Aberrant Megakaryocyte Gene Expression Contributes to Primary Myelofibrosis.

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.

Download Full-text

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

Blood ◽

10.1182/blood.v110.11.1532.1532 ◽

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.

Download Full-text

The Tetraspanin CD9 Is Involved in Primary Myelofibrosis Dysmegakaryopoiesis Through c-Myb Regulation and Stroma Interactions,

Blood ◽

10.1182/blood.v118.21.3834.3834 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3834-3834

Author(s):

Christophe Desterke ◽

Costanza Bogani ◽

Lisa Pieri ◽

Alessandro M. Vannucchi ◽

Bernadette Guerton ◽

...

Keyword(s):

Bone Marrow ◽

Cell Migration ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Actin Polymerization ◽

Primary Myelofibrosis ◽

Membrane Expression ◽

Healthy Donors ◽

Cd34 Cells

Abstract Abstract 3834 Introduction: CD9, a four transmembrane glycoprotein belonging to the tetraspanin family, is suggested to regulate cell motility and adhesion and to play a role in megakaryopoiesis. It has been reported to be a molecular marker of primary myelofibrosis (PMF) being characterized by myeloproliferation, dysmegakaryopoiesis, alterated bone marrow/spleen stroma and extramedullary haematopoiesis. CD9 mRNA has been shown to be overexpressed in CD34+ PMF HPs and its membrane expression level was correlated with platelet counts. Our recent data evidencing an alteration of CD9 expression in PMF megakaryocytes (MK) have encouraged us to investigate whether CD9 participates in the dysmegakaryopoiesis and whether it is involved in the dialogue between MK and stromal cells in PMF patients. Patients and Methods: CD34+ cells were MACS selected from the peripheral blood of PMF patients (n=67) and of unmobilized healthy donors (n=61). Functional studies were performed on MK precursor-derived from CD34+ cells cultured in MK medium with ou without monoclonal antibodies (Syb mAb) or siRNAs targeting CD9. CXCL12-induced MK migration was performed in Boyden chambers. Bone marrow mesenchymal stromal cells (MSC) from healthy donors and PMF patients were cultured in DMEM+10%FCS. Results: Our results showed that CD9 membrane expression was altered on CD34+ cells and on MK precursor-derived from PMF CD34+ cells. Binding of CD9 with Syb mAb restored the in vitro megakaryocyte differentiation process that was altered in patients as shown by an increase in: i) megakaryocytic colony formation in semisolid medium, ii) CD41 and CD62p MK differentiation marker and GATA-1 expression, iii) MK cytoplasmic maturation, iv) apoptotic MK number (reduced AKT phosphorylation and Bcl-XL expression and increased percentage of Annexin+ cells). Activation of CD9 was also associated with regulation of MAPK and AKT-GSK3β pathways whose balance is involved in MK differentiation. Treatment of PMF MK precursors by Syb modulated activation of the MAPK pathway as shown by an increased of p38, JNK and GSK3β phosphorylation and of AP-1 mRNA expression. Taking into account the structure of the tetraspanin molecular network, binding with Syb mAb might also impact the effects associated to the multimolecular complex in which CD9 is involved. This prompted us to study the effects of a molecular silencing of CD9 on the PMF MK differentiation. We showed that, in contrast to the Syb mAb, addition of CD9 siRNA to PMF megakaryocytes reduced their transcriptional program including c-Myb, a transcription factor that is involved in CD9 regulation during megakaryopoiesis. Given the role of CD9 in cell migration, we further investigated whether it could be involved in the megakaryocytic precursor migration observed in patients. We showed that silencing CD9 reduced the CXCL12-dependent megakaryocytic precursor migration as well as the CXCR4 and CXCL12 transcription and that this migration involved actin polymerization. c-Myb siRNA restored CXCR4 and CXCL12 expression and reduced actin polymerization suggesting that CD9 was involved, via c-Myb, in the CXCL12-dependent megakaryocytic precursor migration. Effect of CD9 on cell migration is often interpreted as related to modulation of integrins participating in the integrin/tetraspanin network and of their interaction with mesenchymal stromal cells (MSC). We showed that several genes involving the CD9 partner interactome were over-expressed in MSC from PMF bone marrow as compared to MSC from healthy donors. Preliminary results showing that PMF MK precursors display different behaviour in terms of cell survival and adhesion when co-cultured on bone marrow MSC from PMF patients as compared to healthy donors suggest that interactions between MKs and bone marrow MSC is involved in PMF dysmegakaryopoiesis. Addition of Syb reverses these alterations suggesting the participation of CD9 in the abnormal dialogue between MK and MSC. Conclusion: Our results show a deregulation of CD9 expression in megakaryocytes from PMF patients. They also suggest that CD9 i) participates in PMF dysmegakaryopoieis in terms of MK differentiation and survival and ii) is involved in the increased MK precursor migration through alterations of the CXCL12/CXCR4 axis. Our data further support the role of bone marrow stroma in PMF dysmegakaryopoeisis through CD9 interactions. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Flow Cytometry Analysis of Peripheral Blood CD34+ Cells in Patients with Primary Myelofibrosis

