Flt3 mutations from patients with acute myeloid leukemia induce transformation of 32D cells mediated by the Ras and STAT5 pathways

Blood ◽  
2000 ◽  
Vol 96 (12) ◽  
pp. 3907-3914 ◽  
Author(s):  
Masao Mizuki ◽  
Regina Fenski ◽  
Hartmut Halfter ◽  
Itaru Matsumura ◽  
Rainer Schmidt ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Colony Formation ◽  
Myeloid Leukemia ◽  
Map Kinases ◽  
Rapid Development ◽  
Dominant Negative ◽  
Wild Type ◽  
Flt3 Mutations ◽  
Induced Apoptosis ◽  
Acute Myeloid

Abstract Somatic mutations of the receptor tyrosine kinase Flt3 consisting of internal tandem duplications (ITD) occur in 20% of patients with acute myeloid leukemia. They are associated with a poor prognosis of the disease. In this study, we characterized the oncogenic potential and signaling properties of Flt3 mutations. We constructed chimeric molecules that consisted of the murine Flt3 backbone and a 510-base pair human Flt3 fragment, which contained either 4 different ITD mutants or the wild-type coding sequence. Flt3 isoforms containing ITD mutations (Flt3-ITD) induced factor-independent growth and resistance to radiation-induced apoptosis in 32D cells. Cells containing Flt3-ITD, but not those containing wild-type Flt3 (Flt3-WT), formed colonies in methylcellulose. Injection of 32D/Flt3-ITD induced rapid development of a leukemia-type disease in syngeneic mice. Flt3-ITD mutations exhibited constitutive autophosphorylation of the immature form of the Flt3 receptor. Analysis of the involved signal transduction pathways revealed that Flt3-ITD only slightly activated the MAP kinases Erk1 and 2 and the protein kinase B (Akt) in the absence of ligand and retained ligand-induced activation of these enzymes. However, Flt3-ITD led to strong factor-independent activation of STAT5. The relative importance of the STAT5 and Ras pathways for ITD-induced colony formation was assessed by transfection of dominant negative (dn) forms of these proteins: transfection of dnSTAT5 inhibited colony formation by 50%. Despite its weak constitutive activation by Flt3-ITD, dnRas also strongly inhibited Flt3-ITD–mediated colony formation. Taken together, Flt3-ITD mutations induce factor-independent growth and leukemogenesis of 32D cells that are mediated by the Ras and STAT5 pathways.

Flt3 mutations from patients with acute myeloid leukemia induce transformation of 32D cells mediated by the Ras and STAT5 pathways

Blood ◽  
2000 ◽  
Vol 96 (12) ◽  
pp. 3907-3914 ◽  
Author(s):  
Masao Mizuki ◽  
Regina Fenski ◽  
Hartmut Halfter ◽  
Itaru Matsumura ◽  
Rainer Schmidt ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Colony Formation ◽  
Myeloid Leukemia ◽  
Map Kinases ◽  
Rapid Development ◽  
Dominant Negative ◽  
Wild Type ◽  
Flt3 Mutations ◽  
Induced Apoptosis ◽  
Acute Myeloid

Somatic mutations of the receptor tyrosine kinase Flt3 consisting of internal tandem duplications (ITD) occur in 20% of patients with acute myeloid leukemia. They are associated with a poor prognosis of the disease. In this study, we characterized the oncogenic potential and signaling properties of Flt3 mutations. We constructed chimeric molecules that consisted of the murine Flt3 backbone and a 510-base pair human Flt3 fragment, which contained either 4 different ITD mutants or the wild-type coding sequence. Flt3 isoforms containing ITD mutations (Flt3-ITD) induced factor-independent growth and resistance to radiation-induced apoptosis in 32D cells. Cells containing Flt3-ITD, but not those containing wild-type Flt3 (Flt3-WT), formed colonies in methylcellulose. Injection of 32D/Flt3-ITD induced rapid development of a leukemia-type disease in syngeneic mice. Flt3-ITD mutations exhibited constitutive autophosphorylation of the immature form of the Flt3 receptor. Analysis of the involved signal transduction pathways revealed that Flt3-ITD only slightly activated the MAP kinases Erk1 and 2 and the protein kinase B (Akt) in the absence of ligand and retained ligand-induced activation of these enzymes. However, Flt3-ITD led to strong factor-independent activation of STAT5. The relative importance of the STAT5 and Ras pathways for ITD-induced colony formation was assessed by transfection of dominant negative (dn) forms of these proteins: transfection of dnSTAT5 inhibited colony formation by 50%. Despite its weak constitutive activation by Flt3-ITD, dnRas also strongly inhibited Flt3-ITD–mediated colony formation. Taken together, Flt3-ITD mutations induce factor-independent growth and leukemogenesis of 32D cells that are mediated by the Ras and STAT5 pathways.

