IRS2 Is Dowregulated In Primary MDS Cells and During MDS Erythroid Differentiation

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1886-1886
Author(s):  
Joao Machado-Neto ◽  
Patricia Favaro ◽  
Mariana Lazarini ◽  
Adriana S S Duarte ◽  
Letícia Fröhlich-Archangelo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
High Risk ◽  
Erythroid Differentiation ◽  
Low Risk ◽  
Leukemic Cells ◽  
Granulocytic Differentiation ◽  
Expression Levels ◽  
Cd34 Cells

Abstract Abstract 1886 Myelodysplastic syndromes (MDS) encompass a heterogeneous group of clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis, refractory cytopenia and a tendency to progress towards acute myeloid leukemia (AML). The abnormal differentiation of myeloid and erythroid cells is probably involved in the pathogenesis of MDS. Insulin receptor substrates (IRS) are adaptor proteins that link signaling, from upstream activators, to downstream effectors to modulate normal growth, metabolism, survival and differentiation. IRS2, a member of the IRS family, binds to Insulin Grow Factor 1 receptor (IGF1R) and Erythropoietin receptor (EPOR). It is upregulated and phosphorylated by EPO in normal bone marrow erythroblasts and in UT-7 leukemic cells, as well as in HL60 leukemic cells during granulocytic-differentiation upon DMSO induction. IGF1 signaling is capable of inducing differentiation in several cell types and plays an important role in the regulation of human erythropoiesis. EPO functions primarily as an erythroblast survival factor, and its antiapoptotic actions have been proposed to involve predominantly PI3-kinase and BCL-X pathways. In view of the role of IRS2 in the erythroid and granulocytic differentiation process, we hypothesized that IRS2 might be related to the deficient differentiation of MDS cells and disease progression. The aim of the present study was to characterize the mRNA and protein expression levels of IRS2 in cells of MDS patients and normal donors and to analyze the IRS2 expression levels between low-risk and high-risk of MDS. We also elucidated the expression levels of IRS2 transcripts during erythroid differentiation of CD34+ cells from normal donors and MDS patients. We studied 12 healthy donors and 29 patients with MDS at the time of diagnosis (16 low-risk [RA/RARS] and 13 high-risk [RAEB/RAEBt] according to FAB classification; 15 low-risk [RCUD/RCMD/RARS] and 11 high-risk [RAEB-1/RAEB-2] according to WHO classification; 22 low/INT-1 risk and 7 INT-2/high risk according to IPSS; 25 low risk and 4 intermediate/high risk cytogenetic). RT-PCR was performed in total cell from bone marrow samples for gene expression studies. Protein expression was evaluated by Western blot in bone marrow mononuclear cells from MDS patients or in peripheral blood CD34+ from normal donors. Erythroid-differentiation was performed in CD34+ bone marrow cells from 4 normal donors and 4 MDS patients. IRS2 gene expression was significantly decreased in primary MDS cells compared with normal cells (0.74 [4.06-0.15] vs. 4.71 [11.78-0.66], P<0.0001). Western blot analysis corroborated these findings. According to FAB and WHO classifications, real time RT-PCR demonstrated a significantly lower expression of IRS2 in high-risk MDS samples when compared with low-risk: FAB, 0.34 [0.15–1.56] vs. 1.05 [0.19–4.06], P=0.0172; WHO, 0.30 [0.15–1.44] vs. 1.00 [0.19–4.06], P=0.0204. Based on the IPSS classification and cytogenetic risk group, the expression levels of IRS2 were similar between the low and high-risk groups. During erythroid differentiation, we evaluated IRS2 gene expression of CD34+ cells on days 6, 8 and 12 of culture. On day 12 of normal CD34+ erythroid differentiation, there was an 8.25-fold increased in IRS2 expression, compared to day 6. Interesting, MDS CD34+ cells showed a lower increment in IRS2 transcripts at the same time point (3.89-fold increase only). In conclusion, the down regulation of IRS2 in primary MDS cells and the lower increase in its expression during MDS erythroid differentiation, suggest a role of IRS2 to maintain the effective hematopoiesis. Moreover, IRS2 lower expression in high-risk group, suggests that IRS2 plays a role in the MDS pathophysiology and disease progression. Supported by FAPESP and CNPq. Disclosures: No relevant conflicts of interest to declare.

Let-7a, Mir-17 and Mir-20a Expression Levels in CD34+ Bone Marrow Cells of Patients with Myelodysplastic Syndromes (MDS) Are Associated with Established Prognostic Factors, Supporting Their Implication in the Pathogenesis of the Disease,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3792-3792 ◽  
Author(s):  
Christos K Kontos ◽  
Vassiliki Pappa ◽  
Diamantina Vasilatou ◽  
Maria-Angeliki S Pavlou ◽  
Frida Kontsioti ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Prognostic Factors ◽  
High Risk ◽  
Mirna Expression ◽  
Who Classification ◽  
Low Risk ◽  
Expression Levels ◽  
Cd34 Cells ◽  
Bone Marrow Blasts

