scholarly journals The Cell Surface NADH Oxidase ENOX2 Is Highly Expressed in Chronic Myeloid Leukemia (CML): a Redox Protein As a Secreted Surrogate Biomarker and Druggable Target

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3059-3059
Author(s):  
Seda Baykal ◽  
Maud Voldoire ◽  
Christophe Desterke ◽  
Nathalie Sorel ◽  
Hyacinthe Atchroue Johnson Ansah ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Pattern Matching ◽  
Nicotinamide Adenine Dinucleotide ◽  
Drug Target ◽  
Chronic Phase ◽  
P Value ◽  
Healthy Donors ◽  
Cd34 Cells ◽  
Nadh Ratio

Abstract Ecto-nicotinamide Adenine Dinucleotide Oxidase 2 (ENOX2) or Tumor-Associated Nicotinamide Adenine Dinucleotide Oxidase (tNOX), plays a major role as a membrane hydroxyquinone oxidase catalyzing the conversion of reduced NADH to the oxidized NAD+ .Cellular NAD+/NADH ratio plays a major role in the regulation of several metabolic pathways such cell cycle and apoptosis. The membrane NAD+/NADH ratio has been shown to be involved membrane lipid and sphingomyelin metabolism. Several lines of experimental data indicate that ENOX2, as compared to ENOX1 isoform, is preferentially expressed in cancer cells and ENOX2 is also secreted in the serum of patients with cancer and representing a surrogate marker for cancer progression as well as a drug target. The expression of ENOX2 in CML has not been studied so far. To this end, we have analyzed the expression of ENOX2 by Western blots in 40 patients with CML at diagnosis and compared this expression to that of control cells from healthy donors (n= 30). ENOX2 expression was found to be highly increased ( x 6.6 fold) in primary leukemic cells and this was highly significant ( p= 0.0081). To determine if ENOX2 expression is related directly to BCR-ABL expression, we have analyzed the expression of ENOX2 in UT7 cell line and its BCR-ABL-expressing counterparts UT7-11. As compared to parental UT7, ENOX2 expression was highly increased in UT7 cells expressing either native or T315I-mutated BCR-ABL. This increase was also documented in DOX- inducible cell line BaF/p210 sin1.55 in which activation of BCR-ABL expression correlated with ENOX2 expression. The expression of ENOX2 in CML cells was a tyrosine kinase-dependent event as demonstrated by Western blot experiments showing that ENOX2 expression was reduced UT7/11 cell line treated with Imatinib mesylate (1 microM) for 6, 18 and 24 hours whereas there was no change in ENOX2 expression in similarly treated parental UT7 cell line. As ENOX2 is a protein secreted from tumor cells, we have analyzed the levels of ENOX2 in the plasma of CML patients at diagnosis as compared to controls. A series of 41 patients with CML as compared to plasma from 28 healthy donors were analyzed by ELISA. This analysis showed a highly significant increase of ENOX2 protein levels in the plasma of patients with CML (mean levels of 800 pg/ml in CML versus 500 pg/ml, Mann-Whitney U-Test, p < 0.0001). There was no correlation between ENOX2 levels and leukocyte numbers at diagnosis. In order to determine the potential clinical value of ENOX2 expression in different phases of the disease, ENOX2 expression in CML CD34+ cells was compared to healthy donor samples in microarray dataset GSE 4170. CML patients in chronic phase overexpressed ENOX2 in CD34+ cells as compared to control (two-sided test with Welch correction p-value=0.00054). Gene expression pattern matching correlating ENOX2 in CML CD34+ cells was determined in three phases of the disease. Pavlidis template matching algorithm used with ENOX2 as predictor allowed to discover 301 genes correlated with CML in chronic phase. Unsupervised principal component analysis performed with ENOX2 pattern matching gene expression profile allowed us to discriminate the 3 phases of CML in CD34+ cell compartment in a highly significant manner (p-value 6.75E-15). Functional enrichment performed with ENOX2 pattern matching on Gene Ontology Biological Process database revealed implication of different pathways of cell signaling such as: Rho GTPase, MAPKs, GPCR, RAS and NOTCH pathways, the latter being connected to stem cell biology. This analysis also showed implication of metabolic functions such as carbohydrate homeostasis. Other functionalities that could act on hematopoiesis have been also highlighted by this analysis such as proliferation, integrin-mediated signaling, and circadian rhythm. Finally genes related to angiogenesis have been also found to be implicated in ENOX2 signalling such as placental growth factor (PLGF) also EPHB3 which is a receptor tyrosine kinase implicated in cell migration. Overall these results suggest that ENOX2 pathway plays a major role in the pathogenesis of CML and represents, to the best of our knowledge, the first surrogate secreted tumor marker in CML. ENOX2 expression correlates with disease progression and experiments are underway to determine the use of ENOX2 as a drug target in CML and T315I-mutated CML stem cells. Figure Figure. Disclosures No relevant conflicts of interest to declare.

