The Transcriptional Program of Polycythemia Vera Revelas Altered Expression of Multiple Putative Tumor Suppressor Genes.

Abstract Polycythemia vera (PV) is a myeloproliferative disease characterized by accumulation of erythrocytes and cells of the myeloid and megakaryocyte lineages. Although genes like PRV-1 and PTP-MEG2 have been implicated in the pathology of PV, there is no consensus on their importance in the disease process. Progenitor cells from PV patients can grow in the absence of erythropoietin, and are hypersensitive to a variety of other growth factors. This suggests that polycythemic hematopoietic progenitor cells possess a significantly different genetic program. We tested this idea by molecular profiling hematopoietic progenitor cells (CD34+) from PV specimens and normal donors. We purified CD34+ cells from the marrow of 10 PV patients and harvested total RNA. Biotinylated cRNA was made through two rounds of linear amplification, and hybridized to Affymetrix HGU133A genechips. CD34+ cells from marrow mononuclear cells of 5 normal controls were processed similarly. The resulting datasets were normalized to the median across chips and across genes. Unsupervised hierarchical clustering showed that PV samples had a distinct gene expression profile from the controls. We then performed supervised clustering using a non-parametric t-test (Wilcoxon rank sum test) using the Benjamini and Hochberg multiple testing correction held to a p-value of 0.01 to determine genes that were significantly different between disease and normal samples. Using these stringent criteria, there were 331 genes that reached significance. Strikingly most of these were decreased in expression compared with control CD34+ cells and only 34 genes were upregulated in PV. A 35 gene predictor set was discovered through the use of a k-nearest neighbor metric. This set was 100% accurate for the prediction of PV in a leave one out cross-validation approach. Among these genes are EVI1, a known oncogene and one of only two genes upregulated in PV on this list, and the putative tumor suppressor genes TUSC4 (NPR2), NDRG1 and KLF4. Also among the predictor genes is BAALC, a gene expressed in normal CD34+ cells and known to be a prognostic indicator gene for acute myeloid leukemia.

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Combination treatment in vitro with Nutlin, a small-molecule antagonist of MDM2, and pegylated interferon-α 2a specifically targets JAK2V617F-positive polycythemia vera cells

Blood ◽

10.1182/blood-2012-02-410712 ◽

2012 ◽

Vol 120 (15) ◽

pp. 3098-3105 ◽

Cited By ~ 24

Author(s):

Min Lu ◽

Xiaoli Wang ◽

Yan Li ◽

Joseph Tripodi ◽

Goar Mosoyan ◽

...

Keyword(s):

Polycythemia Vera ◽

Progenitor Cells ◽

Combination Treatment ◽

Colony Formation ◽

P38 Map Kinase ◽

Hematopoietic Progenitor Cells ◽

Interferon Α ◽

Low Doses ◽

Hematopoietic Progenitor ◽

Cd34 Cells

Abstract Interferon (IFN-α) is effective therapy for polycythemia vera (PV) patients, but it is frequently interrupted because of adverse events. To permit the long-term use of IFN, we propose combining low doses of IFN with Nutlin-3, an antagonist of MDM2, which is also capable of promoting PV CD34+ cell apoptosis. Combination treatment with subtherapeutic doses of Peg IFN-α 2a and Nutlin-3 inhibited PV CD34+ cell proliferation by 50% while inhibiting normal CD34+ cells by 30%. Combination treatment with Nutlin-3 and Peg IFN-α 2a inhibited PV colony formation by 55%-90% while inhibiting normal colony formation by 22%-30%. The combination of these agents also decreased the proportion of JAK2V617F-positive hematopoietic progenitor cells in 6 PV patients studied. Treatment with low doses of Peg IFN-α 2a combined with Nutlin-3 increased phospho-p53 and p21 protein levels in PV CD34+ cells and increased the degree of apoptosis. These 2 reagents affect the tumor suppressor p53 through different pathways with Peg IFN-α 2a activating p38 MAP kinase and STAT1, leading to increased p53 transcription, whereas Nutlin-3 prevents the degradation of p53. These data suggest that treatment with low doses of both Nutlin-3 combined with Peg IFN-α 2a can target PV hematopoietic progenitor cells, eliminating the numbers of malignant hematopoietic progenitor cells.