Abstract Abstract 3792 Introduction: MicroRNAs are single, small non-coding RNA molecules of approximately 21–26 nucleotides, which regulate the expression of numerous genes. miRNAs may act either at the post-transcriptional or the post-translational level to repress gene expression; still, upregulation of gene expression has been noticed in some cases as a direct effect of miRNA function. The importance of miRNAs in carcinogenesis is emphasized by the association of cancers with alterations in miRNA expression. Many miRNAs, including let-7a and those of the miR-17-92 cluster (miR-17, miR-20a, etc.), have been shown or are predicted to affect the activities of targeted mRNAs encoding proteins that have oncogenic or anti-oncogenic functions. let-7a downregulates KRAS, while miR-17 and miR-20a downregulate E2F1. Both these proteins are overexpressed in myelodysplastic syndromes (MDS) and have been shown to be involved in the pathobiology of the disease. Purpose: In the current study, we examined the prognostic value of let-7a, miR-17 and miR-20a levels in MDS and their potential as novel molecular biomarkers. Furthermore, we investigated the protein expression levels of validated targets of these three miRNAs in bone marrow CD34+ cells of MDS patients. Material and Methods: We evaluated 43 patients with MDS (34 men, 9 women) with a median age of 73 years (range 45–87). According to WHO classification, 12 patients (27.9%) were diagnosed with RA, 6 (13.9%) RCMD, 8 (18.6%) with RAEB-I, 7 (16.3%) with RAEB-II, 8 (18.6%) with AML, and 2 (4.7%) with CMML. According to IPSS, 13 patients (32.5%) had low risk, 14 (35.0%) intermediate I risk, 6 (15.0%) intermediate II, and 7 (17.5%) high risk disease. WPSS classification was: 8 (23.5%) very low risk, 5 (14.7%) low risk, 8 (23.5%) intermediate, 9 (26.5%) high risk, and 4 (11.8%) very high risk. We isolated CD34+ cells from bone marrow mononuclear cells from MDS patients, as well as from peripheral blood of donors of CD34+ cells for stem cell transplantation, using magnetic beads. Extraction of small RNA-containing total RNA from CD34+ cells was performed and cDNA of let-7a, miR-17 and miR-20a was synthesized using specific primers. miRNA expression levels were determined using quantitative real-time PCR, the TaqMan® chemistry and the relative quantification (2−ΔΔCT) method. The snoRNA RNU48 was used as reference gene. Furthermore, total protein was extracted from CD34+ cells using a lysis buffer and subsequently quantified using the Bradford assay. Western blot analysis was carried out for MYC, E2F1, Cyclin D1 (CCND1), BCL2 and KRAS, while Actin was used as reference protein. Results: In MDS patients, let-7a expression levels were 0.053–506.1 copies/RNU48 copies, while miR-17 and miR-20a expression levels were 0.005–2694.5 and 0.003–3116.7 copies/103RNU48 copies, respectively. No significant differences were found between patients and controls regarding let-7a, miR-17 and miR-20a expression. let-7a underexpression was associated with high (>10%) bone marrow blasts percentage (P =0.036), presence of WHO classification subtypes with poor prognosis (RAEB-I, RAEB-II and AML) (P =0.020), and high IPSS (P =0.037). Furthermore, miR-17 underexpression was related to high (>10%) bone marrow blasts percentage (P =0.008), intermediate and/or high risk karyotype (P =0.018) and high IPSS (P =0.016). Moreover, miR-20a underexpression was associated with high IPSS (P =0.037) and WPSS (P =0.013). Interestingly, protein expression levels of all targets analyzed in the current study were shown to be lower in samples overexpressing let-7a, miR-17 and/or miR-20a, in comparison with the corresponding protein levels noticed in specimens showing lower expression of these three miRNAs. Conclusion: To the best of our knowledge, this is the first study showing that expression levels of let-7a, miR-17 and miR-20a are associated with established prognostic factors in MDS, including IPSS and WPSS. Furthermore, these three miRNAs seem to be implicated in the pathogenesis of the disease, most probably by finely tuning the expression of target proteins that are involved in highly important molecular pathways, therefore affecting key cellular functions, such as cell cycle control, apoptosis, cell proliferation, and regulation of gene expression. Undoubtedly, further studies are needed to confirm the present findings and clarify their association with the pathogenesis of different MDS subgroups. Disclosures: No relevant conflicts of interest to declare.

Downregulation of IRS2 in Myelodysplastic Syndrome (MDS) May Be Related to Impaired Cell Differentiation,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3814-3814
Author(s):  
Joao Machado-Neto ◽  
Patricia Favaro ◽  
Mariana Lazarini ◽  
Adriana S S Duarte ◽  
Leticia Fröhlich Archangelo ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Differentiation ◽  
High Risk ◽  
Peripheral Blood ◽  
Erythroid Differentiation ◽  
Hematopoietic Cell ◽  
Low Risk ◽  
Megakaryocytic Differentiation ◽  
Expression Levels ◽  
Cd34 Cells

