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.

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.

scholarly journals Anti-Tumor Effects of BDH1 in Acute Myeloid Leukemia

2021 ◽  
Vol 11 ◽  
Author(s):  
Fei Han ◽  
Huanhuan Zhao ◽  
Jun Lu ◽  
Weina Yun ◽  
Lingling Yang ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Poor Survival ◽  
Hematopoietic Stem ◽  
Healthy Donors ◽  
Worse Prognosis ◽  
Acute Myeloid

Dysregulation of ketone metabolism has been reported in various types of cancer. In order to find out its role in acute myeloid leukemia (AML) pathogenesis, we first analyzed the expression levels of 10 key genes involved in ketone metabolism in AML blasts and CD34+ hematopoietic stem cells (HSCs) from healthy donors. We found that the expression level of BDH1 was significantly lower in AML than in normal HSCs. The downregulation of BDH1 gene expression in AML cell lines as compared with normal HSCs was further confirmed with real-time RT-PCR. Analysis of TCGA and other database revealed that the downregulation of BDH1 was associated with worse prognosis in AML patients. In addition, we showed that overexpression of BDH1 inhibited the viability and proliferation of AML cells. In contrast, BDH1 knock-down promoted AML cell growth. Collectively, our results suggest the previously unappreciated anti-tumor role of BDH1 in AML, and low BDH1 expression predicts poor survival.

Quantitative RT-PCR Analysis of the MOZ-CBP Fusion Transcript in Therapy-related Acute Myeloid Leukemia With t(8;16)(p11;p13)

2012 ◽  
Vol 34 (5) ◽  
pp. 402-405 ◽  
Author(s):  
Atsushi Fujiki ◽  
Toshihiko Imamura ◽  
Akiyo Furutani ◽  
Waka Hatano ◽  
Daisuke Asai ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Fusion Transcript ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Acute Myeloid

Highly Sensitive Quenching Probe (QProbe) Method Is Useful to Detect c-Kit Mutation and to Predict Relapse of t(8;21)(q22;q22) Acute Myeloid Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2646-2646
Author(s):  
Satoshi Wakita ◽  
Hiroki Yamaguchi ◽  
Yoshio Mitamura ◽  
Fumiko Kosaka ◽  
Koiti Inokuchi ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Stem Cell ◽  
Myeloid Leukemia ◽  
Direct Sequence ◽  
Wild Type ◽  
Kit Mutation ◽  
Highly Sensitive ◽  
Quenching Probe ◽  
Acute Myeloid