FLT3 Mediated MAPK Activation Participates in the Control of Megakaryopoiesis in Primary Myelofibrosis

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1532-1532
Author(s):  
Christophe Desterke ◽  
Hans Hasselbalch ◽  
Dominique Bordessoule ◽  
Heinz Gisslinger ◽  
Alessandro Vannucchi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cultures ◽  
Mononuclear Cells ◽  
Mapk Pathway ◽  
Primary Myelofibrosis ◽  
Mapk Activation ◽  
Specific Chemical ◽  
Mutational Status ◽  
Healthy Donors ◽  
Cd34 Cells

Abstract Myeloproliferation, myelofibrosis, osteosclerosis and neo-angiogenesis are the major intrinsic pathophysiological features of Primary Myelofibrosis (PMF). The myeloproliferation is characterized by an increased number of circulating CD34+ cells with the prominent amplification of “dystrophic” megakaryocytes (MK) through to be responsible for myelofibrosis thought fibrogenic factor release. Comparison of CD34+ and MK cell gene expression profiling between PMF patients and healthy donors revealed a global deregulation of the MAPK pathway genes. This alteration is associated with a modulation of the FLT3 tyrosine kinase gene expression in CD34+ and MK cells from patients, independently of the JAK2V617F mutation presence. Quantification of the FLT3 transcript in mononuclear cells from patients with Polycythemia Vera and Essential Thrombocythemia showed that this over expression is mainly observed in JAK2WT PMF patients. This is associated with a higher proportion of FLT3+CD34+CD41+ cells in the blood of patients. Analysis of FLT3 membrane expression in MK-derived CD34+ cultures revealed that its expression was maintained all along MK differentiation in patients in contrast to healthy donors. Such a higher expression of FLT3 is associated with an increased concentration of its ligand in the platelet rich plasma from patients, independently of their JAK2 mutational status. The role of FLT3 in the regulation of hematopoiesis incited us to analyse whether its alteration could take part in the myeloproliferation and dysmegakaryopoiesis that characterizes PMF. A flow cytometry analysis of FLT3-downstream MAPK activation in PMF CD34+ cells showed a hyperphosphorylation of p38 and JNK as compared to CD34+ cells from normal blood. This phosphorylation was maintained in PMF MK-derived CD34+ cells at day 10. Addition of PD98059, a MAPK inhibitor, induced a dose dependent restoration of the in vitro megakaryopoiesis in PMF as shown by an increase in MK ploidy with apparition of 32N cells associated with a mature cytological aspect and an increase in CD41, CD42a and CD9 MK differentiation marker expression. PD98059 also increased the MK clonogenicity of CD34+ cells from all patients tested (5/5) as compared to healthy donors. Preliminary results using a specific chemical inhibitor of FLT3 in MK-derived CD34+ cell cultures reinforced the involvement of FLT3 in PMF MK differentiation. In presence of FLT3 ligand, the FLT3 mediated MAPK hyperphosphorylation in PMF MK cultures (D6) is reversed by either PD98059 or UO126, another ERK inhibitor and is accompanied by a slight increase in proliferative MK. This effect is not observed in MK cultures from normal CD34+ cells. Surprisingly, ligation of FLT3 by a monoclonal anti-FLT3 antibody in CD34+ cell cultures resulted in an increase MK proliferation. In conclusion, this work shows a deregulation of FLT3 and MAPK pathway in the PMF CD34+ cells and suggests that the persistence of the FLT3 mediated MAPK activation participates in the dysmegakaryopoiesis of PMF patients.

