PHD Domain Deletion Mutations of ASXL1 Promote Myeloid Leukemia Transformation Through Epigenetic Dysregulation and Inhibit Megakaryocytic Differentiation Through the Inactivation of FOSB in K562 Cells.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2393-2393 ◽  
Author(s):  
Rabindranath Bera ◽  
Der-Cherng Liang ◽  
Ming-Chun Chiu ◽  
Ying-Jung Huang ◽  
Sung-Tzu Liang ◽  
...  
Keyword(s):  
Myeloid Leukemia ◽  
Molecular Mechanisms ◽  
Blast Crisis ◽  
K562 Cells ◽  
Specific Cell ◽  
Wild Type ◽  
Megakaryocytic Differentiation ◽  
Deletion Mutations ◽  
Phd Domain

Abstract Abstract 2393 Somatic mutations of ASXL1 gene have been described in patients with myeloid malignancies and were associated with inferior outcomes. ASXL1 mutations have also been detected in myeloid blast crisis of chronic myeloid leukemia (CML) patients. The mechanisms of acute myeloid leukemia (AML) transformation and functional role of ASXL1 mutations in the leukemogenesis remain to be determined. Recently, we identified PHD domain deletion mutations (R693X and L885X) in patients with CML in myeloid blast crisis and/or AML with minimal differentiation (M0). In the present study, we aimed to investigate the role of PHD domain deletion mutations in the pathogenesis of AML transformation. The K562 cells carrying Philadelphia chromosome, serves as a model to study the molecular mechanisms associated with leukemogenesis. Our result showed that R693X/L885X mutations inhibited PMA-treated megakaryocytic differentiation with the change of physiological characteristic features and suppressed the induction of CD61, a specific cell surface marker of megakaryocytes. We also found that FOSB, a member of Fos family of AP-1 transcription factors was down-regulated in K562 cells expressing R693X and L885X compared to wild-type ASXL1 during PMA-mediated megakaryocytic differentiation. Examination of intracellular signaling pathways showed that the mutant ASXL1 protein prevented PMA-induced megakaryocytic differentiation through the inactivation of ERK, AKT and STAT5 which are required for differentiation. Further, ASXL1 depletion by shRNA in K562 cells led to enhanced cell proliferation, increased colony formation and impaired PMA-mediated differentiation. Previous studies in Drosophila had revealed that Asxl forms the protein complexes of both Trithorax and Polycomb groups that are required for maintaining chromatin in both activated and repressed transcriptional states. By using Western blot analysis, we demonstrated that PHD domain deletion mutations of ASXL1 significantly suppressed the transcriptionally repressive mark H3K27 trimethylation, however no effect on methylated H3K4 (H3K4me2 and H3K4me3), an active histone mark in K562 cells. Co-immunoprecipitation analysis revealed that wild-type, but not PHD domain deletion mutations of ASXL1 interact with EZH2, a member of the polycomb repressive complex 2 (PRC2). Importantly, PHD deletion mutations or downregulation of ASXL1 resulted in the suppression of EZH2 in K562 cells. Our study demonstrated that PHD deletion mutations of ASXL1 resulted in a loss-of-function which exhibited direct effects on the proliferation and differentiation and also proposed a specific role for ASXL1 in epigenetic regulation of gene expression in K562 cells. Disclosures: No relevant conflicts of interest to declare.

FLT3 Is Up-Regulated in Blast Crisis of Chronic Myeloid Leukemia and Combination of Small Interference RNA of FLT3 and Gleevec (STI571) Synergistically Induces the Apoptosis in K562 Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4532-4532
Author(s):  
Young Y. Lee ◽  
Kwang-Sung Ahn ◽  
Sung-Soo Yoon ◽  
Jung H. Choi ◽  
Byoung B. Park ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Blast Crisis ◽  
Chronic Phase ◽  
K562 Cells ◽  
Pcr Analysis ◽  
Wild Type ◽  
Small Interference Rna ◽  
Expression Levels ◽  
Significant Difference