Abstract Abstract 2646 Poster Board II-622 Background: t(8;21)(q22;q22) acute myeloid leukemia (t(8;21) AML) show a high rate of complete remission (CR) and prolonged CR duration, especially following consolidation chemotherapy with high-dose cytarabine (HD-ARAC), and are thought to have a better prognosis than other AML patients. Nevertheless, only approximately 50% of patients were alive at 5 years. This suggests that some patients have more aggressive leukemic phenotypes and indicates the need for predictive molecular marker of relapse and treatment optimization with novel and/or more aggressive therapies such as stem cell transplantation.Recently, several groups reported that c-kit mutation (MutKIT) defined an unfavorable subgroup in t(8;21) AML. However MutKIT has been reported approximately 20% at diagnostic samples of core binding factor leukemia. It is possible that MutKIT are more high frequency at relapse sample of t(8;21) AML. In the present study, we analyzed samples collected at diagnosis and relapse to investigate the role of MutKIT s and Flt3 internal tandem duplication (ITD) in t(8;21) AML. Methods: We analyzed MutKIT and Flt3 ITD among 32 t(8;21) AML patients diagnosed between 1991 to 2009 at the Nippon Medical School. All of them were succeeded achieving CR, but 18 patients (56.3%) relapsed, became refractory chemotherapy, and poor prognosis. Exon 8 and 17 of MutKIT were analyzed by direct sequence and QProbe-system (ARKRAY, Inc. Kyoto, Japan). QProbe-system was high sensitivity mutation screening method, detected approximately 1% MutKIT using mutation specific guanine quenching probe (Leukemia Res 32 (2008) 1462–1467). FLT3 ITD was analyzed by PCR amplification. Results: Using direct sequence, MutKITs were found in 5 (18.5%) of the 27 patients at diagnosis (D816V: 3 patients AN822K: 2 patients), and 7 (46.6%) of 15 patients at relapse. Interestingly 3 patients were detected MutKIT at only relapse (D816V: 2 patients AN822K: 1 patient). All mutations found in exon 17 clustered within the A-loop. Next we analyzed to detected very slight amount of mutation by QProbe-system. N822K were newly found of the 3 patients at diagnosis in spite of negative by direct sequence Finally MutKIT were found 8 (29.6%) at diagnosis. Flt3 ITD were found in 2 (9.5%) of the 21 patents at diagnosis, and none of 15 patients at relapse. 2 patients with Flt3 ITD were not accompanied MutKIT. All of N822K newly found by QProbe-system and Flt3 ITD positive patients were relapsed but turn to mutation negative. To evaluate the importance of MutKIT as a predicted relapse, we analyzed the cumulative incidence of relapse (CIR) and relapse free survival (RFS) of these patients with Kaplan-Meier method. The CIR was higher for patients with MutKIT (p=0.040, Wild type c-kit (WtKIT): 45.8% vs MutKIT: 87.5%). In direct sequencing method, The RFS tend to be shorter for patient with MutKIT, but no significant differences (p=0.260; WtKIT: 30 months vs MutKIT: 11 months).On the other hand, in QProbe-system, the median RFS was shorter for patients with MutKIT (p=0.033; WtKIT: 64 months vs MutKIT: 11 months). In addition of FLT3-ITD analysis, median RFS was shorter for patients with MutKIT or FLT3 ITD (p=0.005; Wild type of both genes: 64 months vs MutKIT or FLT3 ITD: 10 months). Additionally, we compared overall survival (OS) of wild type of both genes with MutKIT or FLT3 ITD. The OS tend to be shorter for patient with MutKIT or FLT3 ITD, but no significant differences. Discussion: In this study, we confirmed the prognostic significance of MutKIT in patients with t(8;21) AML when we use the highly sensitive quenching probe method (QProbe-system). We showed that QProbe-system is quite useful in predicting the prognosis of patients with t(8;21) AML and in determining its therapeutic strategy including tyrosine kinase inhibitors and/or up-front stem cell transplantation. We also revealed the important role of MutKIT (46.7%) at relapse, but several questions were still remained. It is unknown for some patients how to gain MutKIT at only relapse and why mutations detected at diagnosis were disappeared at relapse. We need further study to clarify the function and commitment of MutKIT in relapse of t(8;21) AML. Disclosures: No relevant conflicts of interest to declare.

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.

scholarly journals Comprehensive Analysis of CBFβ-MYH11 Fusion Transcripts in Acute Myeloid Leukemia by RT-PCR Analysis

2004 ◽  
Vol 6 (1) ◽  
pp. 22-27 ◽  
Author(s):  
ShriHari S. Kadkol ◽  
Annette Bruno ◽  
Carol Dodge ◽  
Valerie Lindgren ◽  
Farhad Ravandi
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Comprehensive Analysis ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Fusion Transcripts ◽  
Acute Myeloid

PIN1 Blocks Granulocytic Differentiation Via C-Jun in Acute Myeloid Leukemia with CEBPA Mutations.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1205-1205
Author(s):  
John Anto Pulikkan ◽  
Viola Dengler ◽  
Abdul Peerzada ◽  
Stefan Bohlander ◽  
Daniel G. Tenen ◽  
...  
Keyword(s):  
Myeloid Leukemia ◽  
Dominant Negative ◽  
Myeloid Differentiation ◽  
Pcr Analysis ◽  
Wild Type ◽  
Granulocytic Differentiation ◽  
Protein Levels ◽  
Type C ◽  
Cebpa Mutations ◽  
The Stability