A Transcriptomal Profile for Predicting Complete Cytogenetic Response (CCR) in Chronic Phase CML Patients Treated with Imatinib.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1007-1007 ◽  
Author(s):  
Shannon K. McWeeney ◽  
Lucy C. Pemberton ◽  
Chris A. Harrington ◽  
Brian J. Druker ◽  
Michael W. Deininger
Keyword(s):  
Gene Expression ◽  
Functional Annotation ◽  
Chronic Phase ◽  
Cytogenetic Response ◽  
Data Sets ◽  
Data Set ◽  
Independent Validation ◽  
Cd34 Cells ◽  
Learning Set ◽  
Validation Set

Abstract Background: Patients with chronic phase CML who achieve a complete cytogenetic response (CCR) have a low risk of disease progression. Since patients unlikely to achieve CCR may benefit from more aggressive therapy up-front, prediction of response prior to therapy would be useful. Our previous work has demonstrated that the gene expression profiles of unselected leukemia cells from prospective cytogenetic responders and non-responders are similar (Crossman et al. Haematologica90(4):459–64, 2005). Based on the hypothesis that cytogenetic refractoriness may be a property of leukemic progenitor rather than differentiated cells, we explored gene expression profiling of CD34+ cells as a tool for predicting CCR. Methods: Two independent data sets were generated. The first data set (learning set) was based on patients with CML who had either achieved a CCR within 1 year of imatinib therapy (R, n = 24), or remained at least 65% Ph+ (NR, n = 12). The prospectively collected, completely independent validation data set was based on 23 additional subjects using the same criteria (17 R and 6 NR). For the learning set CD34+ cells were isolated by FACS from bone marrow or peripheral mononuclear cells cryopreserved prior to imatinib therapy. For the validation set CD34+ were isolated from fresh cells using immunomagnetic columns. RNA was extracted and samples with ≥5ng of high quality total RNA were amplified and labelled with the Affymetrix two cycle cDNA synthesis and IVT labeling protocol using <20ng input RNA; 10μg of labelled target cRNA were hybridized to Affymetrix HG-U133 Plus 2.0 GeneChip® arrays. For both data sets, low-level analysis was done using Robust MultiArray Average (RMA) and gene-by-gene ANOVA was performed to determine differential expression between NR and R. Both statistical significance and effect size used to filter the ANOVA results. Parameters for the classification algorithms were chosen by nested cross-validation procedures. The classifier generated from the first study was then applied to the blinded validation set. Functional annotation clustering and over-representation analysis was also performed. Results: On the validation set, the classifier had an estimated accuracy rate of 85.7%. Based on transcript annotation analysis, there is significant over-representation of trancripts related to cell cycle, complement and coagulation, and apoptosis, among others. A functional cluster was identified related to regulation of transcription. Transcription factors up-regulated in cells from patients with subsequent cytogenetic refractoriness included ZNF168, MEIS1, KLF2, NFIB, MAF, FOSB and EGR1. In contrast, we found down-regulation of SERPINA1, THBD and PLAUR, a sub-network in the complement and coagulation cascades pathway that is associated with cell adhesion and migration. Conclusions: The transcriptional profiles of CD34+ cell from prospective cytogenetic responders and non-responders are distinct. A classifier based on the learning set predicted cytogenetic response in a totally independent validation set with ∼86% accuracy, making this one of the first prospectively validated classifiers. Examination of functional annotation for the transcripts in the classifier identified several functional clusters that are highly correlated with respect to direction of response (e.g. transcription factors) and may drive the biology of cytogenetic refractoriness.

Transcriptional Changes Induced by Imatinib and Nilotinib in the Chronic Myelogenous Leukemia (CML) Cell Line K562.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4540-4540
Author(s):  
Frank Neumann ◽  
Daniela C. Bruennert ◽  
Anne-Marie Koch ◽  
Ingmar Bruns ◽  
Norbert Gattermann ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Tyrosine Kinase ◽  
Cell Line ◽  
K562 Cells ◽  
Differentially Expressed ◽  
Cd34 Cells ◽  
Number Of Genes ◽  
In Vitro Treatment