Abstract To identify a gene signature for prognostic markers at transition from chronic phase to blast crisis of chronic myeloid leukemia (CML), we have applied Affymetrix Genechips of 22,000 transcripts to analyze total RNA of CML cells from 12 patients with chronic phase and 12 patients with blast crisis. Data analysis using GeneSpring 6.0 generated a list of 143 differentially expressed genes. A total of 89 genes were up-regulated and 54 genes were down-regulated in blast crisis of CML, and vice versa in chronic phase of CML. Array data for 32 genes was validated using quantitative realtime PCR analysis. The expression levels of HSA6591, FLT3, NTE5, RSG1, LAF4, CPA3, ATF, FCGR3A, MYD88, IFIT1, TP73L, DTNA, MDA, and IL18R1 showed statistically significant difference (p < 0.05) between chronic phase and blast crisis. Since CML cells of blast crisis were generally unresponsive to STI571, we further analyzed roles of FLT3 which is known to be a poor prognositic marker in acute myeloid leukemia. For this experiment, K562 cells (CML blast cells) were transfected with small hairpin RNAs (shRNAs), also referred to as small interfering RNAs, to target human FLT3, resulting in the significant inhibition of FLT3 expression at mRNA and protein levels. MTT assay demonstrated that FLT3 knockdown K562 cells by shRNAs were more sensitive to STI571 compared to wild type of K562, although there was no difference at high concentration of STI571 (320 nM) between FLT3 knockdown K562 cells and wild type of K562 cells. The higher expression levels of apoptosis related genes (PARP, caspase-3, Bax) were observed in FLT3 knockdown K562 cells compared to wild type of K562 cells. Thus, RNA interference-directed targeting of FLT3 might be a novel treatment modality in STI571 refractory CML patients.

scholarly journals The Embryonic Program Activated during Blast Crisis of Chronic Myelogenous Leukemia (CML) Implicates a TCF7L2 and MYC Cooperative Chromatin Binding and Represents a Druggable Target

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1632-1632
Author(s):  
Christophe Desterke ◽  
Sarah Pagliaro ◽  
Patricia Hugues ◽  
Annelise Bennaceur-Griscelli ◽  
Ali G Turhan
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Blast Crisis ◽  
Embryonic Stem ◽  
K562 Cells ◽  
The United States ◽  
Myelogenous Leukemia ◽  
P Value ◽  
Chip Sequencing

Chronic myeloid leukemia (CML) is characterized by an inherent genetic instability, which contributes to the progression of the disease towards accelerated and blast crisis (BC). The occurrence of the latter has been hampered by the use of tyrosine kinase inhibitors (TKI), which changed this natural progression, but BC still occurs in patients resistant to TKI. Several cytogenetic (major and minor routes) and genomic (TP53 mutation, p16/INK4A deletions, DNA repair abnormalities such as BRCA1, DNA-PKcs, hnRNP metabolism) events have been reported in the progression towards BC. Previous data have also suggested the involvement of embryonic stem cell program activated in BC cells such as Lin28A. In this work, we have taken advantage of the previously reported gene profiling of BC in a large cohort of patients (Radich et al. 2006) and found a correlation between blast numbers and the involvement of the Transcription Factor 7 like 2 (TCF7L2) in BC. TFC7L2 is a member of the TCF family of proteins that are known to activate WNT target genes such as Cyclin D1. TCF7L2 has been shown to be overexpressed in acute myeloid leukemia (AML) and represents a druggable target (Saenz et al Leukemia 2019). The involvement of TCF7L2 in CML-BC and its interaction with the epigenetic regulators has not been studied so far. The gene correlation study that we have performed using the blast numbers and the expression of TCF7L2 in CD34+ CML cells was found to be highly significant (Pearson test, r = 0.56, p-value=5.2e-4) (Fig.1A). TCF7L2 promoter was classed as active in K562 with ChromHMM Functional genomic analysis. K562 epigenetics peaks of TCF7L2 CHIP-seq were found principally mapped in proximal promoters (39% of the peaks, -3000pb upstream Transcription Starting Sites (TSS), Fig. 1B) and 183 unique peaks matched with promoter of 144 unique genes found to be correlated to the blast number in blood of the CML patients during BC (Fig 1C). This TCF7L2-dependent BC program was characterized to be active because promoters were also found positive for H3K27Ac and negative for H3K27Me3 histone marks, and functionally enriched with binding sites for MYC/MAX interactions (p=1.15e-6). The analysis of CHIP-sequencing of MYC revealed a significant overlapping of TCF7L2 epigenetic program with MYC (fold enrichment: 20.81, p < 2.2e-16). Surprisingly, the TCF7L2 program was found independent of RUNX1 and GATA2 transcriptional program. To determine these potential interactions, we have designed experiments in K562 cell line using the b-catenin activator Lithium Chloride (LiCL2) and the Myc/Max dimerization inhibitor 10058-F4. K562 cells were cultured in the presence of LiCL2 (10mM & 24hours) and the compound 10058-F4 (64µM & 48hours) and the expression of three epigenetic targets was analyzed by Q-RT-PCR in the presence of DMSO controls. The three targets chosen were protein arginine N-methyltransferase (PRMT1), the ATPase/Helicase RUVBL1 and the WD-repeat containing protein WDR77. As expected, after culture with LiCL2, the expression of PRMT1 was increased x 6.3 fold (p=8.49e-13) , that of RUVBL1 by x 1.66 Fold (p=1.67e-6) and that of WDR77 by x 2 fold (p=4.97e) (Fig.1D). On the contrary, the culture of K562 cells in the presence of MYC/MAX inhibitor 10058-F4, decreased the expression of 3 targets as compared to DMSO controls (x 1.6 fold for PRMT1, p=6.52e-5; x2 fold reduction for RUVBL1, p-value=2.71e-5; and x 1.4 fold for WDR77, p =0.0000643). These results show for the first time a cooperative role of TCF7L2 and MYC during blast crisis of CML and provide mechanistic insights into the interactions for the role of MYC in CML blast crisis. In addition they strengthen previous data showing a possible embryonic footprint in the blast development over the hematopoietic differentiation program during progression of the disease and provide a rationale for the pharmacological targeting of BC by the use of MYC/MAX inhibitors such as 10058-F4. Experiments are underway to evaluate the role of these factors and the MYC/MAX inhibitors in primary CML samples. Reference : Radich JP, Dai H, Mao M, Oehler V, Schelter J, Druker B, Sawyers C, Shah N, Stock W, Willman CL, Friend S, Lindsey PS.(2006) :Gene Expression Changes Associated with Progression and Response in Chronic Myeloid Leukemia. Proceedings of the National Academy of Sciences of the United States of America 103 (8): 2794-99. Disclosures Turhan: Incyte: Consultancy, Honoraria; novartis: Honoraria, Research Funding.