Abstract Abstract 3814 Myelodysplastic syndromes (MDS) encompass a heterogeneous group of clonal hematopoietic stem cell disorders, characterized by ineffective hematopoiesis, refractory cytopenia and a tendency to progress towards acute myeloid leukemia. Abnormalities in cell differentiation are probably involved in the pathogenesis of MDS. Insulin receptor substrates (IRS) are adaptor proteins that link signaling from upstream activators to downstream effectors to modulate normal growth, metabolism, survival and differentiation. IRS2, a member of the IRS family, binds to Insulin Grow Factor 1 receptor (IGF1R), the Erythropoietin receptor (EPOR) and Thrombopoietin receptor (MPL). Recently, we reported that IRS2 was downregulated in MDS cells compared to normal cells, and in high risk compared to low-risk MDS. Additionally, IRS2 was significantly upregulated during erythroid differentiation of normal CD34+ cells, but not in MDS CD34+ cells. In view of the role of IRS2 in EPO, IGF1 and TPO signaling, and possibly in erythroid, granulocytic, and megakaryocytic differentiation, we hypothesized that IRS2 might be related to the impaired hematopoiesis of MDS cells. The aim of the present study was to correlate the mRNA expression levels of IRS2 in bone marrow from MDS patients with laboratorial and clinical data. We also elucidated the expression levels of IRS2 during megakaryocytic, granulocytic and erythroid differentiation of established leukemia cell line models. We studied 12 healthy donors and 32 patients with MDS at the time of diagnosis (16 low-risk [RA/RARS] and 13 high-risk [RAEB/RAEBt] according to FAB classification). Gene expression was evaluated by Q-PCR in total cells from bone marrow samples and cell lines. IRS2 protein expression and phosphorylation was evaluated by Western blot and immunoprecipitation. K562 cells were induced to megakaryocytic differentiation by treatment with 20nM PMA for 4 days. NB4 cells were induced to granulocytic differentiation by treatment with 10−6 M ATRA for 4 days. KU812 cells were induced to erythroid differentiation by treatment with 50μM HE and 100 μM HU for 4 days. Spearman correlation analysis showed that IRS2 expression demonstrated a significant positive correlation with peripheral blood neutrophils (r =0.43, P =0.01) and platelets counts (r =0.36, P =0.04), and bone marrow granulocytes percentages (r =0.40, P =0.02). In contrast, a significant negative correlation was observed between IRS2 expression with bone marrow erythroblasts (r =-0.360, P =0.04) and myeloid precursor percentages (grouping myeloblast, promyelocytes, myelocytes, metamyelocytes and blasts) (r =-0.60, P <0.01). There was no correlation between IRS2 expression with age, hemoglobin, number of erythrocytes in the peripheral blood, and number of dysplasias in bone marrow. Finally, since IRS2 is involved with TPO, IGF1 and EPO signaling, and IRS2 expression was correlated with platelet, granulocyte and erythroblast numbers in MDS patients, we monitored IRS2 expression during hematopoietic cell differentiation. Interestingly, a significant increase in IRS2 expression and phosphorylation was observed in K562, NB4 and KU812 after megakaryocytic, granulocytic and erythroid differentiation, respectively. In summary, the lower expression of IRS2 in MDS and its upregulation during differentiation may suggest that defective IRS2 expression in MDS contributes to disease progression and impaired hematopoietic cell differentiation, a hallmark of the disease. Disclosures: No relevant conflicts of interest to declare.

Reduced TET2 expression in Patients with Myelodysplastic Syndromes and Acute Myeloid Leukemia.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2822-2822
Author(s):  
Renata Scopim-Ribeiro ◽  
Joao Machado-Neto ◽  
Paula de Melo Campos ◽  
Patricia Favaro ◽  
Adriana S. S. Duarte ◽  
...  
Keyword(s):  
Gene Expression ◽  
Overall Survival ◽  
High Risk ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Erythroid Differentiation ◽  
Low Risk ◽  
High Risk Disease ◽  
Cd34 Cells ◽  
Acute Myeloid

Abstract Abstract 2822 Introduction: Acquired mutations in TET2 and DNMT3A have been found in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), and may predict a worse survival in these diseases. TET2 mutations are considered to be a loss-of-function mutation and results in decreased 5-hydroxymethylcitosine (5-hmc) levels. In normal CD34+ cells, TET2 silencing skews progenitor differentiation towards the granulomonocytic lineage at the expense of lymphoid and erythroid lineages. Dnmt3a participates in the epigenetic silencing of hematopoietic stem cell regulatory genes, enabling efficient differentiation. Here, we attempted to evaluate the expression of TET2 and DNMT3A in total bone marrow cells from normal donors, patients with MDS and AML, and in CD34+ cells from MDS and normal controls during erythroid differentiation. Materials and Methods: The study included normal donors (n = 21), patients with MDS (n = 43) and AML (n = 42) at diagnosis. All normal donors and patients provided informed written consent and the study was approved by the ethics committee of the Institution. MDS patients were stratified into low and high-risk according to WHO classification (RCUD/RCMD/RARS=31 and RAEB1/RAEB2=12). TET2 and DNMT3A mRNA expression was assessed by quantitative PCR. CD34+ cells from normal donors (n = 9) and low-risk MDS patients (n = 7) were submitted to erythroid differentiation. Cells were collected and submitted to immunophenotyping for GPA and CD71 (days 6 and 12) and q-PCR for TET2 and DNMT3A expression (days 6, 8 and 12). Results of gene expression in normal donors and patients are presented as median, minimum-maximum, and were compared using Mann-Whitney test. Student t test was used for comparison of gene expression during CD34+ erythroid diferentiation. Overall survival was defined from the time of sampling to the date of death or last seen. Univariate analysis for overall survival was conducted with the Cox proportional hazards model. Results: TET2 expression was significantly reduced in both AML (0.62; 0.01–32.69) and MDS (1.46; 0.17–21.30) compared to normal donors (2.72; 0.43–31.49); P<0.0001 and P=0.01, respectively. TET2 expression was also significantly reduced in AML compared to MDS (P=0.0007). MDS patients were stratified into low and high-risk disease, and we still observed a significant reduction in TET2 expression in high-risk (0.73, 0.17–7.25) when compared to low-risk (1.58; 0.48–21.30; P=0.02) patients, but no difference was noted between normal donors vs. low-risk MDS, and high-risk MDS vs. AML. In MDS cohort, the median overall survival was 14 months (range 1–83), increased TET2 expression was associated with a longer survival (HR, 0.44; 95% CI, 0.21–0.91, P=0.03), and, as expected, WHO high-risk disease was associated with a shorter survival (HR, 10.16; 95% CI, 3.06–33.72, P<0.001), even though the confidence interval (CI) was large. TET2 expression did not impact survival in our cohort of AML patients. The erythroid differentiation was effective in cells from normal donors and MDS patients, as demonstrated by the flow cytometry analyses of GPA and CD71. TET2 expression was significantly increased on day 12 of erythroid differentiation, P<0.05. On the other hand, DNMT3A expression was similar between normal donors (0.74; 0.22–1.53), MDS (0.78; 0.26–3.46) and AML (0.95, 0.15–6.46), and during erythroid differentiation, with no impact on survival. Conclusion: These data suggest that decreased TET2 expression may participate in leukemogenesis, and supports the participation of TET2 in the erythroid differentiation of MDS. DNMT3A was not differentially expressed in AML and MDS, indicating that the presence of mutations in this gene may be the predominant mechanism of changes in protein function. We thus suggest that decreased TET2 expression may explain the reduced levels of 5-hmc found in TET2 wild type patients, and may become a predictive marker for outcomes in MDS and other myeloid diseases. Further studies would be necessary to better elucidate the clinical relevance and biologic significance of our findings, and whether the decreased TET2 expression results in hypermethylation in these diseases. Disclosures: Maciejewski: NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding.