Abstract Introduction: Nilotinib is a selective bcr-abl tyrosine kinase inhibitor that is 30-fold more potent than Imatinib in vitro. To examine the molecular and functional effects of Nilotinib and Imatinib we performed gene expression and functional analyses in K562 cells following in vitro treatment with the two tyrosine kinase inhibitors. Particular emphasis was put on 1539 genes which we found to be differentially expressed in primary CD34+ cells from patients with CML in first chronic phase in comparison to CD34+ cells from normal bone marrow (Diaz-Blanco et al., Leukemia 2006). Methods: Affymetrix U133A 2.0 microarrays covering 21.722 probe sets were used to analyse the gene expression profile of 5x107 K562 cells after 24h in vitro treatment with Imatinib (0.5 μM) or Nilotinib (0.05 μM) (half maximal inhibitory concentration, IC 50). FISH analysis confirmed the K562 cell line to be BCR-ABL positive. Gene expression data of the treated cells were compared with the data of untreated cells. In addition, proliferation (Cell Titer 96 AQueous One Solution Cell Proliferation Assay, Promega), apoptosis (Cell Death Detection ELISAPLUS, Roche) and cell cycle (FITC BrdU Flow Kit, BD Pharmingen) assays were performed. A colony assay was performed to see differences in cell growth. Results: Looking at those 1539 differentially expressed genes in K562 cells which distinguish patients with CML from healthy donors, we found that Imatinib led to a significant downregulation of 187 and upregulation of 45 genes. In general, Nilotinib had a more pronounced effect than Imatinib regarding the number of genes affected and the degree of suppression. It caused downregulation of 418 and upregulation of 41 genes. Of note, genes affected by Nilotinib included all genes altered by Imatinib such as those related to bcr-abl signalling (Lyn, BCL2, Myc, PIK3CB, G3BP2). Downregulation of genes involved in cell cycle (CDK2, ORC5L, MCM3, POLE2, CCNG1) was only observed following Nilotinib exposure. The stronger effect of Nilotinib is in line with the results of cell cycle experiments showing that Nilotinib exposed cells had the lowest proportion of actively cycling cells. The proportion of apoptotic K562 cells was 5.5 fold greater following treatment with Nilotinib in comparison to Imatinib after 24 hours. Treatment with either Imatinib or Nilotinib produced a similar apoptotic rate and similar decrease in cell numbers after 96 hours. In the colony forming assay, the controls (K562 cells incubated with DMSO only) displayed strong leukemic growth which was inhibited by both Nilotinib and Imatinib, allowing only small clusters to appear. Conclusion: Nilotinib is apparently more potent than Imatinib with regard to the number of genes downregulated and the degree of their suppression. Many of the suppressed genes are associated with bcr-abl signalling and cell cycle.

Imatinib Induced Molecular Response in CD34+ Hematopoietic Stem and Progenitor Cells of Patients with Chronic Myeloid Leukemia Following the First Week of Therapy

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3231-3231
Author(s):  
Daniela Bruennert ◽  
Akos Czibere ◽  
Ingmar Bruns ◽  
Sabrina Pechtel ◽  
Norbert Gattermann ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Chronic Phase ◽  
Expression Data ◽  
Newly Diagnosed ◽  
Hematopoietic Stem ◽  
Log Reduction ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells ◽  
Ph Chromosome

Abstract Imatinib is a selective bcr-abl tyrosine-kinase inhibitor, successfully used as standard therapy for patients with chronic myelogenous leukemia (CML) in chronic phase. We examined the effect of Imatinib on the gene expression transcriptome pattern of CD34 positive hematopoietic stem and progenitor cells of 6 patients with newly diagnosed CML following the first week of therapy. Affymetrix U133A 2.0 microarrays covering 21.722 probe sets were used to analyze and compare the gene expression profiles of CD34+ cells from peripheral blood of 6 patients with newly diagnosed CML in chronic phase before and 7 days after the initiation of Imatinib first line therapy. One patient was female and 5 male. Their median age was 61 years (range 32–79 years). In addition, quantitative molecular monitoring of bcr-abl was performed using Light Cycler technology. Using a supervised hierarchical cluster analysis of the gene expression data we found that 303 genes were differentially expressed with a lower bound of at least 1.2 fold (−2.58 to −1.2 and 1.2 to 2.09). Of these, 183 genes were down-regulated and 120 genes were up-regulated comparing day 1 with day 7. A considerable number of the down-regulated genes are involved in the regulation of cell cycle (CDC2, CDC20, CCNB1, CCNB2) and DNA replication (TOP2A, POLE2, MCM-complex). In contrast, genes coding for proteins governing adhesion and cell migration were up-regulated (CD44, L-Selectin). The observations of the gene expression data of the aforementioned genes could be confirmed by quantitative real-time PCR. After 10 months of Imatinib therapy (range 6–13 months) all 6 patients reached in median a 3 log reduction (range 1.5–3 log reduction) of their bcr-abl mRNA. Bcr-abl FISH analysis confirmed that the majority of CD34+ cells examined were Ph-Chromosome positive. These data indicate that in the early phase of treatment Imatinib mainly acts as an inhibitor of cell cycle and replication which results in a decreased proliferation. The silencing of the Ph-Chromosome positive cells is associated with an up-regulation of genes coding for adhesion during the first week of therapy.