Role of eIF3a in 4-amino-2-trifluoromethyl-phenyl retinate-induced cell differentiation in human chronic myeloid leukemia K562 cells

Gene ◽  
2019 ◽  
Vol 683 ◽  
pp. 195-209 ◽  
Author(s):  
Ge Li ◽  
Ke Wang ◽  
Yue Li ◽  
Jinging Ruan ◽  
Cong Wang ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
K562 Cells

scholarly journals X chromosome choice occurs independently of asynchronous replication timing

2005 ◽  
Vol 168 (3) ◽  
pp. 365-373 ◽  
Author(s):  
Joost Gribnau ◽  
Sandra Luikenhuis ◽  
Konrad Hochedlinger ◽  
Kim Monkhorst ◽  
Rudolf Jaenisch
Keyword(s):  
X Chromosome ◽  
Molecular Mechanisms ◽  
Replication Timing ◽  
S Phase ◽  
X Inactivation ◽  
Wild Type ◽  
Gene Deletions ◽  
Asynchronous Replication ◽  
Skewed X Inactivation

In mammals, dosage compensation is achieved by X chromosome inactivation in female cells. Xist is required and sufficient for X inactivation, and Xist gene deletions result in completely skewed X inactivation. In this work, we analyzed skewing of X inactivation in mice with an Xist deletion encompassing sequence 5 KB upstream of the promoter through exon 3. We found that this mutation results in primary nonrandom X inactivation in which the wild-type X chromosome is always chosen for inactivation. To understand the molecular mechanisms that affect choice, we analyzed the role of replication timing in X inactivation choice. We found that the two Xist alleles and all regions tested on the X chromosome replicate asynchronously before the start of X inactivation. However, analysis of replication timing in cell lines with skewed X inactivation showed no preference for one of the two Xist alleles to replicate early in S-phase before the onset of X inactivation, indicating that asynchronous replication timing does not play a role in skewing of X inactivation.

scholarly journals Transfected leukocyte integrin CD11b/CD18 (Mac-1) mediates phorbol ester-activated, homotypic cell:cell adherence in the K562 cell line

Blood ◽  
1993 ◽  
Vol 82 (8) ◽  
pp. 2537-2545 ◽  
Author(s):  
DD Hickstein ◽  
E Grunvald ◽  
G Shumaker ◽  
DM Baker ◽  
AL Back ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Activation ◽  
Blast Crisis ◽  
K562 Cells ◽  
Myelogenous Leukemia ◽  
Cell Clones ◽  
A Cell ◽  
Neutrophil Adherence ◽  
The Individual