The New CD95 Ligand Inhibitor APG101 Leads To Decreased Apoptosis and Improved Erythroid Differentiation In Primary CD34+ Cells From Patients With Low Risk Myelodysplastic Syndrome (MDS)

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1562-1562
Author(s):  
Florian Nolte ◽  
Claudia Kunz ◽  
Stephanie Fey ◽  
Julia Obländer ◽  
Susanne Brendel ◽  
...  
Keyword(s):  
Bone Marrow ◽  
High Risk ◽  
Early Stage ◽  
Erythroid Differentiation ◽  
Low Risk ◽  
Cd34 Cells ◽  
Apoptotic Effect ◽  
Risk Patients ◽  
Dose Dependent ◽  
Dose Dependent Effect

Abstract Introduction Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal myeloid malignancies characterized by ineffective hematopoiesis and an increased risk of transformation into acute myeloid leukemia. Particularly early stage MDS are at least in part characterized by an increased apoptosis of myeloid and erythroid progenitors that causes peripheral cytopenia. APG101 is a glycosylated fusion protein consisting of the extracellular domain of human CD95 (Fas receptor) and the Fc domain of human IgG1. APG101 effectively binds to the CD95 ligand (CD95L) expressed on effector cells as well as to functionally active ligand in solution, by that blocking the interaction between CD95 and its ligand. The aim of our study was to evaluate whether APG101 treatment of primary CD34+ reduces the apoptotic rate and improves the differentiation capacity of these cells. Methods Bone marrow cells were obtained during routine bone marrow aspiration after all patients gave their written informed consent. Isolated primary CD34+ cells from 11 MDS patients were cultured in complete supplemented IMDM medium for 6 days with increasing concentrations of APG101 (1 µg/mL, 3 µg/mL, 10 µg/mL, 30 µg/mL, 100 µg/mL, 200 µg/mL, 300 µg/mL). After incubation time, cells were multicolor- stained with the following dye combination: Annexin-FITC + CD235a-PE + CD34-PECy7 + CD71-APC + 7-AAD and analyzed immediately on a flow cytometer (FACSCanto, BD Bioscience, Heidelberg, Germany). Analysis of raw FACS data was done with the FACSDiva software. To analyze the differentiation capacity of CD34+ progenitors, methylcellulose assays were performed in parallel to the aforementioned experiments. However, due to limited cell numbers, colony assays were performed on 9 MDS patients only. Cells were cultured in triplicates with increasing concentrations of APG101 for 14 days. Colonies were counted and the mean number of colonies was determined. Results Treatment of differentiating CD34+ cells with APG101 led to a decreased apoptosis in both CD34+ cells and CD71+ cells, respectively, indicated by decreased Annexin-FITC fluorescence. Interestingly, this effect was particularly seen at low APG101 concentrations (maximum of 10 µg/ml), while the effect was abrogated at higher APG101 concentrations. The anti-apoptotic effect was more pronounced in low risk MDS patients compared to high risk MDS patients. No effect was seen when the CD235a+ fraction of cells was analyzed. With regard to colony formation, an improvement of erythroid differentiation, indicated by an increase in CFU-E, was found in 3 out of 4 low risk patients (less than 5% blasts in the bone marrow). No effect was seen on erythroid differentiation in high risk patients (more than 5% blasts in the bone marrow). Conclusion APG101 shows promising in vitro activity in viable CD34+ cells with regard to inhibition of apoptosis and promotion of differentiation. The observation that the anti-apoptotic effect was more pronounced in low risk MDS patients as compared to high risk MDS patients supports the concept of increased apoptosis particularly in early stage MDS progenitors. Although the numbers in the differentiation experiments are small, we found a promising effect of APG101 on CFU-E formation at lower doses in patients with less than 5% bone marrow blasts. Moreover, the same dose-dependent effect was observed in the apoptosis assays. Since the activation of the CD95 pathway seems not only to be involved in apoptosis induction, but is also required for terminal erythroid differentiation in normal hematopoiesis, this dose-dependent effect might particularly reflect these ambivalent roles of CD95 and its ligand in both MDS and healthy hematopoiesis, repsectively. Disclosures: Kunz: APOGENIX GmbH: Employment. Fricke:APOGENIX GmbH: Employment.