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Treatment with Pegylated Interferon Alpha 2a in Combination with the Bcl-Xl Inhibitor ABT-737 Specifically Targets JAK2V617F Positive Hematopoietic Progenitor Cells From Patients with Polycythemia Vera.

Blood ◽

10.1182/blood.v114.22.3916.3916 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3916-3916 ◽

Cited By ~ 1

Author(s):

Min Lu ◽

Wei Zhang ◽

Daniel Yoo ◽

Dmitriy Berenzon ◽

Yan Li ◽

...

Keyword(s):

Polycythemia Vera ◽

Progenitor Cells ◽

Interferon Alpha ◽

Colony Formation ◽

Conflicts Of Interest ◽

Philadelphia Chromosome ◽

Hematopoietic Progenitor Cells ◽

Low Doses ◽

Hematopoietic Progenitor ◽

Cd34 Cells

Abstract Abstract 3916 Poster Board III-852 Polycythemia vera (PV) is a Philadelphia chromosome negative chronic myeloproliferative neoplasm (MPN) which is characterized by acquisition of a mutation in JAK2 (JAK2V617F). The administration of a pegylated form of interferon-alpha-2a (Peg IFNa-2a) to patients with PV has recently been reported to lead to hematological remissions and a reduction of the JAK2V617F allele burden in most patients receiving this modality of therapy. The mechanism underlying this profound clinical response of PV patients to Peg IFNa-2a has been the subject of a great deal of speculation. In order to evaluate the mechanism by which Peg IFNa-2a affects hematopoiesis in PV patients, CD34+ cells isolated from cord blood and the peripheral blood of patients with PV were cultured in semisolid media in the presence and absence of 200 and 500 U of Peg IFNa-2a. These relatively low doses of Peg IFNa-2a did not alter hematopoietic colony formation by CB CD34+ cells but inhibited PV CFU-GM colony formation by 35% and 50%, and BFU-E colony formation by 60% and 80%, respectively. Furthermore, the hematopietic colonies that formed in the presence of Peg IFNa-2a were composed of far fewer cells than those cultured in the presence of cytokines alone. In addition, individual hematopoietic colonies were plucked and the JAK2 genotype was assessed by nested allele-specific PCR assay. Exposure of PV CD34+ cells to Peg-IFNa-2a (500U) resulted in a reduction in the proportion of JAK2V617F-positive hematopoietic progenitor cells from 81.7±16.3% to 50.3±27.6% (p=0.004). Samples from 81.9% of the PV patients (9 of 11 samples) responded in this fashions to Peg IFNa 2a treatment. We then showed that incubation of PV CD34+ cells but not CB CD 34+ cells with 200 and 500U of Peg IFNa-2a resulted in increased rates of apoptosis by 4.3% and 15.3%, respectively. Erythroblasts and megakayoctes from patients with PV have been previously shown to be characterized by over-expression of the anti-apoptotic proteins Bcl-xL. We then examined if the effects of IFNa-2a could be enhanced by addition of the Bcl-xL inhibitor-ABT-737. After 2 days of treatment, Peg IFNa 2a plus ABT-737 induced significantly greater degree of apoptosis (∼50%) of a JAK2V617F positive erythroleukemia cell line (HEL cells) as compared to treatment with each agent alone, (Peg-IFNa-2a, <5%; ABT-737, 20%). PV CD34+ cells were incubated with Peg IFNa 2a (500 U) alone, ABT-737 (0.25 uM) alone or ABT-737 plus Peg IFNa 2a for 4 days and the numbers of cells were decreased by 35%, 40% and 65 %, respectively; and the corresponding percentage of apoptotic cells was 20%, 15% and 60%, respectively. Western blot analysis showed that the Bcl-xL protein level in PV but not CB mononuclear cells was reduced by treatments with ABT-737 alone or in combination with Peg IFNa 2a. Furthermore, treatment of PV CD34+ cells with ABT-737 plus Peg IFNa 2a (200U) lead to the appearance of a smaller proportion of JAK2 V617F-positive (46.7±26%) hematopoietic progenitor cells as compared to cells incubated with cytokines alone (81.7±17%) or cytokines plus Peg IFNa 2a (69±20%). These data suggest that low doses of Peg IFNa 2a selectively and directly eliminate Jak2V617F hematopietic progenitor cells which likely accounts for the therapeutic responses that have been observed with the use of this agent in the clinic. The enhanced elimination of JAK2V617F hematopoietic progenitor cells observed with the combination of ABT-737 and Peg-IFNa-2a suggests that this strategy might be an even more optimal approach for the treatment of JAK2V617F positive MPN which merits further testing in the clinic. Disclosures: No relevant conflicts of interest to declare.