Blood ◽

10.1182/blood.v112.11.5240.5240 ◽

2008 ◽

Vol 112 (11) ◽

pp. 5240-5240

Author(s):

Archana M Agarwal ◽

Scott James Samuelson ◽

Sergey Preobrazhensky ◽

Charles J. Parker ◽

Kimberly Hickman ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Peripheral Blood ◽

Primary Myelofibrosis ◽

Peripheral Blood Cd34 ◽

Hla Dr ◽

Cd34 Cells ◽

Erythroid Precursors ◽

Normal Controls ◽

Myeloid Progenitors

Abstract Primary myelofibrosis (PMF) is a clonal chronic myeloproliferative disorder characterized by the accumulation of megakaryocytes in the bone marrow (BM), variable degrees of BM fibrosis, tear-drop erythrocytes, increased numbers of CD34+ hematopoietic progenitors in the peripheral blood (PB), and extramedullary hematopoiesis. Since the antigenic properties of the circulating CD34 cells may yield clues to disease pathogenesis and have not been extensively studied, we used five-color flow cytometry to examine these cells from 20 well characterized patients with PMF and 10 normal controls. Bone marrow biopsies, molecular and cytogenetic studies were also reviewed. As expected, the percentages of peripheral-blood CD34 cells were significantly higher in the PMF patients (mean 1.4%, range, range 0.065–7.15) compared to the controls (mean 0.05%, range 0.01–0.57). The mean fluorescence intensity (MFI) values related to HLA-DR expression were increased (more than 3 fold) on the CD34+ cells in 12/20 (60%) PMF patients relative to normal control levels, while increased levels of CD13 were seen in 5/20 (25%) of PMF patients. CD33 and CD117 expression were similar on the CD34+ cells in both groups. Aberrant expression of lymphoid antigens was observed in 6/20 (30%) with CD7, 6/20 (30%) with CD4, and 3/20 (15%) with CD56 on CD34 positive cells in PMF. In the18 cases also studied with antibodies against CD45RA and CD123, the majority of CD34+ CD38 + cells phenotypically resembled megakaryocyte-erythroid precursors (CD45RA−, CD123−) in 5 cases, common myeloid progenitors (CD45RA−, CD123+) in 12 cases, and granulocyte-macrophage progenitors (CD45RA+, CD123 +) in 1 case. JAK2-V617F mutations were detected in 9 of 20 cases, but were present in only 1 of 5 cases showing predominately megakaryocyte-erythroid precursors. The percentage of CD34+ cells also expressing CXCR4 (CD184) appears to be increased in some patients relative to normal controls in contrast to other reported studies. In conclusion, the peripheral blood CD34+, progenitor cells in PMF patients are heterogeneous phenotypically resembling megakaryocyte-erythroid precursors in approximately 30% of cases, and common myeloid progenitors in approximately 70% of cases. In addition, these cells often show phenotypic abnormalities (increased intensity of HLA-DR and CD13 expression) that can be detected with flow cytometry relative to normal peripheral blood CD34+ cells. Patterns of antigen expression in PMF also appear to differ from those reported for CD34 positive cells in other myeloproliferative disorders which may help in early diagnosis and/or monitoring treatment responses.

