell ◽  
Stem Cell Population ◽  
Male Mice ◽  
Self Renewal ◽  
Expression Of Genes

ABSTRACT Male mice lacking expression of Plzf, a DNA sequence-specific transcriptional repressor, show progressive germ cell depletion due to exhaustion of the spermatogonial stem cell population. This is likely due to the deregulated expression of genes controlling the switch between spermatogonial self-renewal and differentiation. Here we show that Plzf directly represses the transcription of kit, a hallmark of spermatogonial differentiation. Plzf represses both endogenous kit expression and expression of a reporter gene under the control of the kit promoter region. A discrete sequence of the kit promoter, required for Plzf-mediated kit transcriptional repression, is bound by Plzf both in vivo and in vitro. A 3-bp mutation in this Plzf binding site abolishes the responsiveness of the kit promoter to Plzf repression. A significant increase in kit expression is also found in the undifferentiated spermatogonia isolated from Plzf −/− mice. Thus, we suggest that one mechanism by which Plzf maintains the pool of spermatogonial stem cells is through a direct repression of kit expression.

scholarly journals Stem cell factor enhances the survival but not the self-renewal of murine hematopoietic long-term repopulating cells

Blood ◽  
1994 ◽  
Vol 84 (2) ◽  
pp. 408-414 ◽  
Author(s):  
CL Li ◽  
GR Johnson
Keyword(s):  
Stem Cell ◽  
Stem Cell Factor ◽  
Cultured Cells ◽  
Stem Cell Differentiation ◽  
The Self ◽  
Isolated Cells ◽  
Self Renewal

The effects of stem cell factor (SCF) have been tested on a murine bone marrow subpopulation (RH123lo, Lin-, Ly6A/E+) that is highly enriched for long-term hematopoietic repopulating cells. SCF maintained cells from this population with long-term repopulating ability for up to 10 days in vitro. However, compared with freshly isolated cells, the level of engraftment in vivo by the cultured cells declined during the in vitro culture period, suggesting that SCF alone was unable to stimulate the self-renewal of long-term repopulating cells. By direct visualization of cultures, only small numbers of cells survived and rarely underwent cell division. However, SCF did directly stimulate proliferation of a population (Rh123med/hi,Lin-,Ly6A/E+) enriched for short-term repopulating cells. These data suggest that stem cell differentiation is associated with the development of mitogenic activity by SCF at least in some progenitor cell populations.

Targeting the Leukemia Initiating Cells in a Mouse T-ALL Model.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3474-3474
Author(s):  
Jessica Tatarek ◽  
Kathleen Cullion ◽  
Michelle Kelliher
Keyword(s):  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Fold Increase ◽  
Thymocyte Development ◽  
Double Positive ◽  
Self Renewal ◽  
Oncogenic Pathways ◽  
Single Positive