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Preferential sequestration in vitro of BCR/ABL negative hematopoietic progenitor cells among cytokine nonresponsive CML marrow CD34+ cells

Bone Marrow Transplantation ◽

10.1038/sj.bmt.1700818 ◽

1997 ◽

Vol 19 (12) ◽

pp. 1213-1221 ◽

Cited By ~ 13

Author(s):

P Veena ◽

K Cornetta ◽

A Davidson ◽

B Agüero ◽

J McMahel ◽

...

Keyword(s):

Progenitor Cells ◽

Hematopoietic Progenitor Cells ◽

Hematopoietic Progenitor ◽

Cd34 Cells

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Wilms' Tumor Gene (WT1) Competes With Differentiation-Inducing Signal in Hematopoietic Progenitor Cells

Blood ◽

10.1182/blood.v91.8.2969.2969_2969_2976 ◽

1998 ◽

Vol 91 (8) ◽

pp. 2969-2976 ◽

Cited By ~ 119

Author(s):

Kazushi Inoue ◽

Hiroya Tamaki ◽

Hiroyasu Ogawa ◽

Yoshihiro Oka ◽

Toshihiro Soma ◽

...

Keyword(s):

Signal Transduction ◽

Tumor Suppressor ◽

Progenitor Cells ◽

Wilms Tumor ◽

Suppressor Gene ◽

Hematopoietic Progenitor Cells ◽

Wt1 Gene ◽

Wild Type ◽

Hematopoietic Progenitor ◽

Oncogenic Function

The WT1 gene is a tumor-suppressor gene that was isolated as a gene responsible for Wilms' tumor, a childhood kidney neoplasm. We have previously reported that the WT1 gene is strongly expressed in leukemia cells with an increase in its expression levels at relapse and an inverse correlation between its expression levels and prognosis, thus making it a novel tumor marker for leukemic blast cells. Furthermore, WT1 antisense oligomers have been found to inhibit the growth of leukemic cells. These results strongly suggested the involvement of the WT1 gene in human leukemogenesis. The present study was performed to prove our hypothesis that the WT1 gene plays a key role in leukemogenesis and performs an oncogenic function in hematopoietic progenitor cells, rather than a tumor-suppressor gene function. 32D cl3, an interleukin-3–dependent myeloid progenitor cell line, differentiates into mature neutrophils in response to granulocyte colony-stimulating factor (G-CSF). However, when transfected wild-type WT1 gene was constitutively expressed in 32D cl3, the cells stopped differentiating and continued to proliferate in response to G-CSF. As for signal transduction mediated by G-CSF receptor (G-CSFR), Stat3α was constitutively activated in wild-type WT1-infected 32D cl3 in response to G-CSF, whereas, in WT1-uninfected 32D cl3, activation of Stat3α was only transient. However, most interesting was the fact that G-CSF stimulation resulted in constitutive activation of Stat3β only in wild-type WT1-infected 32D cl3, but not in WT1-uninfected 32D cl3. Thus, WT1 expression constitutively activated both Stat3α and Stat3β. A transient activation of Stat1 was detected in both wild-type WT1-infected and uninfected 32D cl3 after G-CSF stimulation, but no difference in its activation was found. No activation of MAP kinase was detected in both wild-type WT1-infected and uninfected 32D cl3 after G-CSF stimulation. These results demonstrated that WT1 expression competed with the differentiation-inducing signal mediated by G-CSFR and constitutively activated Stat3, resulting in the blocking of differentiation and subsequent proliferation. Therefore, the data presented here support our hypothesis that the WT1 gene plays an essential role in leukemogenesis and performs an oncogenic function in hematopoietic progenitor cells and represent the first demonstration of an important role of the WT1 gene in signal transduction in hematopoietic progenitor cells.