Recent advances in FLT3 inhibitors for acute myeloid leukemia

2020 ◽  
Vol 12 (10) ◽  
pp. 961-981 ◽  
Author(s):  
Lexian Tong ◽  
Xuemei Li ◽  
Yongzhou Hu ◽  
Tao Liu
Keyword(s):  
Acute Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Myeloid Leukemia ◽  
Clinical Stage ◽  
Wild Type ◽  
New Techniques ◽  
Flt3 Mutations ◽  
Flt3 Inhibitors ◽  
Target Activity ◽  
Acute Myeloid

Fms-like tyrosine kinase-3 (FLT3) mutations occur in approximately 30% of acute myeloid leukemia (AML) cases, suggesting FLT3 as an attractive target for AML treatment. Early FLT3 inhibitors enhance antileukemia efficacy by inhibiting multiple targets, and thus had stronger off-target activity, increasing their toxicity. Recently, a number of potent and selective FLT3 inhibitors have been developed, many of which are effective against multiple mutations. This review outlines the evolution of AML-targeting FLT3 inhibitors by focusing on their chemotypes, selectivity and activity over FLT3 wild-type and FLT3 mutations as well as new techniques related to FLT3. Compounds that currently enter the late clinical stage or have entered the market are also briefly reported.

The Critical Role of Peptidyl-Prolyl cis/trans Isomerase, Pin1 in Acute Myeloid Leukemia with C/EBPalpha Mutations.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2213-2213
Author(s):  
J. Pulikkan ◽  
A. Peer Zada ◽  
M. Geletu ◽  
V. Dengler ◽  
Daniel G. Tenen ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Dominant Negative ◽  
Pcr Analysis ◽  
Wild Type ◽  
Rt Pcr ◽  
Promoter Assay ◽  
Type C ◽  
Acute Myeloid

Abstract CCAAT enhancer binding protein alpha (C/EBPα) is a myeloid specific transcription factor that coordinates cellular differentiation and cell cycle arrest. Loss of C/EBPα expression or function in leukemic blasts contributes to a block in myeloid cell differentiation. C/EBPα is mutated in around 9% of acute myeloid leukemia (AML). The mutations reported in C/EBPα are frame shift mutations and point mutations at basic region Leucine zipper. The mutant form of C/EBPα ie C/EBPα-p30 exhibits dominant negative function over the wild type protein. The role of peptidyl-prolyl cis/trans isomerase, Pin1 in tumorogenesis and its overexpression in many cancers led us to investigate its role in acute myeloid leukemia with C/EBPα mutation. Here we show that Pin1 is upregulated in patients with acute myeloid leukemia by affymetrix analysis. By quantitative Real-Time RT-PCR analysis, we show C/EBPα-p30 could induce Pin1 transcription, while the wild type C/EBPα downregulates Pin1 expression. Luciferase promoter assay for the Pin1 promoter shows that wild type C/EBPα is able to block Pin1 promoter activity. Mean while, C/EBPα-p30 couldn’t block Pin1 promotor activity. By silencing Pin1 by RNA Interference as well as with inhibitor against Pin1 (PiB) we could show myeloid differentiation in human CD34+ cord blood cells as well as in Kasumi-6 cells as assessed by FACS analysis with granulocytic markers. We investigated the mechanism underlying the dominant negative action of C/EBPα-p30 over the wild type protein. We report that Pin1 increases the transcriptional activity of the oncogene c-jun. We also show that c-jun blocks the DNA binding and transactivation of C/EBPα protein as assessed by gel shift assay and promoter assay respectively. We have previously shown that c-jun expression is high in AML patients with C/EBPα mutation and c-jun could block C/EBPα function by protein-protein interaction. Quantitative Real-Time RT-PCR analysis shows that inhibition of Pin1 by the inhibitor PiB downregulates c-jun mRNA expression. In conclusion, inhibition of Pin1 leads to granulocytic differentiation. Our results show Pin1 as a novel target in treating AML patients with C/EBPα mutation.