Abstract Abstract 3474 Poster Board III-411 T cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy that is largely caused by aberrant activation of the TAL1/SCL, LMO1/2 and NOTCH1 oncogenic pathways. Although most patients respond to cytotoxic therapy, 20-30% relapse and currently we cannot distinguish children likely to relapse from those who will respond to therapy. Evidence is emerging that some malignancies are driven by a rare initiating population that is capable of extensive self-renewal and is resistant to conventional chemotherapy. Although the leukemia-initiating cells (L-ICs) have been well documented in AML and CML, it is unclear whether T-ALL is driven by a rare L-IC and whether relapse reflects an inability to eliminate L-ICs. To determine whether T-ALL is driven by a rare L-IC, we utilized a murine model of T-ALL in which expression of the Tal1 and Lmo2 oncogenes arrests thymocyte development via E47/HEB inhibition and 75% of tumors develop spontaneous gain of function mutations in Notch1. We have shown that treatment with γ-secretase inhibitors (GSIs) to inhibit Notch1 activity induces apoptosis of mouse T-ALL cells in vitro and when administered in vivo extends the survival of leukemic mice. We demonstrate that clonal mouse T-ALL tumors are phenotypically heterogeneous, containing immature CD4- and CD8-negative, double negative (DN) 3 and 4 thymic progenitors and differentiated double positive (DP) and/or single positive (SP) leukemic blasts. Importantly, the DN3 or DN4 progenitors are maintained upon serial transplantation of the tumor into syngeneic recipient mice. Injection of serial dilutions of murine T-ALL cells reveal that the tumors are also functionally heterogeneous; with 1/5000-1/50,000 tumor cells exhibiting leukemia initiating activity. We found the CD44-, CD25+ DN3 progenitors enriched in disease potential, whereas mice injected with DP leukemic blasts failed to develop leukemia. Consistently, our preleukemic studies reveal a 3.5-fold increase in the percentage of Notch1 active, DN3/4 thymic progenitors, raising the possibility that Notch1 drives L-IC growth. Collectively, these studies suggest that activation of the Notch1-c-Myc pathway may confer self-renewal capabilities on committed thymic progenitors. The effects of Notch inhibitors on mouse L-IC survival and activity will be discussed. Disclosures: No relevant conflicts of interest to declare.

Methylation and Silencing of miRNA-124 In Hematopoietic Cells by EVI1

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2476-2476
Author(s):  
Vitalyi Senyuk ◽  
Kavitha Premanand ◽  
Peng Xu ◽  
Francesca Cattaneo ◽  
Jerome Dickstein ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Hematopoietic Cells ◽  
Cyclin D3 ◽  
Self Renewal ◽  
Stem Loop ◽  
Cpg Dinucleotides

Abstract Abstract 2476 The inappropriate expression of EVI1 is associated with aggressive myelodysplastic syndromes (MDS) and acute myeloid leukemia. We previously showed that in vivo EVI1 causes an MDS-like disease in mice and that in vitro it alters cell cycling, self-renewal, and differentiation pathways of primary bone marrow (BM) cells. It is likely that these alterations contribute to the oncogenic potential of EVI1. MicroRNAs (miRNA) are small noncoding RNAs that are 21 to 25 nt in length that up- and down-regulate gene expression during cell development, proliferation, differentiation, and apoptosis. MiRNAs play a critical role in solid and hematopoietic cancers. We found that in hematopoietic cells the expression of several miRNAs is significantly altered by EVI1. Among them, miRNA-124 is strongly downregulated in EVI1-expressing primary BM and 32Dcl3 cells. To evaluate the role of miRNA-124 in primary murine BM cells, we isolated Lin- cells and infected the cells with the empty vector, EVI1, or both EVI1 and miRNA-124. We confirmed that EVI1 alters cellular pathways regulating self-renewal, S1 phase entry, and differentiation. More importantly, however, we found that when miRNA-124 is co-expressed with EVI1, the expression of proteins such as Cyclin D3 and BMI-1, that control some of these pathways, is normalized. These results suggest that the effects of EVI1 are mediated by miRNA-124 repression. We hypothesized that the down-regulation of miRNA-124 could be associated with inappropriate DNA methylation of the miRNA. Both in man and mouse, there are three known alleles of miRNA-124. In the mouse, the alleles are located on chromosome bands 2H4, 3A1, and 14D1. Analysis of the three alleles indicates that miRNA-124-3 harbors the larger number of CpG dinucleotides, including 24 CpG within a stretch of 190 bp upstream of and overlapping the stem-loop start-site. By using the bisulfite DNA sequencing method, we found that in BM cells the number of methylated CpG dinucleotides in this region is significantly increased after EVI1 expression. The DNA methylation is mostly centered in two clusters, which we named cluster I and cluster II. These data clearly show that miRNA-124 is a critical target of EVI1 in human diseases. Disclosures: No relevant conflicts of interest to declare.

