Stat5 Is Essential for BCR-ABL-Transformed Chronic Myeloid Leukemia (CML) Associated with Increased CCN3 Gene Expression.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3271-3271
Author(s):  
Bing-Mei Zhu ◽  
Mark Wickre ◽  
Risu Na ◽  
Peter Klover ◽  
Cyril Martin ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Disease Model ◽  
Gene Profiling ◽  
Growth Factor Signaling ◽  
Downstream Target ◽  
Cellular Processes ◽  
Ccn3 Expression

Abstract Abstract 3271 Poster Board III-1 Introduction: Signal transducers and activators of transcription5 (Stat5) proteins are involved in many cellular processes through mediated cytokine, hormone, and growth factor signaling. But its role in disease pathogenesis has not been fully elucidated. Recently, activation of BCR-ABL has been reported to regulate a novel gene, CCN3 in cell lines and primary cells derived from chronic myeloid leukemia (CML) patients. To investigate the function of Stat5 in CML initiation and maintenance and determine the downstream target genes on Jak-Stat5 pathway, we developed a BCR-ABL - induced CML - like disease model by using retro-viral infection in Cre-mediated Stat5 knockout transgenic mice and analyzed the progress of CML. We also used Stat5 knockout (Stat5 KO) mice to perform gene profiling. Results: Our study showed that loss of Stat5 resulted in increased survival rate and remission of CML. Microarray analysis showed that CCN3 expression was down-regulated in KL cells derived from Stat5 KO mice. BCR-ABL-activated Stat5 increased expression level of CCN3 in CML cells. We further determined that Stat5 binds to CCN3 promoter region in IL-3 stimulated 32D cells and BCR-ABL-induced CML cells. Conclusions: Our study suggested that Stat5 is essential for BCR-ABL transformed CML and that CCN3 is involved in normal hematopoiesis and CML development. Further study will be necessary to uncover the function of CCN3 and more targets of Stat5 pathway in CML development and discover the therapeutic significance. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Phosphorylation-Dependent Differences in CXCR4-LASP1-AKT1 Interaction between Breast Cancer and Chronic Myeloid Leukemia

Cells ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 444 ◽  
Author(s):  
Elke Butt ◽  
Katrin Stempfle ◽  
Lorenz Lister ◽  
Felix Wolf ◽  
Marcella Kraft ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Kinase Inhibitor ◽  
Binding Partner ◽  
Downstream Target ◽  
Cellular Processes ◽  
Direct Binding ◽  
Chemokine Receptor 4 ◽  
Inhibitor Treatment

The serine/threonine protein kinase AKT1 is a downstream target of the chemokine receptor 4 (CXCR4), and both proteins play a central role in the modulation of diverse cellular processes, including proliferation and cell survival. While in chronic myeloid leukemia (CML) the CXCR4 is downregulated, thereby promoting the mobilization of progenitor cells into blood, the receptor is highly expressed in breast cancer cells, favoring the migratory capacity of these cells. Recently, the LIM and SH3 domain protein 1 (LASP1) has been described as a novel CXCR4 binding partner and as a promoter of the PI3K/AKT pathway. In this study, we uncovered a direct binding of LASP1, phosphorylated at S146, to both CXCR4 and AKT1, as shown by immunoprecipitation assays, pull-down experiments, and immunohistochemistry data. In contrast, phosphorylation of LASP1 at Y171 abrogated these interactions, suggesting that both LASP1 phospho-forms interact. Finally, findings demonstrating different phosphorylation patterns of LASP1 in breast cancer and chronic myeloid leukemia may have implications for CXCR4 function and tyrosine kinase inhibitor treatment.

Chronic Myeloid Leukemia with Deletions on Der(9) Shows Mir-199b Downregulated Expression.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1083-1083
Author(s):  
Francesco Albano ◽  
Luisa Anelli ◽  
Antonella Zagaria ◽  
Alessandra Pannunzio ◽  
Antonella Russo Rossi ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Target Genes ◽  
Expression Level ◽  
Regulation Of Transcription ◽  
Fish Analysis ◽  
Biological Processes ◽  
Relative Expression ◽  
Genomic Deletions ◽  
Mirnas Expression