Bcl-2 Protects against p53-Induced Apoptosis through Hdm2

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5333-5333
Author(s):  
Line Wergeland ◽  
Kevin B. Spurgers ◽  
Eystein Oveland ◽  
Torill Høiby ◽  
Manel Cascallo ◽  
...  
Keyword(s):  
Dna Damage ◽  
Acute Myeloid Leukemia ◽  
Protein Level ◽  
Myeloid Leukemia ◽  
Proteasome Inhibitors ◽  
Wild Type ◽  
Inhibitory Molecule ◽  
Wild Type P53 ◽  
Induced Apoptosis ◽  
Acute Myeloid

Abstract Hdm2 is up-regulated in several malignancies including sarcomas and acute myeloid leukemia, where it counteracts the anti-proliferative and pro-apoptotic effect of wild type p53. The anti-apoptotic protein Bcl-2 is often elevated in many tumors with wild type p53 and serves to block p53-induced apoptosis. We demonstrate that the protein level of Hdm2 positively correlates with the level of Bcl-2 and follows the Bcl-2 level in different cell systems. Over-expression of Bcl-2 protects Hdm2 from DNA-damage induced degradation in a dose dependant manner. In addition, modulation of Bcl-2 by shRNA knockdown reduced the Hdm2 protein level in parallel. Consequently, treatment of AML cells with the Bcl-2 small inhibitory molecule HA14-1 attenuated the level of Hdm2. The Bcl-2 level, but not the DNA damage induced Hdm2 degradation, was affected by disruption of the E3 ubiquitin ligase activity of Hdm2. In addition, the DNA-damage induced Hdm2 down-regulation was blocked by disrupted E1 ubiquitin-activation, defect polyubiquitination and by proteasome inhibitors. Finally, we show that Bcl-2 protection from p53-induced cell death requires co-expression of Hdm2 in double null p53/mdm2 mouse embryonic fibroblasts. Our results indicate that Bcl-2 regulates the Hdm2 level and that Hdm2 is a key mediator in Bcl-2 inhibition of p53-induced apoptosis. This is of particular therapeutic interest for cancers displaying elevated Hdm2 and Bcl-2, like sarcoma and acute myeloid leukemia.

A Critical Role of YB-1 In NPMc-Induced Acute Myeloid Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3149-3149
Author(s):  
Yoko Ogawara ◽  
Takuo Katsumoto ◽  
Takeshi Uchiumi ◽  
Kimitoshi Kohno ◽  
Issay Kitabayashi
Keyword(s):  
Acute Myeloid Leukemia ◽  
Colony Formation ◽  
Myeloid Leukemia ◽  
Fetal Liver ◽  
Hematopoietic Cells ◽  
Wild Type ◽  
Embryonic Lethal ◽  
Hoxa Genes ◽  
Fetal Liver Cells ◽  
Acute Myeloid