Targeting of a Novel MNK-eIF4E-b-Catenin Axis in Blast Crisis Chronic Myelogenous Leukemia Inhibits Leukemia Stem Cell Function

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 963-963 ◽  
Author(s):  
Sharon Lim ◽  
Tzuen Yih Saw ◽  
Sandy Chang ◽  
Min Zhang ◽  
Matthew Robert Janes ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Function ◽  
Nuclear Translocation ◽  
Blast Crisis ◽  
Mrna Translation ◽  
Self Renewal ◽  
Nsg Mice ◽  
Cd34 Cells

Abstract Abstract 963 Blast crisis (BC) chronic myeloid leukemia (CML) is characterized by expansion of a granulocyte macrophage progenitor-like population (GMPs) that has acquired self-renewal capacity, a feature not seen in normal or chronic phase (CP) GMPs. The ability to self-renew is thought to be mediated by b-catenin activation, and may contribute to disease persistence, as well as act as a reservoir for resistance. The mechanisms contributing to b-catenin activation remain obscure, and will need to be identified to improve the control of BC. In this study, we investigated the role of the mRNA translation machinery in mediating b-catenin-mediated self-renewal, since our prior work had implicated aberrant mRNA translation in drug-resistance and BC pathophysiology (Ly et al. Cancer Research 2003; Prabhu et al. Oncogene, 2007; Zhang et al. MCB, 2008). Using immunofluorescence (IF), we first confirmed that BC GMPs have activated nuclear b-catenin compared to GMPs isolated from normal cord blood, and that this was associated with increased eIF4E expression and phosphorylation at Ser209. Next, using biochemical and genetic approaches in CML cell lines (K562 and KCL22), we demonstrated that eIF4E overexpression was sufficient to increase b-catenin activity (as measured by IF for nuclear b-catenin, b-catenin reporter assays, and expression of b-catenin-regulated genes). By expressing phospho-mutant forms of eIF4E (S209A, S209D), we also found that the increase in b-catenin transcriptional activity is dependent on phosphorylation of at Ser209. In line with these observations, siRNA-mediated knockdown or pharmacologic (CGP57380) inhibition of the MNK1/2 kinases (which mediate in vivo eIF4E phosphorylation) prevented the increased b-catenin activity induced by eIF4E overexpression. Mechanistically, we found that eIF4E activated b-catenin signaling via a two-step mechanism. First, eIF4E overexpression increased total cell b-catenin. Second, eIF4E phosphorylation facilitated b-catenin nuclear translocation. The latter step was associated with increased b-catenin phosphorylation at Ser552, a site known to be involved in nuclear translocation, and directly regulated by AKT. Consistent with this model, siRNA-mediated knockdown or small molecule inhibition of AKT (AKT Inhibitor IV) prevented eIF4E-mediated increases in b-catenin transcriptional activity. The importance of eIF4E phosphorylation on b-catenin activation and the self-renewal capacity of primary BC GMPs cells was assessed next. First, we showed that treatment with CGP57380, but not imatinib or dasatinib, inhibited eIF4E phosphorylation, as well as prevented accumulation of active nuclear b-catenin in BC GMPs. Next, we evaluated the effect of MNK1/2 inhibition on the stem cell function of BC cells using both in vitro and in vivo assays. In an in vitro serial replating assay, we showed that CGP57380 impaired the ability of CD34+ BC cells (including those carrying T315I mutation), but not normal CD34+ cells, to serially replate for more than 8 weeks in methylcellulose. Interestingly, treatment with either imatinib or dasatinib only partially impaired the ability of BC CML to serially replate. Next, we found that in vitro treatment of BC CD34+ CML cells, but not normal cord blood CD34+ cells, with CGP57380 retarded their ability to engraft NSG mice. Finally, we developed an in vivo serial transplantation assay for assessing the leukemia stem cell (LSC) function of patient-derived BC GMPs. Here, we injected either BC GMPs or BC CD34+ CML cells intrafemorally into 8- to 10-week old sublethally irradiated NSG mice. Following engraftment, mice were treated with vehicle, CGP53780 (40 mg/kg/d), or dasatinib (5mg/kg/d) for three consecutive weeks. Following treatment, human CD34+ cells were isolated from the mice, and transplanted into a second recipient mouse. At 16 weeks, we found that in vivo treatment with CGP57380, but not dasatinib, prevented BC cells from serially transplanting NSG mice. In summary, our results demonstrate that: 1. eIF4E is overexpressed and phosphorylated at Ser209 in BC, but not normal, GMPs; 2. eIF4E phosphorylation activates b-catenin signalling in BC GMPs; 3. MNK inhibition prevents eIF4E phosphorylation and b-catenin signalling in BC GMPs; and 4. MNK inhibition prevents BC GMPs from functioning as LSCs. Our studies suggest that pharmacologic inhibition of the MNK1/2 kinases may be therapeutically useful in BC CML. Disclosures: No relevant conflicts of interest to declare.