Aberrant Megakaryocyte Gene Expression Contributes to Primary Myelofibrosis.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2867-2867
Author(s):  
Laure Gilles ◽  
Christy Finke ◽  
Terra L Lasho ◽  
Animesh Pardanani ◽  
Ayalew Tefferi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Clinical Trial ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Primary Myelofibrosis ◽  
Gene Set Enrichment Analysis ◽  
Gene Expression Program ◽  
Healthy Donors ◽  
Cd34 Cells ◽  
Expression Program

Abstract Abstract 2867 Primary myelofibrosis (PMF) is a clonal hematologic malignancy, which results from the transformation of a pluripotent hematopoietic progenitor cell. A major consequence of this transformation is increased hematopoiesis and an overproduction of abnormal blood cells. PMF is associated with bone marrow fibrosis, extramedullary hematopoiesis, increased numbers of circulating CD34+ cells, splenomegaly, and a propensity to evolve to AML. Patients also display anemia and thrombocytopenia and harbor abnormal, immature megakaryocytes (Mks) in their bone marrow and spleen. PMF patients can present well known mutations including JAK2V617F (65%), MPL (10%), TET2 (17%), CBL (6%), IDH (4%,), which are not specific to the disease and are also present in polycythemia vera, essential thrombocythemia and AML. We hypothesize that the genetic events associated with PMF, including MPL and JAK2 mutations, contribute to defects in Mk maturation, but that additional changes are needed to explain the striking abnormalities seen in PMF relative to the other myeloproliferative diseases. Although there have been studies to examine the aberrant gene expression program of CD34+ cells of PMF patients, we chose to examine the changes that occur in gene expression specifically in Mks as a way to better understand their abnormal differentiation and to determine their contribution to the disease. Primary CD34+ cells from PMF patients and healthy donors were cultivated in serum free media supplemented with recombinant TPO, BSA, liposomes, insulin and transferrin to support the growth of Mks. After 10 days of differentiation, we evaluated the cultures for proliferation, apoptosis and differentiation by flow cytometry. We found that PMF specimens gave rise to a lower percentage of mature (CD41+CD42+) cells as compared to healthy donors, but showed, a lower ploidy level, a greater proliferation and increased survival. These observations are consistent with the clinical observations that PMF bone marrow is characterized by an increased number of immature, dysplastic Mks. We used flow cytometry to collect two populations of cells for analysis: immature CD41+CD42− Mks, and CD41+CD42+ mature MKs. After sorting, we extracted RNA and performed whole genome microarray analysis with Illumina Human HT12-v4 arrays on cohorts of PMF and control specimens. Gene expression data were analyzed by GeneSpring and Gene Set Enrichment Analysis (GSEA). We found that the CD41+CD42− MKs derived from PMF progenitors showed reduced expression of GATA1 as compared to control cells, as expected based on previous study by Dr. Alessandro Vannuchi. GeneSpring analysis revealed that myeloid transcription factors, including CEBPa, GFI1, and SPI1 (PU.1), which are not expressed in normal MKs, are strikingly and significantly overexpressed in PMF samples. Moreover, c-myb, which regulates the erythroid/Mk cell fate decision, FOG-1 and AML1, are also overexpressed in PMF Mks. This aberrant myeloid gene expression program in PMF Mks is reminiscent of a similar defect we observed in Mks with reduced expression of GATA-1 and GATA-2. We predict that reduced levels of GATA-1 protein in PMF Mks, as reported by Dr. Alessandro Vannucchi and colleagues, is in part responsible for the aberrant growth and differentiation of the PMF Mks. Our data support the model that PMF Mks are defective in their ability to properly regulate expression of hematopoietic regulators. Further analysis by GSEA revealed that hematopoietic and cytokine pathways are among those that are highly enriched in PMF Mks. We recently reported that the molecules dimethylfasudil (diMF) and MLN9237 are able to selectively increase ploidy, Mk surface marker expression, and apoptosis of malignant Mks. We treated Mks derived from PMF progenitor cells with diMF and observed a high increase in polyploidization accompanied with a reduction of Mks proliferation. Thus, diMF is able to partially restore Mk differentiation of PMF cells, supporting the testing of polyploidy inducers in myelofibrosis patients. Disclosures: Pardanani: Sanofi-Aventis: Clinical trial support Other; YM BioSciences: Clinical trial support, Clinical trial support Other; Bristol-Myers Squibb: Clinical trial support, Clinical trial support Other.