Abstract Transcription factor CCAAT enhancer binding protein α (C/EBPα) is the master regulator of myeloid differentiation programme. Tumor suppressor function of C/EBPα is shown by the deregulation of C/EBPα in AML by various mechanisms. Mutations in the CEBPA gene are reported in around 9% of patients with acute myeloid leukemia (AML). The CEBPA mutations reported are frame shift mutation at N-terminal domain, which results in C/EBPα-p30 and point mutations at the basic region Leucine zipper. The mutant form of C/EBPα (C/EBPα-p30) displays dominant negative function over the wild type protein and induces AML in mouse model. The mechanism for the dominant negative action of C/EBPα-p30 in AML is poorly understood. Peptidyl-prolyl cis/trans isomerase, PIN1 binds to and isomerizes specific pSer/Thr-Pro motifs. Recent studies show that PIN1 is overexpressed in many types of cancers and it acts as a crucial catalyst for oncogenesis. Here we investigated the role of PIN1 in AML with CEBPA mutations. We report C/EBPα-p30 could induce PIN1 mRNA and protein levels as revealed by quantitative Real-Time RT-PCR analysis and western blot analysis, respectively. Affymetrix mRNA expression analysis shows that PIN1 mRNA is upregulated in patients with AML. Interestingly, the wild type C/EBPα down regulates PIN1 mRNA and protein levels. Chromatin immunoprecipitation assays demonstrate that induction of C/EBPα-p30 results in recruitment of E2F1 in the PIN1 promoter. Silencing PIN1 with inhibitor against PIN1 (PiB) in AML blast cells with CEBPA mutation and in Kasumi-6 cells leads to myeloid differentiation as assessed by myeloid markers by FACS analysis and quantitative Real-Time RT-PCR analysis. Furthermore, PIN1 inhibition by PiB leads to upregulation of wild type C/EBPα protein level. Next, we investigated the mechanism through which the C/EBPα-p30 blocks the wild type protein. c-Jun expression has been shown to be high in AML patients with CEBPA mutation and c-Jun blocks C/EBPα function by proteinprotein interaction. In vivo ubiquitination assay demonstrates that PIN1 increases the stability of the c-Jun protein by inhibiting c-Jun ubiquitination. We also show that c-Jun blocks granulocytic differentiation mediated by C/EBPα as assessed by myeloid specific markers. In summary, C/EBPα-p30 induces PIN1 expression and increases the stability of c-Jun and c-Jun blocks the wild type C/EBPα. Our data suggest the inhibition of PIN1 could be a potential strategy in the treatment of AML patients with CEBPA mutation.

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.

BEX1, a Marker for Acute Myeloid Leukemia Cell Lines with MLL Rearrangements.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3013-3013
Author(s):  
Hilmar Quentmeier ◽  
Wilhelm G. Dirks ◽  
Robert Geffers ◽  
Cord C. Uphoff ◽  
Hans G. Drexler
Keyword(s):  
Gene Expression ◽  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Hox Genes ◽  
Expression Profiles ◽  
Pcr Analysis ◽  
Wild Type ◽  
Acute Leukemias ◽  
Acute Myeloid

Abstract Patients with acute leukemias carrying MLL rearrangements have a poor prognosis. The tumor cells show characteristic gene expression profiles with increased levels of selected HOX genes (1). We have shown that acute lymphoblastic leukemia (ALL) cell lines with and without MLL rearrangements could likewise be recognized by analysis of HOX gene expression (2). In contrast, MLL wild-type (MLLwt) and mutant (MLLmu) acute myeloid leukemia (AML) cell lines could not be distinguished by analysis of HOX genes, because even wild-type cell lines had a high expression background (2). It was our aim to find out whether MLLwt and MLLmu AML cell lines could be discriminated on the basis of gene expression - other than HOX genes. We performed gene expression analysis with pooled RNAs of MLLmu (n=8) and MLLwt (n=8) cell lines applying high density oligonucleotide Genechips from Affymetrix (HG-U133A). Defensin alpha4 (83x), defensin beta1 (32x), cathepsinG (9x) and FLT3 (7x) genes were overexpressed in MLLmu cell lines, stabilin1 (82x) and galectin10 (55x) in MLLwt cell lines. PCR analysis with individual (non-pooled) cDNAs of the 16 cell lines showed that none of the above genes was exclusively expressed by MLLmu or MLLwt cells. Thus, pooling RNAs has a major disadvantage: many PCRs have to be performed to establish faithful expression profiles for individual samples. BEX1 finally proved to be a gene that was exclusively overexpressed in one group of cell lines. It had been an interesting candidate already after oligonucleotide chip analysis, being both overexpressed (18x) in MLLmu cell lines, and - in contrast to the genes listed above - not a marker of myeloid differentiation. BEX1 is reportedly expressed in brain, testis and ovary, but not in peripheral blood leukocytes, lymph node and bone marrow (3). By RT-PCR analysis we showed that 7/8 MLLmu and 0/8 MLLwt cell lines expressed BEX1. Screening a panel of 54 hematopoetic cell lines gave the same result: BEX1 expression was restricted to MLLmu AML cell lines: 8/11 (73%) MLLmu AML cell lines expressed BEX1, but 43 other hematopoetic cell lines (including Hodgkin′s disease, anaplastic large cell lymphoma, ALL and MLLwt AML cell lines) tested negative. BEX1 expression may depend on the type of MLL rearrangement or the histological background of the cells, as MLLmu ALL cell lines (0/5) also tested negative. It has been shown in the mouse system, that BEX family members may be involved in cell signalling processes. Thus it will be interesting to elucidate whether BEX1 also participates in proliferative/antiapoptotic signalling processes in MLLmu AML cell lines.