Drug Screening of Primary Human Pediatric Pre-B Cell Acute Lymphoblastic Leukemia (pre-B ALL) Cells in Their Stromal Niche

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4295-4295
Author(s):  
Jae-Hung Shieh ◽  
Tsann-Long Su ◽  
Jason Shieh ◽  
Malcolm A.S. Moore
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
Culture System ◽  
Lymphoblastic Leukemia ◽  
Nsg Mice ◽  
Cd34 Cells ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Abstract 4295 Pre-B cell acute lymphoblastic leukemia (pre-B ALL) is the most common leukemia in children and is treatable. However, no in vitro nor in vivo models are available to investigate their pathophysiology other than a number of established cell lines that grow in the absence of any cytokine dependence or stromal interaction. We developed a serum-free MS-5 cell (a murine bone marrow stromal cell line) co-culture system that is capable of expanding human primary pre-B ALL CD34+CD19+ cells in vitro. To define a population of pre-B ALL initiating cells, our study reveals that a sorted CD34bright population displays a slow proliferation and maintains a high % of CD34+ cells. In contrast, CD34dim cells/CD34− cells fraction shows a higher proliferation but expanded cells lost CD34 antigens. A group of alkylating molecules (BO-1055, -1090, 1099, -1393 and -1509) was evaluated for proliferation of the pre-B ALL CD34+ cells, the pre-B ALL CD34− cells, human mesenchymal stem cells (hMSC), murine MSC (MS-5 cells and Op9 cells), human bone marrow derived endothelial cells (BMEC), and human cord blood (CB) CD34+ cells, as well as for a week 5 cobblestones area forming (CAFC) assay with CB CD34+ cells. BO-1055 molecule efficiently suppressed the growth of pre-B ALL CD34+ cells (IC50 = 0.29 μM) and CD34− cells (IC50 = 0.31 μM). In contrast, IC50 of BMEC, MSC, CB CD34+ cells and CAFC are >10, >25, 8, and >5 μM, respectively. Pre-B ALL cells expressing green fluorescent protein (GFP) and luciferase (GFP-Lu-pre-B ALL) were created, and a xenograft of the GFP-Lu-pre-B ALL cells to NOD/SCID IL2R gamma null (NSG) mice was established. The in vivo effect of BO-1055 to the GFP-Lu-pre-B ALL cells in NSG mice is under investigation. Our stromal culture system supports primary pre-B ALL cells and closely recapitulates the growth of primary human pre-B ALL cells in their niche in vivo. Based on this co-culture system, we identified BO-1055 as a potential therapeutic agent with an excellent toxicity window between pre-B ALL cells and normal tissues including BMEC, MSC and hematopoietic progenitor/stem cells. The in vitro stromal co-culture system combined with the xenograft model of GFP-Lu-pre-B ALL cells provides an efficient and powerful method to screen new drugs for pre-B ALL therapy. Disclosures: No relevant conflicts of interest to declare.

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