Characterization of gene expression of CD34+ cells from normal and myelodysplastic bone marrow

Blood ◽  
2002 ◽  
Vol 100 (10) ◽  
pp. 3553-3560 ◽  
Author(s):  
Wolf-K. Hofmann ◽  
Sven de Vos ◽  
Martina Komor ◽  
Dieter Hoelzer ◽  
William Wachsman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
High Risk ◽  
Bone Marrow Cells ◽  
Low Risk ◽  
Control Group ◽  
Expression Data ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Marrow Cells

Gene patterns of expression in purified CD34+ bone marrow cells from 7 patients with low-risk myelodysplastic syndrome (MDS) and 4 patients with high-risk MDS were compared with expression data from CD34+ bone marrow cells from 4 healthy control subjects. CD34+ cells were isolated by magnetic cell separation, and high-density oligonucleotide microarray analysis was performed. For confirmation, the expression of selected genes was analyzed by real-time polymerase chain reaction. Class membership prediction analysis selected 11 genes. Using the expression profile of these genes, we were able to discriminate patients with low-risk from patients with high-risk MDS and both patient groups from the control group by hierarchical clustering (Spearman confidence). The power of these 11 genes was verified by applying the algorithm to an unknown test set containing expression data from 8 additional patients with MDS (3 at low risk, 5 at high risk). Patients at low risk could be distinguished from those at high risk by clustering analysis. In low-risk MDS, we found that the retinoic-acid–induced gene (RAI3), the radiation-inducible, immediate-early response gene (IEX1), and the stress-induced phosphoprotein 1 (STIP1) were down-regulated. These data suggest that CD34+cells from patients with low-risk MDS lack defensive proteins, resulting in their susceptibility to cell damage. In summary, we propose that gene expression profiling may have clinical relevance for risk evaluation in MDS at the time of initial diagnosis. Furthermore, this study provides evidence that in MDS, hematopoietic stem cells accumulate defects that prevent normal hematopoiesis.

Myelodysplastic Syndromes Are Characterized by Gene Expression Changes in Hematopoietic Stem Cells and Alterations in Hematopoietic Stem Cell and Myeloid Progenitor Composition.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1762-1762
Author(s):  
Christopher Y. Park ◽  
Wendy W Pang ◽  
Peter L Greenberg ◽  
Irving L. Weissman
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
High Risk ◽  
Low Risk ◽  
Hematopoietic Stem ◽  
Myeloid Progenitor ◽  
Transcriptional Changes ◽  
Equity Ownership ◽  
Myeloid Progenitors

Abstract Abstract 1762 Poster Board I-788 The myelodysplastic syndromes (MDS) represent a heterogeneous group of disorders characterized by peripheral cytopenias due to impaired hematopoietic differentiation. To date, most data characterizing the immature hematopoietic compartment in MDS have relied on evaluation of CD34+ bone marrow cells, which are a heterogeneous population containing a predominance of oligo- and unilineage-potent progenitors and few hematopoietic stem cells (HSC). In this study we show that MDS are disorders of HSC, evidenced by the presence of recurrent cytogenetic alterations, including -5q, -7, and -20q, in highly purified HSC (Lin-CD34+CD38-CD90+CD45RA-) by FISH. Because MDS HSC harbor cytogenetic changes, we sought to better characterize the molecular basis of MDS HSC function by performing whole transcriptome analysis of highly purified HSC and committed myeloid progenitor populations from low-risk (n=8) and high-risk (n=2) MDS patients. When compared to control HSC from healthy patients (n=10), MDS HSC showed broad transcriptional changes. Using the significance analysis of microarrays (SAM) algorithm and Ingenuity Pathways Analysis software, we identified 3,258 differentially expressed genes (FDR < 0.1) with increased expression of genes positively associated with cell growth and proliferation (p < 0.001) and increased expression of inflammatory response genes (p < 0.015). Interestingly, while MDS common myeloid progenitors (CMP, Lin-CD34+CD38+CD123+CD45RA-) showed increased expression of cell death-related genes when compared to normal CMP (p < 0.001), neither MDS HSC nor multipotent progenitors (MPP, Lin-CD34+CD38-CD90-CD45RA-) showed significant differential expression of these genes when compared to their normal counterparts. To assess the cellular and developmental correlates of HSC/committed progenitor transcriptional changes, we evaluated by flow cytometry the frequency of HSC and committed myeloid progenitors in bone marrow aspirates from 35 low-risk MDS, 6 high-risk MDS and 32 healthy patient samples (range 4-84 yo). Low-risk MDS bone marrow samples showed significantly increased numbers of HSC compared to normal bone marrow samples (+3-fold change, p < 0.03). In addition, myeloid progenitor composition was frequently altered in low-risk MDS patients, with decreased percentages of granulocyte-macrophage progenitors (GMP, Lin-CD34+CD38+CD123+CD45RA+) when expressed as a percentage of total myeloid progenitors [including GMP, CMP and megakaryocyte-erythroid progenitors (MEP, Lin-CD34+CD38+CD123loCD45RA-)] (-2.3-fold change, p < 1e-6). This altered myeloid progenitor profile was highly specific to MDS, even when MDS patient samples were compared to a group of control bone marrow samples from non-MDS patients exhibiting at least one cytopenia (n=34, p < 1e-5), allowing for the distinction of MDS samples from non-MDS cytopenias with 0.89 sensitivity and 0.89 specificity. Together, these data indicate that MDS HSC exhibit significantly altered gene expression profiles and suggest that gene expression changes in MDS HSC induce the altered developmental fate decisions and transcriptional changes observed in MDS committed myeloid progenitors. These data also demonstrate that the changes in MDS myeloid progenitor composition may provide a novel, flow cytometric method for distinguishing MDS from other hematologic conditions that mimic MDS. Finally, these studies indicate that molecular characterization of MDS phenotypes may require evaluation of purified hematopoietic progenitors in order to account for the differential effect of MDS-associated changes on specific hematopoietic progenitor populations. Disclosures Weissman: Amgen: Equity Ownership; Cellerant Inc.: Founder; Stem Cells Inc.: Equity Ownership, Founder; U.S. Patent Application 11/528,890 entitled “Methods for Diagnosing and Evaluating Treatment of Blood Disorders.”: Patents & Royalties.