Interferon α Has Varied Effects On CD34+ Cells From Patients With Polycythemia Vera

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2840-2840
Author(s):  
Min Lu ◽  
Seungyeul Yoo ◽  
Lijuan Xia ◽  
Xiaoli Wang ◽  
Yan Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Immune Response ◽  
Colony Formation ◽  
Cellular Response ◽  
P Value ◽  
Type I ◽  
Signature Genes ◽  
Cd34 Cells ◽  
Ifn Γ

Abstract Prolonged therapy with pegylated interferon a (Peg-IFNα 2a) leads to hematological and complete molecular remissions in 70% and 17% of patients with polycythemia vera (PV) , respectively (Quintas-Cardama et al, Blood 2013). We have previously shown that PV CD34+ cells are more responsive to Peg-IFNα 2a than normal CD34+ cell. The type I IFN receptor 1 and 2 were shown to be expressed by a greater number of by PV CD34+ cells than normal(N) CD34+ cells (p=0.01 and p=0.002, respectively). The effects of Peg-IFNα 2a on PV hematopoietic stem cells(HSCs) was next evaluated by incubating PV CD34+ cell for 7 days with Peg-IFNα 2a (200ng/ml) followed by their transplantation into NSG mice. The degree of human cell chimerism following the transplantation of MPN CD 34+ cells was reduced by 50 -90% and the JAK2V617F allele burden by 40 -80%. Treatment of N CD34+ cells with Peg-IFNα 2a reduced donor chimerism by only 20%. We next examined the effects of increasing doses of Peg-IFNα 2a on CD34+ cells from 11 PV patients and 5 N controls. In 4 out of 10 PV cases the IC50 of Peg-IFNα 2a was less than 200ng/ml while in the remainder of cases the IC50 was greater. Low doses of IFNa were capable of eliminating JAK2V617F+ hematopoietic colonies in these IFNα sensitive patients while higher doses of IFNα were required to achieve the same effect in the other patients. PV and N CD34+ cells were then profiled using Illumina Gene expression arrays. In total, 32 intensity data files were generated, each containing 47,231 features, corresponding to 12,388 unique genes. At p-value <0.05 386 genes were down-regulated in PV; these genes were enriched for biological processes related to immune response including the IFN-γ-mediated signaling pathway (p=0.0002), the response to IFN-gamma (p=0.004), and the cellular response to IFN-γ (p=0.0004). The 715 up-regulated genes in PV were enriched for pathways involving glycolysis (p=9.4×10-05), cellular response to stress (p=0.006), and catabolic processes. The gene expression patterns of CD34+ cells incubated with and without INFα were next analyzed. At pairwise t-test p-value <0.001, 315 genes were differentially expressed (223 up-regulated and 92 down-regulated by INFα). Up-regulated genes were enriched for INFα functions and immune response including: response to type I IFN (p=9.0×10-49), innate immune response (2.6×10-45), response to virus (7.5×10-40). Among the 223 up-regulated genes, half were previously known as IFN regulated genes (IRGs). The individual response (IR) of genes to IFN was then defined as: IRi=log (exp ressioni @IFN/exp ressioni@control) IR patterns were remarkably consistent within N samples while large inter-patient variations were observed within the PV samples. Significantly positive IRs were observed for 75 genes and negative IRs for 117 genes within PV as compared to N samples (p value<0.01). The 75 positively responsive genes to IFNa overlapped with 16 down-regulated PV signature genes (p=1.1×10-10) while the negatively responsive of genes overlapped with 41 up-regulated PV signature genes (p=2.2×10-24).These data indicate that the action of IFNa is associated with the alteration of the expression of specific PV signature genes. The varied inhibitory effect of Peg-IFNα 2a on PV colony formation was then correlated with the IR of individual genes. The IRs of OAS2 and RPS24 showed particularly high variance and were related to colony formation. OAS2 (2'-5'-oligoadenylate synthetase 2) is an INF-induced, dsRNA-activated antiviral enzyme which plays a critical role in cellular innate antiviral response but also influences apoptosis, cell growth, differentiation and gene regulation. The IR of this gene was directly related to the inhibitory actions of IFNa (p=0.0011). By contrast, the IR of RPS24 (40S ribosomal protein S24), was inversely correlated to the IFNα response (p=0.0038). Mutations in RPS24 are associated with Diamond-Blackfan anemia. The strong correlation between the IR of these 2 genes with the inhibitory effects of IFNα suggests that their response ratio might be useful as therapeutic biomarker. These data indicate that the IFNα receptor is up-regulated in PV CD34+ cells and that IFNα treatment eliminates PV stem cells and its sensitivity against individual patient PV HSC/HPC varies. The patterns of differentially expressed genes following IFNα treatment may prove useful in determining its mechanism of action and predicting IFNα patient response. Disclosures: No relevant conflicts of interest to declare.