Download Full-text

PI3K/AKT Activity and Functional Relevance in Deguelin Induced Apoptosis in MDS Cell Line.

Blood ◽

10.1182/blood.v114.22.4977.4977 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4977-4977

Author(s):

Denise Carvalho Rezende ◽

Lorena Pacheco ◽

Gustavo Loureiro ◽

Elisa Kimura ◽

Laila Silva ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Cell Line ◽

Jurkat Cells ◽

Akt Activation ◽

Constitutive Activation ◽

Healthy Donors ◽

Cd34 Cells ◽

Induced Apoptosis

Abstract Abstract 4977 Introduction PI3K/AKT signalling pathway is involved in cell growth, proliferation and apoptosis. PI3K/AKT constitutive activation is observed in several solid tumors and leukemic cells. Inhibition of PI3K/AKT activity using specific inhibitor, LY294002, results in apoptosis. Deguelin is a natural product isolated from the leguminous, Mundulea sericea, with antitumorigenic effect in vitro and in vivo. The inhibition effect of Deguelin in PI3K/AKT signalling pathway in leukaemia cells was also observed in vitro. Aims We evaluated PI3K/AKT activation using p-AKT expression by flow cytometry in P39 cell line and CD34+ hematopoietic progenitors. PI3K/AKT activity inhibition mediated by deguelin and its proapoptotic effect was tested in P39 cell line. Material and Methods The high-risk MDS cell line P39 and leukaemia cell lines, Jurkat and HL60, were maintained in RPMI1640 and FBS 10%. Jurkat and HL60 were used as positive and negative control for p-AKT expression, respectively. Cell lines and CD34+ cells from bone marrow healthy donors (n=5) were assessed for p-AKT expression. Flow cytometry detection of intracellular Ser 473 p-AKT was performed after permeabilization and fixation with a saponin based solution with Tween20 and FACSlysing solution (Becton Dickinson-BD). An alexa-fluor 488-conjugated rabbit antibody to Ser 473 p-AKT (Cell Signalling Technology) was employed. A double immunostaining procedure using CD45-PerCP and CD34+PE was performed to analyse p-AKT expression in CD34+ cells from bone marrow healthy donors. After incubation, cells were analysed on a FACSCalibur (BD). The p-AKT activity was determined using Kolmogorov-Smirnov test (D). Difference between groups was analyzed using the Mann Whitney test. For treatment and determination of apoptosis, cells were exposed to deguelin (at concentrations of 10-300nM) and LY294002 (15-50uM) for 24-48h. To determine apoptotic changes, cells were stained withy Annexin-V/FITC and propidium iodide and examined on a FACScalibur (BD). Results Jurkat cells showed constitutive PI3K/AKT activation with a mean D value for p-AKT expression of D=0,84 ± 0,02. P39 cells also showed a constitutive PI3K/AKT activation with p-AKT expression as high as in Jurkat cells (mean D= 0,76±0,07). P-AKT expression was significant lower in CD34+cells from health donors (D=0,38±0,06) than in Jurkat and P39, respectively (p=0,0043 and p=0,015). Inhibition of p-AKT was observed using different concentrations of LY294002 (15-50uM) in Jurkat cells and apoptosis was observed after 24 and 48h when cells were treated with 50uM. For P39, although apoptosis was observed, no p-AKT inhibition was detected after LY294002 treatment. Treatment with deguelin induced apoptosis in Jurkat via p-AKT inhibition. No effect on apoptosis or p-AKT expression was observed in P39 cell line after deguelin treatment. Conclusions PI3/AKT constitutive activation was observed in P39 cell line and suggests that PI3/AKT can play a role in MDS progression. Deguelin effect in PI3K/AKT pathway can be cell specific, as observed in Jurkat but not in P39 cell line. Thus, deguelin in combination with other agents should be tested as a potential therapeutic option for MDS via PI3K/AKT inhibition. Disclosures No relevant conflicts of interest to declare.