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Putative Tumor Suppressor Genes EGR1 and BRSK1 Are Mutated in Gastric and Colorectal Cancers

Oncology ◽

10.1159/000450616 ◽

2016 ◽

Vol 91 (5) ◽

pp. 289-294 ◽

Cited By ~ 7

Author(s):

Eun Ji Choi ◽

Nam Jin Yoo ◽

Min Sung Kim ◽

Chang Hyeok An ◽

Sug Hyung Lee

Keyword(s):

Tumor Suppressor ◽

Tumor Suppressor Genes ◽

Colorectal Cancers ◽

Suppressor Genes ◽

Putative Tumor Suppressor

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Orderly Process of Sequential Cytokine Stimulation Is Required for Activation and Maximal Proliferation of Primitive Human Bone Marrow CD34+ Hematopoietic Progenitor Cells Residing in G0

Blood ◽

10.1182/blood.v90.2.658 ◽

1997 ◽

Vol 90 (2) ◽

pp. 658-668 ◽

Cited By ~ 63

Author(s):

Amy C. Ladd ◽

Robert Pyatt ◽

Andre Gothot ◽

Susan Rice ◽

Jon McMahel ◽

...

Keyword(s):

Cell Cycle ◽

Bone Marrow ◽

Phase I ◽

Progenitor Cells ◽

Phase Ii ◽

Hematopoietic Progenitor Cells ◽

Hematopoietic Progenitor ◽

Cd34 Cells ◽

Cytokine Stimulation ◽

Phase Iv

Abstract Bone marrow (BM) CD34+ cells residing in the G0 phase of cell cycle may be the most suited candidates for the examination of cell cycle activation and proliferation of primitive hematopoietic progenitor cells (HPCs). We designed a double simultaneous labeling technique using both DNA and RNA staining with Hoechst 33342 and Pyronin Y, respectively, to isolate CD34+ cells residing in G0(G0CD34+ ). Using long-term BM cultures and limiting dilution analysis, G0CD34+ cells were found to be enriched for primitive HPCs. In vitro proliferation of G0CD34+ cells in response to sequential cytokine stimulation was examined in a two-step assay. In the first step, cells received a primary stimulation consisting of either stem cell factor (SCF), Flt3-ligand (FL), interleukin-3 (IL-3), or IL-6 for 7 days. In the second step, cells from each group were washed and split into four or more groups, each of which was cultured again for another week with one of the four primary cytokines individually, or in combination. Tracking of progeny cells was accomplished by staining cells with PKH2 on day 0 and with PKH26 on day 7. Overall examination of proliferation patterns over 2 weeks showed that cells could progress into four phases of proliferation. Phase I contained cytokine nonresponsive cells that failed to proliferate. Phase II contained cells dividing up to three times within the first 7 days. Phases III and IV consisted of cells dividing up to five divisions and greater than six divisions, respectively, by the end of the 14-day period. Regardless of the cytokine used for primary stimulation, G0CD34+ cells moved only to phase II by day 7, whereas a substantial percentage of cells incubated with SCF or FL remained in phase I. Cells cultured in SCF or FL for the entire 14-day period did not progress beyond phase III but proliferated into phase IV (with <20% of cells remaining in phases I and II) if IL-3, but not IL-6, was substituted for either cytokine on day 7. G0CD34+ cells incubated with IL-3 for 14 days proliferated the most and progressed into phase IV; however, when SCF was substituted on day 7, cells failed to proliferate into phase IV. Most intriguing was a group of cells, many of which were CD34+, detected in cultures initially stimulated with IL-3, which remained as a distinct population, mostly in G0 /G1 , unable to progress out of phase II regardless of the nature of the second stimulus received on day 7. A small percentage of these cells expressed cyclin E, suggesting that their proliferation arrest may have been mediated by a cyclin-related disruption in cell cycle. These results suggest that a programmed response to sequential cytokine stimulation may be part of a control mechanism required for maintenance of proliferation of primitive HPCs and that unscheduled stimulation of CD34+ cells residing in G0 may result in disruption of cell-cycle regulation.