Trans-Cinnamaldehyde Induces Granulocytic Differentiation and Impairs Survival and Migration of Acute Myeloid Leukemic Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5035-5035
Author(s):  
Liqiong Liu ◽  
Ping Wang ◽  
Xingbin Wang ◽  
Junxia Gu ◽  
Xiaoqing Li ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mononuclear Cells ◽  
Leukemic Cells ◽  
Migration And Invasion ◽  
Granulocytic Differentiation ◽  
Hl60 Cells ◽  
Hematopoietic Malignancies ◽  
Cd34 Cells ◽  
Induced Apoptosis ◽  
Acute Myeloid

Abstract Trans-cinnamaldehyde (TCA), the major active component of oil isolated from the stem bark of Cinnamomum cassia traditionally used to treat dyspepsia, gastritis and inflammatory disease world widely, has been shown to inhibit proliferation and promote apoptosis in a number of cancer cells. However, the functional roles TCA plays in hematopoietic system have not been fully investigated. In this study, we show the effects of TCA on acute promyelocytic leukemia (APL) cell line HL60 and primary bone marrow mononuclear cells (BMMNC) as well as bone marrow stromal cells (BMSC) from acute myeloid leukemia (AML) patients. We found that TCA affected HL60 cells in a time-and dose-dependent fashion. Low concentration of TCA (20 μM) arrested HL60 cells at G0/G1 phase at 72 h without significant apoptosis. Middle concentration of TCA (60 μM) accumulated HL60 cells at G2/M at 24 h with increased apoptosis when the treated time was prolonged. Both low and middle concentrations of TCA induced HL60 cells to differentiate toward mature granule cells characterized with up-regulation of CD11b on cells accompanied by decreased c-Myc protein and increased p27 protein. Consistently, the expression and cellular distribution of p16 and Cdc6 were also significantly changed in differentiated HL60 cells treated with TCA. On the other hand, high concentration of TCA (100 μM) rapidly inhibited NF-kappaB activity and induced apoptosis in HL60 cells. Importantly, TCA induced apoptosis of AML CD34+ cells and suppressed colony formation of AML BMMNC, while its cytotoxicity on normal BMMNC was minor. In addition, TCA synergized with AraC to kill AML BMMNC and AML CD34+ cells. Finally, TCA also decreased CXCR4 expression on HL60 cells, consistent with it ability to depress migration and invasion of HL60 cells induced by rhSDF-1α as well as the adhesion of HL60 cells to AML BMSC. Of note, TCA also impaired survival and SDF-1α secretion of AML BMSC, which may further suppress the interaction of HL60 with AML BMSC. Taken together, our data show that TCA is an effective agent for the treatment of hematopoietic malignancies, not only being the direct inducer of terminal differentiation and apoptosis of acute myeloid leukemic cells, but attenuating the protective effect of AML BMSC on leukemia cells via inhibiting SDF-CXCR4 axis, which highlights the potential of TCA to be a promising therapeutic agent for hematopoietic malignancies treatment.

Molecular Characterization of the Leukemic Niche in Chronic Myeloid Leukemia (CML) and Evaluation of a Leukemia / Niche Cross-Talk

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 908-908
Author(s):  
Djamel Aggoune ◽  
Nathalie Sorel ◽  
Sanaa El Marsafy ◽  
Marie Laure Bonnet ◽  
Denis Clay ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
Mrna Expression ◽  
Expression Pattern ◽  
Myeloid Leukemia ◽  
Gene Expression Pattern ◽  
Fold Increase ◽  
Leukemic Cells ◽  
Cd34 Cells