Abstract Abstract 3149 Frameshift mutations in Nucleophosmin gene (NPM) are the most frequent abnormality in acute myeloid leukemia (AML), found in approximately 30% of all cases and 50% of patients with normal karyotype (NK) AML. NPM mutations result in an aberrant cytoplasmic localization of NPM protein (NPMc) through a loss of nucleolar localization signal accompanied by acquisition of new nuclear export signal. NPM mutations are heterozygous, so the other wild-type allele is consistently retained. NPMc binds to wild-type NPM through oligomerization domain and impairs its activity by delocalizing to the cytoplasm. It was reported that the NPM-null mice are early embryonic lethal and defective in primary hematopoiesis, suggesting important roles of NPM in early hematopoiesis. However, the molecular mechanism by which NPMc exerts its leukemogenic potential has never been established. Here we show that ectopic expression of NPMc, but not wild type (WT) NPM, in mouse bone marrow (BM) cells enhanced their colony formation activity in methylcellulose media. Increased expression of HoxA7, 9 and 10 genes were observed in cells expressing NPMc but not in those expressing WT NPM. It has been reported that the expression levels of HOXA genes are upregulated in various types of AML including NPMc+ AML. Since overexpression of HoxA9 immortalizes hematopoietic progenitor cells, our findings suggest that up-regulation of HoxA genes are involved in NPMc-mediated leukemogenesis. To clarify roles of NPMc in leukemogenesis, we purified the NPM protein complex and identified Y box-binding protein 1 (YB-1) as a binding partner for NPM. YB-1 belongs to the cold shock family and functions in gene transcription and RNA processing. YB-1 strongly bound to WT NPM but not to NPMc. In addition, interaction between YB-1 and NPM was impaired in the presence of NPMc. YB-1-deficient mice were embryonic lethal and their fetal liver were small. YB-1-deficient yolk sac cells showed decreased colony-forming activity, and decreased number of hematopoietic cells were observed when AGM region of YB-1-deficeint embryo were cultured on OP9 cells. Furthermore, expression of Hoxa9 was decreased in fetal liver cells derived from YB-1 knockout mice. To investigate the roles of YB-1 in NPMc-associated leukemogenesis, WT and YB-1-null E14.5 fetal liver cells were infected with retrovirus expressing NPMc. Analyses of colony-forming activity and mRNA expression showed that YB-1 was essential for NPMc-induced increases in colony formation activity as well as in expression of HoxA genes. However, YB-1 was not necessary for colony formation activity induced by other AML-associated fusion genes, such as AML1-MTG8 and MLL-AF10. These data indicate that YB-1 is specifically required for NPMc-induced leukemogenic transformation of hematopoietic cells. Disclosures: No relevant conflicts of interest to declare.

Selective FLT3 Inhibitor FI-700 Neutralizes Mcl-1 and Enhances p53-Mediated Apoptosis in Acute Myeloid Leukemia (AML) Cells with Activating Mutations of FLT3 Via Mcl-1/Noxa Axis.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2597-2597
Author(s):  
Kensuke Kojima ◽  
Marina Konopleva ◽  
Twee Tsao ◽  
Michael Andreeff ◽  
Hiroshi Ishida ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Activating Mutations ◽  
Phosphatidylserine Externalization ◽  
Flt3 Inhibitor ◽  
Flt3 Mutations ◽  
Flt3 Inhibitors ◽  
Induced Apoptosis ◽  
Apoptotic Effects ◽  
Acute Myeloid