Abstract The CD11b/CD18 leukocyte integrin molecule mediates diverse neutrophil adherence-related functions, including cell:cell and cell:extracellular matrix attachments. To study the individual role of this leukocyte integrin in cell adherence in hematopoietic cells, we expressed the CD11b/CD18 complex on the surface of K562 cells, a cell line derived from an individual with chronic myelogenous leukemia in blast crisis. We used an amphotrophic retroviral vector designated LCD18SN, harboring the complete coding sequence for the CD18 subunit, to transfer the CD18 cDNA into K562 cells and select stable cell lines. The CD11b subunit in the expression plasmid pREP4 was transfected into these K562/CD18 cells by electroporation and stable cell clones were selected. These K562 cells possessed RNA and intracellular protein for each subunit, and they expressed the CD11b/CD18 heterodimer on the cell surface. When CD11b/CD18 expressing K562 cells were stimulated with phorbol myristate acetate (50 ng/mL) for 24 to 48 hours, these K562 cells formed dense cell:cell aggregates. This homotypic aggregation required both activation of the CD11b/CD18 complex and the induction of the counter- receptor for CD11b/CD18 on the conjugate cell. This cell line will (1) enable the structure-function relationships between cell activation and homotypic adherence to be assessed, (2) provide the opportunity to identify accessory molecules required for activation of the CD11b/CD18 complex, and (3) facilitate the identification of novel ligands for the CD11b/CD18 complex.

scholarly journals Molecular biology of bcr-abl1–positive chronic myeloid leukemia

Blood ◽  
2009 ◽  
Vol 113 (8) ◽  
pp. 1619-1630 ◽  
Author(s):  
Alfonso Quintás-Cardama ◽  
Jorge Cortes
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Kinase Inhibitors ◽  
Current Knowledge ◽  
Blast Crisis ◽  
Chronic Phase ◽  
Oncogenic Signaling ◽  
Stem Cell Quiescence ◽  
Oncogenic Signaling Pathways

Abstract Chronic myeloid leukemia (CML) has been regarded as the paradigmatic example of a malignancy defined by a unique molecular event, the BCR-ABL1 oncogene. Decades of research zeroing in on the role of BCR-ABL1 kinase in the pathogenesis of CML have culminated in the development of highly efficacious therapeutics that, like imatinib mesylate, target the oncogenic kinase activity of BCR-ABL1. In recent years, most research efforts in CML have been devoted to developing novel tyrosine kinase inhibitors (TKIs) as well as to elucidating the mechanisms of resistance to imatinib and other TKIs. Nonetheless, primordial aspects of the pathogenesis of CML, such as the mechanisms responsible for the transition from chronic phase to blast crisis, the causes of genomic instability and faulty DNA repair, the phenomenon of stem cell quiescence, the role of tumor suppressors in TKI resistance and CML progression, or the cross-talk between BCR-ABL1 and other oncogenic signaling pathways, still remain poorly understood. Herein, we synthesize the most relevant and current knowledge on such areas of the pathogenesis of CML.

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.

PTEN Regulates VEGF, VEGFR1 Expression and Its Clinical Significance in Myeloid Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1001-1001
Author(s):  
Zhiyong Cheng ◽  
Lin Pan ◽  
Xiaoling Guo ◽  
Xuejun Zhang ◽  
Fuxu Wang
Keyword(s):  
Clinical Significance ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
K562 Cells ◽  
Hebei Province ◽  
High Expression ◽  
Pten Gene ◽  
Wild Type ◽  
Akt Phosphorylation ◽  
Pten Mrna