Download Full-text

High Expression of Carcinoembryonic Antigen-Related Cell Adhesion Molecule(CEACAM) 6 In Primary Myelofibrosis

Blood ◽

10.1182/blood.v116.21.4116.4116 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4116-4116

Author(s):

Hans Carl Hasselbalch ◽

Vibe Skov ◽

Thomas Stauffer Larsen ◽

Mads Thomassen ◽

Caroline Riley ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Cell Adhesion ◽

Adhesion Molecule ◽

Cell Adhesion Molecule ◽

Primary Myelofibrosis ◽

P Value ◽

P Values ◽

Related Cell ◽

Cd34 Cells

Abstract Abstract 4116 Introduction: Carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 6, also known as non-specific cross-reacting antigen (NCA) or CD66c is a glycosylphosphoinositol (GPI)-linked cell surface protein and a member of the CEA family of proteins. Structurally, this protein shares close homology with CEACAM1, CEACAM7 and CEACAM8. Functionally, CEACAM6 has been implicated in cell adhesion, cellular invasiveness and metastatic behaviour of tumour cells. Expression of CEACAM6 protein has been found in a variety of normal human tissues, including myeloid cells. A key pathophysiological feature of primary myelofibrosis (PMF) is changes in the bone marrow micromilieu, progressive accumulation of connective tissue, pronounced neovascularisation and altered stromal cell adhesion. Consequently, CD34+ cells escape the bone marrow and seed extramedullarily. Being involved in cell adhesion, cellular invasiveness, angiogenesis, and inflammation – all key processes in the pathophysiology of PMF – we hypothesized that CEACAM6 might play an important role in these processes in patients with myelofibrosis. We have assessed gene expression of several CEA genes in patients with PMF and related neoplasms in order to elucidate the significance of CEACAM6 and other members of the CEA family of proteins in these disorders. Patients and Methods: Gene expression microarray studies have been performed on whole blood from control subjects (n=21) and patients with ET (n =19), PV (n=41), and PMF (n=9). Gene expression profiles were generated using Affymetrix HG-U133 2.0 Plus microarrays recognizing 54.675 probe sets (38.500 genes). Total RNA was purified from whole-blood and amplified to biotin-labeled aRNA and hybridized to microarray chips. Results: 20.439, 25.307, 17.417, and 25.421 probe sets were identified to be differentially expressed between controls and patients with ET, PV, PMF, and CPMNs as a whole, respectively (false discovery rate (FDR) adjusted p values < 0.05). Several CEACAM-genes were significantly deregulated. In PMF patients, the CEACAM genes 1, 6 and 8 were significantly upregulated with the highest upregulation of CEACAM6 and CEACAM8 (fold change (FC) 12.5 and 14.0, respectively and FDR adjusted p values 7.71 × 10-7 and 1.48 × 10-5, respectively). Only the CEACAM21 gene was significantly downregulated (FC -1.3 and FDR adjusted p-value 4.14 × 10-7) whereas the other CEACAM-genes tested (3, 4, 5, 7, 19) displayed no significant changes as compared to controls. In ET patients, the CEACAM genes 3, 6, and 7 were significantly upregulated (FC 1.2, 1.8, and 1.1, respectively) and FDR adjusted p values < 0.03). The CEACAM21 gene was significantly downregulated (FC -1.3 and FDR adjusted p-value 0.0004). In PV patients, the CEACAM genes 1, 3, 6 and 7 were significantly upregulated (FC 1.7, 1.2, 1.7, and 1.1, respectively) and FDR adjusted p values 0.0002, 0.0009, 0.0002, and 0.03, respectively. The CEACAM21 gene was significantly downregulated (FC -1.3 and FDR adjusted p-value 4.14×10-5) All other CEACAM-genes showed no significant changes in either ET or PV as compared to controls.When comparing controls with non-PMF-patients, a significant upregulation of the CEACAM genes 1, 3, 6, and 7 were recorded in non-PMF patients (FC 1.5, 1.2, 1.7, and 1.1, respectively; FDR adjusted p values 0.001, 0.0002, 0.002, and 0.02, respectively). The CEACAM19 and CEACAM21 genes were significantly downregulated (FC - 1.1 and -1.4, respectively; adjusted p-values 0.008 and 3.46 × 10-8). Discussion and Conclusions: Using global gene expression profiling, we have found a pronounced deregulation of CEACAM genes, involving highly significant upregulation of the CEACAM genes 6 and 8 in PMF (FCs 12.0 and 14.0, respectively). Upregulation of CEACAM6 was seen in both ET, PV and PMF by far the highest levels being recorded in PMF-patients. Of note, significant upregulation of CEACAM8 (FC 14) was only seen in patients with myelofibrosis. The elevated expression of CEACAMs genes in ET, PV, and PMF may solely reflect neutrophil activation being most exaggerated in patients with PMF in whom the highest CEACAM6 and 8 expression patterns were recorded. Alternatively, the highly elevated gene expression of CEACAM6 and 8 in PMF are molecular markers of clonal expansion and myelofibrotic transformation, implying enhanced proteolytic activity and egress of CD34+ cells into the circulation. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Dynamics of Bcl-2, Bax and ACE Expression In CD34+ Cells of Peripheral Blood and Bone Marrow In Patients with Acute Leukemia During Induction Therapy