Abstract Abstract 2597 Poster Board II-573 Introduction: Activating mutations of the Fms-like tyrosine kinase-3 gene (FLT3) occur in approximately 30–40% of acute myeloid leukemia (AML) patients. FLT3 mutations confer numerous oncogenic properties, including dysregulated proliferation, resistance to apoptosis and a block in differentiation. FLT3 mutations result in abnormal activation of the downstream pathways, including signal transducer and activator of transcription 5 (STAT5), mitogen-activated protein kinase kinase (Mek)/extracellular signal–regulated kinase (Erk) and phosphatidylinositol-3 kinase (PI3K)/Akt. Activation of these downstream effectors has been thought to allow leukemia cells to evade apoptosis. Targeting of FLT3 mutations is a promising approach to overcome the dismal prognosis of acute myeloid leukemia (AML) with activating FLT3 mutations. Current trials are combining FLT3 inhibitors with p53-activating conventional chemotherapy. The mechanisms of cytotoxicity of FLT3 inhibitors are poorly understood. We investigated the interaction of FLT3 and p53 pathways after their simultaneous blockade using the selective FLT3 inhibitor FI-700 and the MDM2 inhibitor Nutlin-3 in AML. Results: FI-700 induced G1-phase cell cycle arrest and apoptosis as evidenced by increased sub-G1 DNA content and phosphatidylserine externalization in FLT3/ITD MOLM-13 (FLT3-ITD, wild-type (wt)-p53) and MV4-11NR (FLT3-ITD, mutated-p53) AML cells. FI-700 did not affect cell cycle distribution patterns nor did it induce apoptosis in FLT3/WT OCI-AML-3 (FLT3/WT, wt-p53) and HL-60 (FLT3/WT, del (del)-p53). Wt-p53 MOLM-13 and OCI-AML-3 cells were susceptible to Nutlin-induced apoptosis. FI-700 augmented Nutlin-induced Bax activation, mitochondrial membrane potential (MMP) loss, caspase-3 activation and phosphatidylserine externalization in MOLM-13 cells. FI-700 rapidly reduced Mcl-1 levels in FLT3/ITD cells, mainly by enhancing proteasomal Mcl-1 degradation. Levels of other Bcl-2 family proteins examined did not change significantly. Mcl-1 levels were only modestly reduced upon Nutlin treatment. The FI-700/Nutlin-3 combination profoundly reduced Mcl-1 levels. Immunoprecipitation/ immunoblotting results suggested that the drug combination results in a profound decrease in Mcl-1-bound Bim. FI-700 enhanced doxorubicin-induced apoptosis in FLT3/ITD MOLM-13 and MV4-11NR cells, suggesting that FI-700 can enhance both the p53-dependent and the p53-independent apoptotic effects of doxorubicin. Finally, cooperative apoptotic effects of FI-700/Nutlin-3 were seen in primary AML cells with FLT3/ITD. Conclusion: FLT3 inhibition by FI-700 immediately reduces anti-apoptotic Mcl-1 levels and enhances Nutlin-induced p53-mediated mitochondrial apoptosis in FLT3/ITD-expressing AML cells via the Mcl-1/Noxa axis. FLT3 inhibition, in combination with p53-inducing agents, might represent a potential therapeutic approach in AML with FLT3/ITD. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Induced Apoptosis Investigation in Wild-type and FLT3-ITD Acute Myeloid Leukemia Cells by Nanochannel Electroporation and Single-cell qRT-PCR

2016 ◽  
Vol 24 (5) ◽  
pp. 956-964 ◽  
Author(s):  
Keliang Gao ◽  
Xiaomeng Huang ◽  
Chi-Ling Chiang ◽  
Xinmei Wang ◽  
Lingqian Chang ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Single Cell ◽  
Myeloid Leukemia ◽  
Leukemia Cells ◽  
Wild Type ◽  
Qrt Pcr ◽  
Acute Myeloid Leukemia Cells ◽  
Induced Apoptosis ◽  
Acute Myeloid

Myc Drives Chromosomal Gain in Acute Myeloid Leukemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 792-792
Author(s):  
Letetia Jones ◽  
Sabina Sevcikova ◽  
Vernon Phan ◽  
Sachi Jain ◽  
Angell Shieh ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Selective Pressure ◽  
Rapid Development ◽  
Chromosome 15 ◽  
Wild Type ◽  
Trisomy 8 ◽  
Leukemic Transformation ◽  
Myc Gene ◽  
Acute Myeloid