Abstract Abstract 908 There is growing evidence that the bone marrow microenvironment could participate to the progression of chronic myeloid leukemia (CML). Recent data show indeed that placental growth factor (PGF) expression is highly induced in stromal cells from CML patients although they are not part of the leukemic clone as they are Ph1-negative (Schmidt et al, Cancer Cell 2011). It is possible that leukemic cells instruct the niche components via extracellular or contact signals, transforming progressively the “normal niche” into a functionally “abnormal niche” by inducing aberrant gene expression in these cells, similar to the pattern that has been identified in cancer-associated fibroblasts (CAF). In an effort to identify the differential gene expression pattern in the CML niche, we have undertaken two strategies of gene expression profiling using a Taqman Low Density Arrays (TLDA) protocol designed for 93 genes involved in antioxidant pathways (GPX, PRDX, SOD families), stromal cell biology (Collagen, clusterin, FGF, DHH), stem cell self-renewal (Bmi1, MITF, Sox2) and hematopoietic malignancies (c-Kit, hTERT, Dicer, beta-catenin, FOXO3). The first strategy consisted in the analysis of mesenchymal stem cells (MSCs) isolated from the bone marrow of newly diagnosed CP-CML patients (n=11). As a control, we have used MSCs isolated from the bone marrow of age-matched donors (n=3). MSCs were isolated by culturing 6–8.106 bone marrow mononuclear cells in the presence of b-FGF (1 ng/ml). At 2–3 weeks, cells were characterized by the expression of cell surface markers (CD105+, CD90+) and by their potential of differentiation towards osteoblastic, chondrocytic and adipocytic lineages. The second strategy aimed to study the potential instructive influence of leukemic cells in the gene expression program of normal MSC after co-culture with either the UT7 cell line expressing BCR-ABL (3 days) or with CD34+ cells isolated from CP-CML at diagnosis (5 days) as compared to co-culture with cord blood CD34+ cells. After culture, CD45-negative MSC were cell-sorted and analyzed by TLDA. All results were analyzed using the StatMiner software. Results: TLDA analysis of gene expression pattern of MSC from CML patients (n=11) as compared to normal MSCs (n=3) identified 6 genes significantly over-expressed in CML-MSC: PDPN (10-Fold Increase), V-CAM and MITF (∼8 Fold increase), MET, FOXO3 and BMP-1 (∼ 5 Fold increase). To confirm these results we have performed Q-RT-PCR in a cohort of CML-MSC (n= 14, including the 11 patients as analyzed in TLDA) as compared to normal MSC. High levels of PDPN (Podoplanin, ∼8 fold increase), MITF (Microphtalmia Associated Transcription factor, 4-Fold) and VCAM (Vascular Cell Adhesion Protein, 2 fold increase) mRNA were again observed on CML MSCs. Our second strategy (co-culture of normal MSC with BCR-ABL-expressing UT7) revealed an increase of IL-8 and TNFR mRNA expression in co-cultured MSCs (∼5-fold ) whereas there was a major decrease in the expression of DHH (∼ 25-fold) upon contact with BCR-ABL-expressing cells. No modification of the expression of PDPN, MITF or VCAM was noted in normal MSC after this 3-day co-culture strategy using UT7-BCR-ABL cells. Current experiments are underway to determine if primary CD34+ cells from CML patients at diagnosis could induce a specific gene expression pattern in normal MSC after 5 days of co-culture. PDPN is a glycoprotein involved in cell migration and adhesion, acting downstream of SRC. It has been shown to promote tumor formation and progression in solid tumor models and is highly expressed in CAFs. MITF is a bHLH transcription factor involved in the survival of melanocyte stem cells and metastatic melanoma. Finally, high VCAM1 mRNA expression by MSCs from CML patients could be involved in increased angiogenesis known to be present on CML microenvironment. In conclusion, our results demonstrate an abnormal expression pattern of 3 important genes (PDPN, MITF and VCAM1) in MSC isolated in CP-CML patients at diagnosis. The mechanisms leading to an increased mRNA expression (instructive or not instructive by leukemic cells) and their relevance to CML biology are under evaluation. Our results, confirming previous data, suggest strongly the existence of a molecular cross-talk between leukemic cells and the leukemic niche. The elucidation of such aberrant pathways in the microenvironment could lead to the development of “niche-targeted” therapies in CML. Disclosures: Turhan: Novartis, Bristol Myers Squibb: Honoraria, Research Funding.

Identification of Defects in the Transcriptional Program during Differentiation of CD34+Cells Selected from Patients with Both, Low- and High-Risk Myelodysplastic Syndromes.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 201-201 ◽  
Author(s):  
Martina Komor ◽  
Saskia Gueller ◽  
Sven de Vos ◽  
Oliver G. Ottmann ◽  
Dieter Hoelzer ◽  
...  
Keyword(s):  
Gene Expression ◽  
High Risk ◽  
Stress Responses ◽  
Low Risk ◽  
Transcriptional Analysis ◽  
Transcriptional Program ◽  
Hematopoietic Stem ◽  
Specific Differentiation ◽  
Cd34 Cells ◽  
Transcriptional Pattern