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Allogeneic blood stem cell transplantation: peripheralization and yield of donor-derived primitive hematopoietic progenitor cells (CD34+ Thy- 1dim) and lymphoid subsets, and possible predictors of engraftment and graft-versus-host disease

Blood ◽

10.1182/blood.v86.7.2842.2842 ◽

1995 ◽

Vol 86 (7) ◽

pp. 2842-2848 ◽

Cited By ~ 189

Author(s):

M Korbling ◽

YO Huh ◽

A Durett ◽

N Mirza ◽

P Miller ◽

...

Keyword(s):

Body Weight ◽

Progenitor Cells ◽

Peripheral Blood ◽

Allogeneic Transplantation ◽

Hematopoietic Progenitor Cells ◽

Donor Blood ◽

Hematopoietic Progenitor ◽

Cd34 Cells ◽

The Mean ◽

Single Bone

Abstract Apheresis-derived hematopoietic progenitor cells have recently been used for allogeneic transplantation. Forty-one normal donors were studied to assess the effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF) (12 micrograms/kg/d) on the peripheralization of hematopoietic progenitor cells and lymphoid subsets. The white blood cell, polymorphonuclear cell (PMNC), and lymphocyte concentrations at the peak of rhG-CSF effect in the donor's peripheral blood (PB) exceeded baseline by 6.4-, 8.0-, and 2.2-fold, respectively. Corresponding concentrations of PB CD34+ cells and primitive subsets such as CD34+ Thy-1dim, and CD34+ Thy-1dim CD38- cells increased by 16.3-fold, 24.2-fold, and 23.2-fold, respectively in eight normal donors. The percentage of CD34+ Thy-1dim and CD34+ Thy- 1dim CD38- cells among CD34+ cells increased as well, suggesting an additional peripheralization effect of rhG-CSF on primitive CD34+ subsets. The preapheresis PB CD34+ and CD34+ Thy-1dim cell concentrations were predictive of their corresponding apheresis yield per liter of donor blood processed PB lymphoid subsets were not significantly affected by rhG-CSF treatment. The mean apheresis-derived yield of CD34+, CD34+ Thy-1dim, and CD34+ Thy-1dim CD38- cells per kilogram of recipient body weight and per liter of donor blood processed was 48.9 x 10(4) (n = 41), 27.2 x 10(4) (n = 10), and 1.9 x 10(4) (n = 10), respectively. As compared with 43 single bone marrow (BM) harvest, the CD34+ cell yield of peripheral blood progenitor cell allografts of 41 normal donors exceeded that of BM allografts by 3.7- fold and that of lymphoid subsets by 16.1-fold (CD3+), 13.3-fold (CD4+), 27.4-fold (CD8+), 11.0-fold (CD19+), and 19.4-fold (CD56+CD3-). All PBPC allografts were cryopreserved before transplantation. The mean recovery of CD34+ cells after freezing, thawing, and washing out dimethylsulfoxide was 86.6% (n = 31) and the recovery of lymphoid subsets was 115.5% (CD3+), 121.4% (CD4+), 105.6% (CD8+), 118.1% (CD19+), and 102.4% (CD56+CD3-). All donors were related to patients: 39 sibling-to-sibling, 1 parent-to-child, and 1 child-to-parent transplant. Thirty-eight transplants were HLA fully identical, two transplants differed in one and two antigens. Engraftment occurred in 38 recipients; two patients died too early to be evaluated, and one patient did not engraft. The lowest CD34+ cell dose transplanted and resulting in complete and sustained engraftment was 2.5 x 10(6)/kg of recipient body weight.(ABSTRACT TRUNCATED AT 400 WORDS)

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