Abstract Acute Myeloid Leukemia (AML) is a disease characterized by diverse genetic pathogenesis, including both balanced and unbalanced chromosomal aberrations. Much is known regarding the pathogenic effects of balanced rearrangements in AML, whereas our understanding of how unbalanced aberrations contribute to leukemia is more limited. The balanced t(15;17) chromosomal rearrangement is a nearly constant feature of acute promyeloctyic leukemia (APL), a subtype AML. The translocation fuses the promyelocytic leukemia gene (PML) to the retinoic acid receptor α gene (RARA). Trisomy 8 is the most common secondary karyotypic lesion observed in APL, and it has been speculated but not proven that the MYC gene contributes to this chromosomal gain. We previously reported that mouse chromosome 15, which contains the mouse Myc gene in a region syntenic to human chromosome 8q24, is commonly gained in the MRP8 PML-RARA mouse model of APL. We now report our work to assess the hypothesis that increased MYC cooperates with PML-RARα to accelerate disease and that gain of MYC/Myc drives +8 in humans and +15 in mice. Expressing MYC with a retroviral vector in PML-RARA bone marrow led to the rapid development of APL-like leukemias (3 months vs. 8.5 months with PML-RARA alone). Chromosome 15 was not gained in any of the leukemias, although 70% had other clonal karyotypic abnormalities. This finding suggests that when MYC is overexpressed, there is no selective pressure to gain chromosome 15, supporting our hypothesis that Myc is driving this gain. We also generated PML-RARA mice haploinsufficient for Myc to examine the effect of decreasing MYC levels. The median latency among leukemic animals was 258 days for mice with PML-RARA and two wild-type Myc alleles, whereas the latency was increased to 339 days for PML-RARA Myc haploinsufficient mice. Hence, lower MYC expression served as a check on leukemic transformation. Furthermore, the majority of the leukemias that arose in Myc haploinsufficient mice had gained wild-type Myc. These data demonstrate a selective pressure for Myc gain. Additional experiments showed that as MYC expression increases there is a decrease in both latency and genetic complexity of leukemias that arise, that MYC and PML-RARα interact to disrupt myeloid differentiation in vivo and that although MYC cooperates with PML-RARα to cause leukemia, additional events are required for completing transformation even at high levels of MYC. Altogether our studies of increased and decreased MYC expression in PML-RARA mice show a strong correlation between MYC dosage and leukemic transformation. Our results suggest that agents that target MYC might be useful for the treatment of AML.

Prevalence and Prognostic Significance of FLT3 Mutations in Acute Myeloid Leukemia: Association of ITDs with Poor Outcome in Patients with Normal Cytogenetics.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2017-2017
Author(s):  
Michela Palmisano ◽  
Emanuela Ottaviani ◽  
Tiziana Grafone ◽  
Nicoletta Testoni ◽  
Stefania Paolini ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Myeloid Leukemia ◽  
Prognostic Impact ◽  
Wild Type ◽  
Poor Prognostic Factor ◽  
Flt3 Mutations ◽  
Blast Cells ◽  
Normal Cytogenetics ◽  
Acute Myeloid

Abstract Acute Myeloid Leukemia (AML) is a difficult disease to treat, and better treatments are needed. Molecular targeted therapy represents a novel therapeutic approach. Activating mutations of FMS-like tyrosine kinase 3 (FLT3) are present in approximately one third of patients with de novo AML and have been implicated in its pathogenesis. The leukemic blasts of most AML patients have the internal tandem duplications (ITDs) in the juxtamembrane region or point mutations in Asp835 and Iso836 codons in the activation loop of the kinase domain (TKD) of the FLT3 receptor. Both mutations result in constitutive FLT3 receptor activity and may play a significant role in leukemogenesis. In this study we have analyzed the incidence and type of FLT3 mutations in a large series of newly diagnosed AML patients. Furthermore, we have evaluated the prognostic impact of FLT3 mutations. The FLT3/ITD was determined by polymerase chain reaction (PCR). The mutations of D835 and I836 codons were determined by PCR followed by restriction enzyme digestion (PCR-RFLP). For the estimation of the statistic significance of the differences in the clinical-biological characteristics, between the mutated patients and wild-type patients, it has been used the Student’s test t for independent data. The probabilities of overall survival (OS) and disease free survival (DFS) were analysed by Kaplan-Meier method; the differences of OS and DFS, between the mutated patients and wild-type patients, were assessed using the log-rank test. Both FLT3/ITD and FLT3/TKD mutations were found in 15%. Dual mutations were found in 2% of 126 patients. Among the FAB subtypes of AML, the rate of FLT3 aberrations was higher in M4 (27%) and M5 (26%). FLT3/ITD was associated to leukocytosis (106.8x10(e)9/l vs 30x10(e)9/l in FLT3-wt, p=0.015) and high percentage of circulating blast cells (82% vs 42% in FLT3-wt, p<0.0001). Differently, FLT3/TKD mutations were not associated with high white blood cells count and blast cells percentage. FLT3 mutations were more prevalent in patients with normal karyotype (51%). In this group, DFS and OS were significantly inferior for patients with FLT3/ITD than patients withouth mutations (0 vs 5, p=0.0032; 5 vs 9, p=0.049, respectively). We have identified the FLT3/ITD as an independent poor prognostic factor in AML patients with normal cytogenetics. Therefore, targeting FLT3 mutations represents a potential therapeutic target for AML. These results suggest that new treatment modalities, such as therapy with a FLT3 tyrosine kinase inhibitor, are clearly needed for this group of patients with “standard risk” profile.