Blood ◽

10.1182/blood.v116.21.4840.4840 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4840-4840

Author(s):

Elena E. Khodunova ◽

Elena N. Parovichnikova ◽

Irina V. Galtzeva ◽

Sergey M. Kulikov ◽

Valentin G. Isaev ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Drug Resistance ◽

Acute Leukemia ◽

Induction Therapy ◽

Peripheral Blood ◽

Acute Leukemias ◽

Healthy Donors ◽

Cd34 Cells ◽

Blast Cells

Abstract Abstract 4840 The causes of drug resistance in acute leukemias (AL) have been studied very intensively and the key research was done on Bcl-2 family proteins. Last studies have showed that high level Bcl-2 expression in acute leukemia is really associated with drug resistance andpoor prognosis [Haematologica 2007, U. Testa]. It was demonstrated that lower Bax/Bcl-2 ratio (<0,3) was associated with FAB M0-M1 classes (p=.00001), poor-risk cytogenetics and poor prognosis [Blood 2003, G. Poeta]. But there were no studies on the dynamic evaluation of Bcl2 and Bax expression on CD34+ cells during chemotherapy. Renin-angiotensin system and angiotensin concertin enzyme (ACE) influence on leukogenesis is extensively investigated. It was reported that ACE expression on blast cells is high [Leuk Lymphoma 2006, S. Aksu]. Recent publications indicate that primitive hematopoietic precursors have different characteristics regarding ACE: CD34+ACE+cells transplanted into NOD/SCID mice contribute 10-fold higher numbers of multilineage blood cells than their CD34+ACE- counterparts and contain a significantly higher incidence of SCID-repopulating cells than the unfractionated CD34+ population [Blood 2008, V. Jokubaitis]. But it's still unknown how CD34+ACE+ cells in AL behave on and after chemotherapy. We have studied the dynamics of Bcl-2 and Bax expression by flow cytometry in CD34+ cells of peripheral blood (PB) and bone marrow (BM) in pts with AL. PB and BM samples were collected before treatment, on days +8, +36, only PB - on day + 21. Bcl-2 and Bax were detected on CD34+ cells by flow cytometry using specific monoclonal antibodies: CD34 (8G12, BD), Bcl-2 (100, BD), Bax (2D2, Santa Cruz). ACE (9B9, BD) expression was also evaluated. We calculated 10 000 cells in each sample. 10 pts were included in the study: 4 AML, 6 ALL. The control group comprised 4 healthy donors. At time of diagnosis CD34+ cells number in BM was 38,7%± 9,75, in PB - 38,3%± 8,14 in AL pts, not differing much in AML and ALL, and indicating blast cells population. CD34+ cells numbers in BM and PB of healthy donors were 1,35% and 0,23%, respectively. After induction therapy and WBC recovery (days +36-38) CD34+ cells