Abstract Abstract 1001 Poster Board I-23 Phosphatase and tensin homology deleted on chromosome ten (PTEN) as a novel tumor suppressor gene, plays an important role in regulating proliferation, apoptosis, invasion and migration of many cancer cells. PTEN also modulates angiogenesis mediated by vascular endothelial growth factor (VEGF) via down-regulating the activity of PI3K/Akt pathway in many solid tumors. However, in myeloid leukemia, the effects of PTEN on VEGF and VEGFR1 (FLT1) mediated angiogenesis, migration, invasion of leukemia cells and its clinical significance are still unknown. Therefore, in the present study, we investigated the effect of PTEN on the activity of PI3K/Akt and VEGF/FLT1 pathways. Wild type PTEN gene was transfected into K562 cells, a cell line establish from a chronic myelogenous leukemia in blast crisis, to induce high expression of wild-type PTEN gene and protein by the cells. The correlation between the expression levels of PTEN and VEGF/FLT1 and its clinical significance in myeloid leukemia patients were also observed. We found that the expression reconstitution of wild-type PTEN had significance effect on inhibiting proliferation, migration and invasion ability of K562 cells via down-regulation of Akt phosphorylation and inhibition of VEGF/FLT1 expression. In myeloid leukemia patients, a negative correlation was found between the expression level of PTEN mRNA and that of VEGF and FLT1 mRNA. Low expression of PTEN mRNA and high expression of VEGF and FLT1 mRNA indicated a higher tendency of extramedullary disease in acute myeloid leukemia patients. Taken together, our findings indicated that PTEN could modulate the function of VEGF/VEGFR signaling pathway via down regulating Akt phosphorylation and that PTEN would be a candidate target for the treatment of myeloid leukemia. Disclosures: Pan: Nature science foundation of Hebei Province: Research Funding; Research Fund for the Doctoral Program of Higher Education of China: Research Funding; Emphases follow up pregram of Health Bureau of Hebei Province: Research Funding.

Identification of Rab5 as a Gene Involved in Chronic Myeloid Leukemia (CML) Progression.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3470-3470
Author(s):  
Daniela Cilloni ◽  
Monica Pradotto ◽  
Francesca Messa ◽  
Francesca Arruga ◽  
Enrico Bracco ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Western Blot ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Blast Crisis ◽  
Small Gtpases ◽  
Loss Of Function ◽  
Imatinib Resistance ◽  
Protein Levels

Abstract Abstract 3470 Poster Board III-358 The role of Bcr-Abl in the pathogenesis of Chronic Myeloid Leukemia (CML) is well established, however, the mechanisms leading to CML progression remain poorly understood. By using our model of transgenic Drosophila Melanogaster (Dm) for human Bcr-Abl driven CML we have identified Rab5 as a gene involved in the regulation of CML progression. The Rab5 is a member of gene family small GTPases which are involved in the regulation of vesicular transport. Lately several important reports have linked some members of the Rab family to invesivness and migration of cancer cells. Rab5 is associate with alpha-integrin subunits and modulates their endosomal traffic and subcellular localization. We have observed that a loss of function of Rab5 gene have induced a worsening of the CML phenotype generated by hBcr-Abl expression. In contrast, Rab gain of function rescued Bcr-Abl phenotype. The aim of the study was to evaluate the expression of Rab5 in CML cells to better understand if a potential correlation with progression, which has been observed in the model, could be confirmed in patients. Methods Rab5 gene expression was measured by Real Time PCR in 90 samples from 80 CML patients (32 PB and 58 BM). Among those, 53 are collected at diagnosis (19 of 53 patients have been enrolled in TOPS study). In addition, 9 samples from in CP patients have been collected at the time of imatinib resistance, 7 in accelerated phase and 11 in BC. In 14 patients, genes expression was analyzed during remission as, well. In parallel, 21 healthy donors (10 PB and 11 BM) have been evaluated. Rab5 protein expression was investigated by Western Blot and Immunofluorescence. We have also utilized K562 transfected with Rab5 plasmid, which we have generated to gain insight about the effects of Rab5 on cell proliferation and apoptosis. Results Rab5 transfection and overexpression in K562 significantly reduced proliferation and affected apoptosis. We found that in CML patients Rab5 expression levels were significantly decreased in either BM or PB (p<0.001 and p<0.0001) as compared to healthy subjects. Furthermore, in blast crisis samples we have found Rab5 transcripts levels to be further decreased. In contrast, at the time of remission, the transcript levels were comparable to normal values. Our preliminary analysis of samples from TOPS trial have shown a trend that Rab5 levels are lower among those patients achieving MMR by 12 months, when compared to the group of patients non achieving MMR on 400 mg, but that difference was not statistically significant (p=0.2). Among those randomized to receive imatinib 800 mg the difference was statistically significant with a median value among those achieving MMR of 1.27 vs 2.14 in the group without MMR (p=0.04). The protein levels have been analyzed by Western Blot and immunofluorescence and allow us to show detectable levels of Rab5 in samples collected at remission, but undetectable levels in course of active CML disease. Although preliminary, our results show a significant decrease of Rab5 expression in blast crisis samples, when compared to CP CML and healthy volunteers, which suggest a role of Rab5 in slowing down or suppressing a progression. Surprisingly, among CP CML patients the responders to TKI therapy have been detected to express a lower level of Rab5 than non responders. We are conducting further studies to better explain these data, which we find intriguing and suggesting that molecular factors involved in the regulation of CML progression could be uncoupled from the mechanisms regulating response to TKI therapy. Supported by Novartis Oncology, Clinical Development, TOPS Clinical Correlative Studies Network Disclosures No relevant conflicts of interest to declare.