Molecular profiling of CD34+ cells identifies low expression of CD7, along with high expression of proteinase 3 or elastase, as predictors of longer survival in patients with CML

Blood ◽  
2006 ◽  
Vol 107 (1) ◽  
pp. 205-212 ◽  
Author(s):  
Agnes S. M. Yong ◽  
Richard M. Szydlo ◽  
John M. Goldman ◽  
Jane F. Apperley ◽  
Junia V. Melo
Keyword(s):  
Gene Expression ◽  
Kinase Inhibitors ◽  
Cox Regression ◽  
Fusion Gene ◽  
Chronic Phase ◽  
High Expression ◽  
Proteinase 3 ◽  
Cd34 Cells ◽  
Microarray Screening ◽  
Indolent Disease

Abstract Although most patients with chronic myeloid leukemia (CML) have the same initial molecular abnormality, the BCR-ABL fusion gene, the duration of chronic phase (CP) varies widely. To identify the possible molecular basis of this heterogeneity, we studied CD34+ cells collected at diagnosis from 68 patients with CML-CP. By using oligonucleotide microarray screening, we performed gene-expression profiling on 2 subsets of patients, one comprising patients with an “aggressive disease” who developed blastic transformation (BT) within 3 years of diagnosis (n = 10) and, at the other extreme, patients with an “indolent disease” whose BT occurred 7 or more years from diagnosis (n = 9). This screening revealed 20 genes differentially expressed in patients with aggressive and indolent disease, which were validated by quantitative reverse transcriptase/polymerase chain reaction (Q-RT/PCR). A multivariate Cox regression model identified the combination of low CD7 expression with high expression of proteinase 3 or elastase as associated with longer survival in the complete cohort of 68 patients. This differential pattern of gene expression probably reflects the intrinsic heterogeneity of the disease; if so, assessing expression levels of selected genes at diagnosis may be valuable in predicting duration of survival in patients treated with imatinib and the newer tyrosine kinase inhibitors.

scholarly journals A gene expression signature of CD34+ cells to predict major cytogenetic response in chronic-phase chronic myeloid leukemia patients treated with imatinib

Blood ◽  
2010 ◽  
Vol 115 (2) ◽  
pp. 315-325 ◽  
Author(s):  
Shannon K. McWeeney ◽  
Lucy C. Pemberton ◽  
Marc M. Loriaux ◽  
Kristina Vartanian ◽  
Stephanie G. Willis ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Chronic Phase ◽  
Gene Expression Signature ◽  
Cytogenetic Response ◽  
Expression Array ◽  
Cd34 Cells ◽  
Risk Patients ◽  
Validation Set