number in AL pts decreased dramatically in BM to 3,83%±1,51 (p=0,001) and in PB to 0,98%± 0,29 (p=0,0001), indicating the efficacy of chemotherapy. The dynamics of Bcl-2, Bax and ACE expression on CD34+ cells of BM and PB in AL pts are presented in fig.1-6 As seen in the fig.1,2 CD34/Bcl-2 expression in BM is significantly higher (p=0,04) and in PB is similar in AL pts at the diagnosis comparing with donors. It's also worth to note that BM and PB CD34+ cells in donors had different expression characteristics of Bcl-2 demonstrating much higher level of antiapoptotic marker in PB cells. On the contrast CD34+ AL cells in BM and PB had similar characteristics regarding CD34/Bcl-2 expression. This expression level decreased substantially in BM at day +36 comparing with day 0 (p=0,04), but it never reached the donors level remaining extremely high and supposing the persistence of antiapoptotic activity in CD34+ cells in AL pts. It did not change at all during chemotherapy in PB cells, being identical to donors characteristics. The fig.2,3 demonstrate that, CD34/Bax expression in BM is almost 3-times higher (p=0,14) and in PB is twice lower (p=0,02) in AL pts in comparison with donors. It's interesting that CD34/Bax expression in leukemic BM and PB cells looks very similar, when in donors we had very low expression in BM and high - in PB. This fact demonstrates the heterogeneity of donor CD34+cells in BM and PB and points that leukemia CD34+cells in BM and PB are rather similar in Bax expression. Chemotherapy caused the significant augmentation of CD34/Bax expression in PB on day +8 (p=0,01) and near significant on day +21 (p= 0,09) showing the increased level of “dieing” cells in PB after cytostatic influence. The fig. 5,6 show that CD34/ACE coexpression in BM cells of AL pts and donors did not differ much at any time of evaluation. But CD34/ACE expression in PB cells of AL pts was much lower (p=0,02) than in donors and substantially increased at day +36 almost reaching the donor level. We may conclude that Bcl-2, Bax, ACE expression on CD34+ cells in AL pts and donors significantly differs, the dynamics of expression in AL while chemotherapy shows critical changes in CD34/Bcl-2 expression in BM, CD34/Bax and CD34/ACE in PB. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Defect in Ribosome Biogenesis Contributes to Impaired Megakaryopoiesis in Primary Myelofibrosis

Blood ◽

10.1182/blood.v124.21.4585.4585 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4585-4585 ◽

Cited By ~ 1

Author(s):

Laure Gilles ◽

Ahmet Dirim Arslan ◽

Katerina Konstantinoff ◽

Maureen McNulty ◽

Lasho Terra ◽

...