Abstract MicroRNAs (miRNAs) are small, single stranded non-coding RNAs, 19–24 nucleotides long, involved in crucial biological processes, including differentiation, apoptosis and proliferation. Recent evidence indicates that miRNAs may play an important role in tumorigenesis; changes in miRNAs expression level were identified in many types of human hematological and solid malignancies. To date, several papers have reported the occurrence of genomic deletions flanking the breakpoint on der(9)t(9;22) in 10%–18% of patients with chronic myeloid leukemia (CML). The most probable consequence of der(9) deletions is the loss of tumour suppressor genes, conferring a proliferative advantage to the Philadelphia-positive clone. On the other hand, two miRNAs, namely miR219-2 and miR-199b, are found to map centromeric to the ABL gene within the chromosomal region at 9q34 that is frequently lost in CML patients with der(9) deletions. In this study, we investigated the loss of miR-219-2 and miR-199b by fluorescence in situ hybridization (FISH) analysis with specific bacterial artificial chromosome (BAC) probes in 68 CML cases bearing der(9) deletions. We further evaluated miR-219-2 and miR- 199b expression levels by quantitative real-time polymerase chain reaction (qRT-PCR) experiments in cases showing deletions of at least one of these miRNAs. Depending on RNA sample availability, miRNAs expression level was evaluated in 7 and in 5 CML cases with miR-219-2 and miR-199b deletions, respectively. Statistical analysis of the relative expression results was performed by the Relative Expression Software Tool (REST). To explore the predicted miR-199b target genes, the miRGen targets database (http:// www.diana.pcbi.upenn.edu/cgi-bin/miRGen/v3/Targets.cgi) was queried; this interface provides integrated data of four widely used target prediction programs (miRanda, PicTar, TargetScan, DIANA-microT). FISH experiments revealed the loss of miR-219-2 and miR-199b in 17 (25%) and 10 (15%) out of 68 patients. The miR-199b expression study showed a downregulation in the analyzed group of 5 CML cases with miR-199b deletion as compared to a pool of 10 patients without deletions. The expression level of the miR-199b was 0.279 and the difference between the two groups was statistically significant (p= 0.028). On the contrary, the miR- 219-2 analysis did not reveal a detectable expression level in the examined patients. There were 26 predicted miR-199b target genes, involved in several biological processes such as signal transduction (Protein phosphatase inhibitor 2, PPP1R2), regulation of transcription (Hepatic leukaemia factor, HLF), chromosome organization and biogenesis (Zinc finger protein 238, ZNF238), cell proliferation (Mitogen-activated protein kinase 11, MAP3K11) and DNA repair (UV excision repair protein RAD23 homologue B, RAD23B). Among the CML patients evaluable for the response to the treatment, all cases with the miR-199b deletion were resistant to IFN-a and imatinib therapy. In conclusion, our data demonstrate a crucial role for miR-199b in CML cases bearing der(9) deletions. This miR-199b downregulation could influence the expression level of different target genes modifying important cellular pathways. Further analysis of miR-199b target genes will be needed to shed light on the link between miRNAs and CML.

ABCG2 C.421C>A Is Associated with Higher Trough Imatinib Plasma Levels in Patients with Chronic Myeloid Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 110-110
Author(s):  
Naoto Takahashi ◽  
Masatomo Miura ◽  
Stuart A Scott ◽  
Kenichi Sawada
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Plasma Levels ◽  
Myeloid Leukemia ◽  
Drug Transport ◽  
Conflicts Of Interest ◽  
Nucleotide Polymorphisms ◽  
Wild Type ◽  
Large Patient

Abstract Abstract 110 [Background] Despite the excellent efficacy of imatinib for the treatment of chronic myeloid leukemia (CML), trough imatinib plasma levels can vary widely among patients. This may be due, in part, to inter-individual variation in imatinib metabolism and drug transport efficacy. To investigate the role of genetic variation in the pharmacokinetics of imatinib, we analyzed common single nucleotide polymorphisms within important imatinib pathway genes including ABCG2 (BCRP), ABCB1 (MDR1), ABCC2 (MRP2), CYP3A5, and SLC22A1 (OCT1) in 67 CML patients treated with imatinib. In addition, trough imatinib plasma levels were determined using high-performance liquid chromatography-tandem mass spectrometry. [Results] Distinct imatinib pharmacokinetics were identified in association with ABCG2 c.421C>A (p.Q141K; rs2231142) genotype. Specifically, the presence of the variant c.421A allele was significantly (p=0.024) associated with higher imatinib concentrations [median Cmin/Dose 2.70 (range: 1.50-8.30) ng/ml/mg; n=25] compared to patients with the wild-type ABCG2 (c.421C/C) genotype [median Cmin/Dose 2.27 (range: 0.37-5.30) ng/ml/mg; n=42]. ABCG2 is an efflux transporter for many xenobiotics, including imatinib, and is expressed at high levels in the human liver. Previous studies indicate that c.421A causes a 40% reduction in imatinib transport in vitro when compared to the wild-type genotype. Our data suggest that CML patients with ABCG2 c.421A allele may have deficient ABCG2 activity in vivo, resulting in reduced hepatic excretion of imatinib. Of note, although less common among Africans and individuals of European decent, the ABCG2 c.421C>A allele occurs at a high frequency in the Japanese (0.311) and Han Chinese (0.289) populations. [Conclusion] The association of ABCG2 c.421C>A with imatinib pharmacokinetics may explain why some Japanese CML patients administered less than 400 mg/day of imatinib have clinically sufficient trough imatinib plasma levels. Prospective studies are warranted to confirm the association between ABCG2 genotype and imatinib pharmacokinetics in large patient populations. Disclosures: No relevant conflicts of interest to declare.