scholarly journals The Oncogenic Role of N6-Methyladenosine Reader Protein IGF2BP3 in Acute Myeloid Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1334-1334
Author(s):  
Huilin Huang ◽  
Hengyou Weng ◽  
Mingli Sun ◽  
Huizhe Wu ◽  
Zhenhua Chen ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Viability ◽  
Progenitor Cells ◽  
Myeloid Leukemia ◽  
Poor Prognosis ◽  
Expression Level ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Acute Myeloid

Abstract RNA N6-Methyladenosine (m6A) modification is an abundant modification of internal mRNAs in eukaryotes and some viruses, which is dynamically and reversibly fine-tuned during normal and pathological bioprocesses. Recent studies have shown that m6A methyltransferases, METTL3 and METTL14, play important roles in maintaining self-renewal capacity of hematopoietic stem/progenitor cells (HSPCs) and promoting acute myeloid leukemia (AML) development (Barbiori et al., Nature, 2017; Vu et al., Nature Method, 2017; Weng et al. Cell Stem Cell, 2018). The m6A demethylase, FTO, was also shown to promote leukemic cell transformation and leukemogenesis in various type of AML (Li et al., Cancer Cell, 2017). However, little is known about the functions of m6A readers in malignant hematopoiesis. We recently reported that Insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) is an specific m6A binding protein, which recognize m6A transcripts through the K Homology (KH) domains to stabilize and promote translation of its target mRNAs (Huang et al., Nature Cell Biology, 2018). In analysis of TCGA AML dataset (n=157), we found that a higher expression level of IGF2BP3 is significantly associated with a poor prognosis in AML patients (p<0.001; Median overall survival: IGF2BP3-High vs. IGF2BP3-Low = 11 months vs. 31 months). In addition, we analyzed our in-house microarray profiling of 113 AML patient samples and found that IGF2BP3 is highly expressed in mononuclear cells (MNC) from MLL-rearranged leukemia patients as compared to those from healthy donors (p<0.05) or non-MLL-rearranged leukemic patients (p<0.001). Consistent with the overexpression of IGF2BP3 in human MLL-rearranged AML, MLL-AF9 or MLL-AF10 transformed mouse hematopoietic stem/progenitor cells (HSPCs; herein mouse lineage negative (Lin-) bone marrow cells) showed a >10 fold increase in expression level of Igf2bp3, compare to the non-transformed counterpart HSPCs. Furthermore, in analysis of 562 samples from adult patients with AML (GSE37642), we found that within cytogenetically normal human AML, patients carrying FLT3-ITD mutation showed a significantly higher level of IGF2BP3 expression than those without FLT3-ITD mutation (p<0.01). To investigate the potential oncogenic role of IGF2BP3 in AML, we cotransduced mouse Lin- BM progenitor cells with MLL-AF9 and three individual shRNAs targeting Igf2bp3 or a scrambled control shRNA and performed colony-forming/replating assays. Knockdown of Igf2bp3 significantly (p<0.05) reduced the colony-forming capacity of MLL-AF9-transduced HSPCs to 20-50% of that of the control group. Conversely, forced expression of wild-type IGF2BP3 significantly (p<0.05) promoted colony formation of MLL-AF9-transduced Lin- BM progenitor cells. Such promotion was almost completely impaired when KH3-4 domain of IGF2BP3 was mutated or when Mettl14 was depleted, suggesting that IGF2BP3 exerts its oncogenic function as an m6A reader through an m6A-dependent mechanism. We further used human leukemia cell lines to investigate the function of IGF2BP3 in human AML cells. Silencing of IGF2BP3 by two shRNAs significantly inhibited cell viability and proliferation and induced cell apoptosis (p<0.01) in MonoMac6 AML cell line which harbors the t(9;11) translocation. In Molm13 and MV4-11 AML cells which are heterozygous and homozygous for the FLT3-ITD mutation, respectively, a further decrease of cell viability and increase of apoptotic cells upon IGF2BP3 knockdown was observed compared to MonoMac6 with wild-type FLT3. Mechanically, through cross-linking immunoprecipitation sequencing (CLIP-seq), we showed that IGF2BP3 targets mRNAs in cell cycle, DNA replication and protein synthesis pathways. Taken together, these results demonstrated the oncogenic role of the new m6A reader protein IGF2BP3 in AML. Given the fact that expression of IGF2BP3 correlates with an overall poor prognosis in AML, IGF2BP3 is likely a promising therapeutic target for AML treatment. Disclosures No relevant conflicts of interest to declare.

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