Molecular Mechanism Regulating Foxo In Leukemia Initiating Cells of Chronic Myeloid Leukemia.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3391-3391
Author(s):  
Kazuhito Naka ◽  
Takayuki Hoshii ◽  
Yuko Tadokoro ◽  
Takako Ooshio ◽  
Yukio Kondo ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Chronic Myeloid Leukemia ◽  
Nuclear Localization ◽  
Myeloid Leukemia ◽  
Cellular Localization ◽  
Molecular Mechanisms ◽  
Imatinib Treatment ◽  
Cytoplasmic Localization ◽  
Wild Type ◽  
Imatinib Resistant

Abstract Abstract 3391 Chronic myeloid leukemia (CML) is caused by a defined genetic abnormality that generates BCR-ABL, a constitutively active tyrosine kinase. Although the development of imatinib, a small molecule inhibitor of ABL, represents a breakthrough in the treatment of CML, major part of patients treated in chronic phase CML are not off therapy due to resistance or intolerance. Recent studies have suggested that imatinib is a potent inhibitor against differentiated leukemia cells, but does not deplete leukemia-initiating cells (LICs) responsible for recurrence of CML. To date, therapeutics that can eradicate CML LICs, however, have remained under investigation. To overcome these clinical problems, here we studied the molecular mechanisms regulating maintenance of imatinib-resistant CML LICs by forkhead transcription factor Foxo3a. We first generated a mouse CML model by using retroviral induction of BCR-ABL-ires-GFP gene into mouse immature hematopoietic cells, and the cells were subsequently transplanted into irradiated recipient mice. These experiments showed that CML LICs were highly enriched in c-Kit+Lin−Sca-1+ (KLS+) population in BCR-ABL+ CML cells. Serial transplantation experiments for CML LICs originated from Foxo3a-deficient mice and littermate wild-type mice indicated that Foxo3a-deficiency reduced lethality of recipient mice at third transplantation. Although recipients that transplanted with wild-type LICs developed CML and acute lymphocytic leukemia (ALL) at third transplantation, we did not observe development of ALL or CML in recipients of Foxo3a deficient LICs after 45 days post-third transplantation, suggesting that the Foxo3a deficient LICs lose their potential to generate malignancies. In addition, a combination of Foxo3a deficiency and imatinib treatment led to efficient depletion of CML in vivo, indicating that Foxo3a plays an essential role for the maintenance of imatinib-resistant CML LICs (Naka et al., Nature 463, 676–680, 2010). Interestingly, when we examined sub-cellular localization of Foxo3a transcription factor in the CML LICs, we found two CML LIC populations; one population was the cells with nuclear localization of Foxo3a (Foxo3a transcription factor is active) and the other population was the cells with cytoplasmic localization of Foxo3a (Foxo3a is inactive). To understand the molecular mechanisms regulating Foxo3a in CML LICs, we next evaluated the activity of upstream BCR-ABL, PI3K, PDK1, and Akt signaling pathway by fluorescence immunohistochemistry. BCR-ABL activity that was determined by phosphorylation levels of CrkL, a down-stream target of BCR-ABL, was detected in almost all of the CML LICs. However, unexpectedly, phosphorylation levels of Akt in the CML LICs with nuclear localization of Foxo3a appeared to be lower than that in the CML LICs with cytoplasmic localization of Foxo3a, despite it is widely believed that BCR-ABL induces activation of Akt signal. Consistent with Akt phosphorylation status, we detected low levels phosphorylation of PDK1 and PI3K p85, upstream regulators for Akt, in the CML LICs with nuclear localization of Foxo3a. Interestingly, expression levels of the cell proliferation antigen Ki67 were lower in the CML LICs with nuclear Foxo3a than that in the CML LICs with cytoplasmic Foxo3a. These results suggest that Foxo3a responsible for maintenance of imatinib-resistant CML LICs may be regulated by molecular mechanisms that are involved in dormancy in CML LICs. Disclosures: No relevant conflicts of interest to declare.

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