Keyword(s):

Ribosome Biogenesis ◽

Hematologic Malignancy ◽

Primary Myelofibrosis ◽

Gene Set Enrichment Analysis ◽

Hematopoietic Progenitor Cell ◽

Pcr Analysis ◽

Hematopoietic Progenitor ◽

Knock Down ◽

Healthy Donors ◽

Cd34 Cells

Abstract Primary myelofibrosis (PMF) is a clonal hematologic malignancy resulting from the transformation of a pluripotent hematopoietic progenitor cell leading to increased hematopoiesis and overproduction of abnormal blood cells. PMF is characterized by bone marrow (BM) 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 BM and spleen. Numerous mutations are associated with PMF including JAK2, CALR, MPL, TET2, CBL, ASXL1, and IDH. The presence of abnormal Mks in the BM of PMF patients is an important characteristic of the disease, as these cells are believed to contribute to the fibrosis.Here, we performed an analysis of Mk differentiation from individuals with PMF compared to healthy individuals as a way to better understand the mechanism leading to their defects. CD34+ cells obtained from PMF and healthy donors were cultivated in presence of thrombopoietin to induce Mks differentiation. Under these conditions, we observed that the proliferative capacity of CD34+ cells from PMF patients was increased as compared to normal CD34+ cells. At the same time, the percentage of mature Mks obtained was reduced. Semi-solid culture conditions confirmed the proliferative advantage of the PMF CD34+ cells. To investigate the mechanism responsible for this phenotype, we performed genome wide expression analysis of sorted Mks from patients versus controls. Nine PMF patients with different mutations (5 JAK2V617F, 1 MPLW515L, 2 CALR and 1 triple negative) and 4 healthy donors were included in the study. Gene Set Enrichment Analysis (GSEA) analysis revealed that the top scoring pathways included the RPS14 signature, ribosome biogenesis, rRNA metabolic process, and rRNA processing. We first studied the RPS14 geneset, which consists of a group of genes that were up-regulated in CD34+ cells after knock-down of RPS14, a component of the small ribosome subunit. Using shRNA against RPS14, we discovered that its knock down leads to a significant reduction in the development of mature Mks from normal CD34+ cells. The microarray data also revealed changes in the expression of several myeloid genes. CEBPa and GFI-1 were over-expressed in PMF Mks, whereas GFI-1b and PF4, which are associated with Mk differentiation, were down-regulated. Surprisingly the expression of GATA1, a master regulator of megakaryopoiesis, was not changed. However, consistent with previous report, we discovered that GATA1 protein level was reduced in PMF Mks. GATA1 deficient Mks are impaired in their maturation, so we reasoned that the reduction in GATA1 contributes to the defects in PMF. Indeed, we found that overexpression of GATA1 in PMF CD34+ cells rescued Mk differentiation. To understand how GATA1 protein but not mRNA, is reduced, we knocked down RPS14 in the JAK2 mutant SET2 megakaryoblastic cell line. As predicted, we observed a reduction in GATA1 protein, but not mRNA. By ribosomal profiling, we saw that the shRPS14 led to an abnormal ribosomal profile with a reduction of the 40s peak. Finally, by RT-PCR analysis of the ribosomal and polysomal fractions, we found that GATA1 mRNA was less abundant in the polysomal fraction in cells expressing the shRPS14. This finding is consistent with recent data showing that alterations in ribosomal genes cause a decrease in GATA1 protein in Diamond-Blackfan Anemia. Although we identified the RPS14 gene signature, we did not observe changes in RPS14 expression or in any of the ribosomal subunits. Since our GSEA analysis also identified deficiencies in three ribosomal biogenesis pathways, we looked for genes present in all three signatures. NOP58, a ribonucleoprotein required for ribosome biogenesis, was significantly reduced in all samples of PMF Mks. To link the reduction in NOP58 expression with PMF associated mutations, we expressed MPLW515L or the CALR type II mutant in hematopoietic progenitor cells and detected a reduction in NOP58 expression. Our findings suggest that the activation of JAK/STAT signaling leads to a reduction in NOP58, which contributes to defects in ribosome assembly, decreased GATA1 expression and subsequent impairment in Mk differentiation. This work reveals, for the first time, that defective ribosomes contribute to the pathogenesis of PMF and suggests new avenues for therapeutic intervention. Disclosures Stein: Incyte Corporation: Honoraria, Speakers Bureau; Sanofi Oncology: Honoraria.

Download Full-text