Abstract In chronic-phase chronic myeloid leukemia (CML) patients, the lack of a major cytogenetic response (< 36% Ph+ metaphases) to imatinib within 12 months indicates failure and mandates a change of therapy. To identify biomarkers predictive of imatinib failure, we performed gene expression array profiling of CD34+ cells from 2 independent cohorts of imatinib-naive chronic-phase CML patients. The learning set consisted of retrospectively selected patients with a complete cytogenetic response or more than 65% Ph+ metaphases within 12 months of imatinib therapy. Based on analysis of variance P less than .1 and fold difference 1.5 or more, we identified 885 probe sets with differential expression between responders and nonresponders, from which we extracted a 75-probe set minimal signature (classifier) that separated the 2 groups. On application to a prospectively accrued validation set, the classifier correctly predicted 88% of responders and 83% of nonresponders. Bioinformatics analysis and comparison with published studies revealed overlap of classifier genes with CML progression signatures and implicated β-catenin in their regulation, suggesting that chronic-phase CML patients destined to fail imatinib have more advanced disease than evident by morphologic criteria. Our classifier may allow directing more aggressive therapy upfront to the patients most likely to benefit while sparing good-risk patients from unnecessary toxicity.

Differential Sensitivity of Normal and CML-Derived CD34+ Cells to Inhibition of SHP-2 Gene Expression by RNA Interference (RNAi).

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2778-2778
Author(s):  
Michaela Scherr ◽  
Karin Battmer ◽  
Anuhar Chaturvedi ◽  
Beate Schultheis ◽  
Arnold Ganser ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Interference ◽  
Functional Genomics ◽  
Cell Lines ◽  
Mammalian Cells ◽  
Chronic Phase ◽  
Differential Sensitivity ◽  
Shrna Expression ◽  
Gm Csf ◽  
Cd34 Cells

Abstract RNA interference (RNAi) has rapidly evolved into an efficient tool for functional genomics in a variety of organisms. Stable expression of shRNA (short hairpin RNA) driven by pol III promoters upon retro- or lentiviral gene transfer can induce long-term gene silencing in mammalian cells, including human hematopoietic cells. We recently demonstrated that lentivirus mediated anti bcr-abl RNAi can specifically silence bcr-abl gene expression, inhibit oncogene driven cell proliferation, and eradicate leukemic cells depending on the dose of lentivirus-mediated shRNA expression (Scherr et al. Gene Therapy 2004). Since effective depletion requires a threshold of lentiviral integrations into target cell genomes, the risk of insertional mutagenesis may limit the therapeutic value of this approach. We therefore applied lentivirus-mediated RNAi for functional genomics in purified primary normal and CD34+ cells from chronic phase CML patients harvested at initial diagnosis. Several SHP-2 shRNAs were generated according to established rules and were functionally evaluated using a bicistronic reporter system as described earlier. Effective shRNA expression cassettes were subsequently cloned into lentiviral plasmids encoding RFP to track lentiviral transduction. Transduction of K562, U937, NB-4 and TF-1 cells with lentiviral supernatants results in a reduction of SHP-2 mRNA and protein by more than 90 %. Interestingly, anti-SHP-2 shRNA induced almost complete depletion of RFP+ cells in all four cell lines, demonstrating that SHP-2 expression is essential for proliferation and survival in these cells. We next transduced normal and CML-derived CD34+ cells with a puritiy of &gt; 95% with control and anti-SHP-2 lentiviruses, and stimulated methylcellulose cultures of the cells with high (GM-CSF: 20 ng/ml; IL-3: 10 ng/ml) or low (GM-CSF: 0.2 ng/ml; IL-3: 0.1 ng/ml) cytokine concentrations. This assay relies on the fact that colony formation of CML-CFU is mediated by both cytokine receptor and bcr-abl signaling. Therefore differential numbers of transduced, i.e. RFP+ colonies under different cytokine stimulations reflect the role of the RNAi-target in normal or malignant CFU. Whereas anti-SHP-2 RNAi did not reduce the proliferation of normal transduced CFU (n=5), proliferation of CFU from CML patients was specifically reduced between 50 to 85 % under low cytokine concentration (n= 9). These data suggest that primary normal cells are more resistant to inhibition of SHP-2 gene expression than leukemic cell lines and CD34+ cells from CML patients and identify SHP-2 as a potential target for anti bcr-abl therapy.

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