Significant Role of Peptidyl-Prolyl cis/trans Isomerase, Pin1 in Acute Myeloid Leukemia with C/EBPα Mutations.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 55-55 ◽  
Author(s):  
John Anto Pulikkan ◽  
Viola Dengler ◽  
Abdul A. Peer Zada ◽  
Mulu Gelutu ◽  
Daniel G. Tenen ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Mrna Expression ◽  
Myeloid Leukemia ◽  
Dominant Negative ◽  
Pcr Analysis ◽  
Wild Type ◽  
Promoter Assay ◽  
Type C ◽  
Acute Myeloid

Abstract Transcription factor CCAAT enhancer binding protein α (C/EBPα) is crucial for the differentiation of granulocytes. Experimental data from animal models as well as patient samples suggest that loss of function or expression of C/EBPα provides a platform on which acute myeloid leukemia (AML) develops. C/EBPα is mutated in around 9% of acute myeloid leukemia. The mutations reported in C/EBPα are frame shift mutations at N-terminal domain and point mutations at basic region Leucine zipper. The mutant form of C/EBPα ie C/EBPα-p30 exhibits dominant negative function over the wild type protein. Peptidyl-prolyl cis/trans isomerase, Pin1 binds to and isomerizes the peptidyl-prolyl bond in specific phosphorylated Ser/Thr-Pro motifs. A growing number of studies show that Pin1 is overexpressed in many cancers and has significant role in tumorigenesis. In the present study we investigated the role of Pin1 in acute myeloid leukemia with C/EBPα mutation. Here we report C/EBPα-p30 could induce Pin1 transcription as assessed by quantitative Real-Time RT-PCR analysis. Affymetrix mRNA expression analysis show that Pin1 is upregulated in patients with acute myeloid leukemia. Silencing of Pin1 could overcome the dominant negative action of the C/EBPα-p30 over the C/EBPα-p42 transactivation capacity as analyzed by promoter assay. By silencing Pin1 with inhibitor against Pin1 (PiB), we could show myeloid differentiation in Kasumi-6 cells by FACS analysis with granulocytic specific markers. Western blot analysis shows that Pin1 inhibition by PiB could upregulate wild type C/EBPα protein level. Luciferase promoter assay for the Pin1 promoter shows that C/EBPα-p30 induces Pin1 promoter activity in association with E2F1. Mean while, wild type C/EBPα interferes with transactivation of the Pin1 promoter and downregulates Pin1 mRNA expression. We investigated the mechanism underlying the dominant negative action of C/EBPα-p30 over the wild type protein. We have previously shown that c-Jun expression is high in AML patients with C/EBPα mutation and c-Jun could block C/EBPα function by protein-protein interaction. Quantitative Real-Time RT-PCR analysis shows that overexpression of Pin1 induces c-Jun mRNA expression, while inhibition of Pin1 by the inhibitor PiB downregulates c-Jun mRNA expression. We show that c-Jun blocks the DNA binding and transactivation of wild type C/EBPα protein as assessed by gel shift assay and promoter assay respectively. In conclusion, inhibition of Pin1 leads to granulocytic differentiation of human myeloid cells. Our findings suggest inhibition of Pin1 as a novel strategy in treating AML patients with C/EBPα mutation.

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