Abstract The development of MDS is suggested to follow a multistep pathogenesis and is characterized by accumulation of molecular defects of the hematopoietic stem/progenitor cells. To detect alterations within the transcriptional program in MDS derived CD34+ cells during lineage-specific differentiation, CD34+ bone marrow cells were selected from healthy individuals (n=3) and patients with low-risk (IPSS, n=3) or high-risk (n=4) MDS and stimulated in vitro with EPO, TPO or G/GM-CSF to induce lineage-specific differentiation. Lineage-determined cells were harvested and if necessary purified by immunomagnetic beads at days 4, 7 and 11 for gene expression profiling. Gene expression was analyzed by oligonucleotide microarrays (HG-U133A, Affymetrix, Santa Clara, CA). The experiments were done in triplicates for each of the time points and each of the conditions. First, we identified 260 genes with a significant expression pattern associated with normal lineage-specific differentiation. These continuously up- or down-regulated genes are considered to be part of a specific genetic program of normal hematopoietic cells during lineage-specific differentiation. In MDS, 57% of these genes showed a different expression from the normal transcriptional pattern. Thirteen of 24 genes up-regulated during normal erythropoiesis were opponently expressed in MDS containing putative new erythro-specific genes like two GTPase activator proteins, RAP1GA1 and ARHGAP8, which regulate small Rho GTPases. Fourteen of 22 continuously up-regulated genes during normal granulopoietic development displayed a significantly different expression in MDS containing the putative candidate desmocollin 2, a gene which is involved in intercellular cell-adhesion. Delta-like 1 (DLK1) is known to be overexpressed in stem cells from patients with myelodysplastic syndrome. The role of DLK1 in normal hematopoiesis is still not defined. We found DLK1 with increasing expression during normal megakaryopoiesis but reverse expression during megakaryopoiesis in MDS. Interestingly, in erythropoiesis from both, high- and low risk MDS we found overexpression of Bladder cancer overexpressed (BLOV1) and Apoptosis inhibitor 5 (API5, which acts as a cellular survival factor by inhibiting apoptosis after growth factor withdrawal). These genes are not expressed in normal erythropoiesis. Furthermore, we identified the gene for a novel v-maf-like protein F, MafF-like (v-maf: musculoaponeurotic fibrosarcoma oncogene homolog F) to be significantly downregulated exclusively in low-risk MDS. MafF belongs to a basic leucine-zipper(bZIP)-transcription factor family normally involved in multiple physiological processes including hematopoiesis and stress responses. Our data provide the first comprehensive transcriptional analysis of differentiating human CD34+ cells derived from normal individuals compared to MDS. It gives new insights to understand the alteration of differentiation and proliferation of MDS derived CD34+ cells. In particular, the study could identify the gene encoding for the MafF-like protein that acts as a transcriptional regulator of normal hematopoiesis to be significantly down-regulated in low-risk MDS.

Erythropoiesis Is Highly Stimulated in CD34+ Cells in Low-Risk Myelodysplastic Syndromes (MDS) with an Improper Mitochondrial Function.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 473-473
Author(s):  
Ramin Tehranchi ◽  
Rosangela Invernizzi ◽  
Boris Zhivotovsky ◽  
Bengt Fadeel ◽  
Ann-Mari Forsblom ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Erythroid Differentiation ◽  
Low Risk ◽  
Refractory Anemia ◽  
Continuous Increase ◽  
Atp Production ◽  
Cd34 Cells ◽  
Significant Difference ◽  
Cyt C

Abstract The apoptosis of early erythroblasts from patients with low-risk MDS, refractory anemia (RA) and RA with ringed sideroblasts (RARS), is mediated through a constitutive cytochrome c (cyt c) release from mitochondria (Tehranchi etal, 2003). Moreover, mature erythroblasts in RARS, but not in RA, show mitochondrial accumulation of aberrant ferritin (MtF) (Cazzola etal, 2003). This study aimed at further describing the pathophysiology of ineffective hematopoiesis in low-risk MDS, by studying cyt c release and MtF expression during erythroid differentiation and mitochondria ATP production in MDS bone marrow cells. We assessed freshly isolated CD34+ cells and day 4–14 erythroblasts from RARS, RA and normal bone marrow (NBM). CD34+ cells from all individuals were negative for MtF. NBM showed only few positive cells (0–4%, d4–14), and RA erythroblasts a median of 3% (0–8%) MtF+ cells. RARS erythroblasts, on the contrary, showed an early increase in MtF+ cells and a continuous increase during the culture period (d4=10%, d7=17%, d14=19%). There was a positive correlation between MtF expression and cyt c at day 14 ( r2=0.8). There was no significant difference in mitochondria ATP production between RARS, RA and NBM (all complexes or cyt -dependent complex IV). We found a significant over-expression (mRNA) of the pro-apoptotic genes for cyt c, Bid and Bax at day 0. Moreover, genes involved in erythroid differentiation were significantly up-regulated in MDS CD34+ cells: 6-fold for GATA-1 and 23-fold for β-globin; p&lt;,0005 for both. GATA-1 and β-globin expression increased during normal erythroid maturation, but in MDS erythroblasts GATA-1 declined and β-globin showed only a weak increase. Comparing RARS with RA, the former showed both higher expression of the β-globin and GATA-1 genes, and a higher degree of cyt c release and MtF expression. This indicates that the cellular abnormalities leading to erythroid apoptosis as well as efforts to compensate for these defects are present at the stem cell level in RARS. G-CSF that reduces cyt c release in MDS erythroblasts (RARS&gt;RA) showed no effect at all on ATP production or cyt c mRNA. Moreover, G-CSF tended to increase MtF expression in some RARS erythroblast cultures, indicating that it allows survival of pro-apoptotic MDS erythroblasts rather than addressing the cause of apoptosis. In conclusion, the aberrant MtF expression of RARS erythroblasts occurs at a very early stage of erythroid differentiation and is paralleled by an up-regulation of genes involved in erythroid differentiation. Alternative mechanisms may be involved in RA pathogenesis, since these cells show cyt c release but only moderate MtF expression, and less gene up-regulation.

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