Results of Imatinib Dose Escalation After 36 Months of Follow-up in Chronic Myeloid Leukemia Patients with Failure or Sub-Optimal Response According to 2006 EuropeanLeukemia Net (ELN) Criteria.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3302-3302
Author(s):  
Massimo Breccia ◽  
Fabio Stagno ◽  
Roberto Latagliata ◽  
Paolo Vigneri ◽  
Laura Cannella ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Dose Escalation ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Standard Dose ◽  
Acquired Resistance ◽  
Chronic Phase ◽  
Cytogenetic Response ◽  
Molecular Response

Abstract Abstract 3302 Poster Board III-190 Introduction Imatinib mesylate (IM) given at a daily dose of 400 mg currently represents the gold standard of care for patients with chronic myeloid leukemia (CML) in chronic phase (CP). European LeukemiaNet (ELN) guidelines propose IM dose escalation to rescue those CML patients with either suboptimal response or drug resistance. We report on the long-term efficacy of IM dose escalation in 74 patients with CP-CML after suboptimal response or failure to IM conventional dose. Patients and methods Median age was 50 years (range 19-85), there were 52 males and 22 females. Thirteen patients were classified as hematologic failure (10 primary and 3 secondary), 57 patients as cytogenetic resistance (24 primary and 33 acquired). Three patients escalated the dose for cytogenetic suboptimal response and one patient for molecular suboptimal response at 18 months. Fifty-four received IM dose escalation from 400 to 600 mg and 20 patients from 400 to 800 mg. Results Overall, after a median follow-up of 36 months, 68/74 (91.8%) patients maintained or achieved a complete haematologic response (CHR); this was maintained in all patients who escalated the dose for cytogenetic failure or suboptimal response. A major cytogenetic response (MCyR) was achieved in 41 patients (72%) who escalated the dose for cytogenetic failure and in 6/13 (46%) patients who escalated imatinib for hematologic failure (p=0.002). Overall, complete cytogenetic responses (CCR) were achieved in 27 (37%) out of 74 CML patients: of the 13 hematologic failure patients, only 5 achieved CCyR: all patients had prior acquired resistance to imatinib. Of the 57 cytogenetic failure, 22 reached CCR: this response was obtained in 27% of the primary cytogenetic resistant, and in 50% of the acquired cytogenetic resistant patients (p=0.02). Three patients who escalated the dose for cytogenetic suboptimal response obtained CCR and complete molecular response (CMR), whereas one patient who escalated the dose for molecular suboptimal response at 18 months did not obtain CMR. Median time to cytogenetic response was 3.5 months. Cytogenetic responses occurred in 37/50 patients who escalated the dose to 600 mg and in 10/20 patients who escalated to 800 mg daily (p=0.234). CMR was obtained in 10 patients: in 7 patients who escalated the dose for cytogenetic failure and in 3 patients who escalated imatinib for suboptimal cytogenetic response. Estimated 2 year-progression free survival (PFS) and overall survival (OS) is 87% and 85% respectively. Sixteen patients (21.6%) experienced toxicities and had temporarily IM interruption. Conclusions Imatinib dose escalation can induce sustained responses in a subset of patients with cytogenetic resistance and a prior suboptimal cytogenetic response to standard-dose imatinib, whereas it appears less effective in haematologic failure patients or in molecular sub-optimal responders. The availability of second generation TKI should be taken into account in these letter categories of patients. Disclosures No relevant conflicts of interest to declare.

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.

Quantitative Phosphoproteomics Identified a New Syk-Lyn-Axl Signalling Pathway Involved In Resistance to Nilotinib In Chronic Myeloid Leukemia Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3376-3376
Author(s):  
Romain Gioia ◽  
Cedric Leroy ◽  
Claire Drullion ◽  
Valérie Lagarde ◽  
Serge Roche ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Dependent Manner ◽  
Stable Isotope Labelling ◽  
Resistant Cells

Abstract Abstract 3376 Nilotinib has been developed to overcome resistance to imatinib, the first line treatment of chronic myeloid leukemia (CML). To anticipate resistance to nilotinib, we generate nilotinib resistant CML cell lines in vitro to characterize mechanisms and signaling pathways that may contribute to resistance. Among the different mechanisms of resistance identified, the overexpression of the Src-kinase Lyn was involved in resistance both in vitro, in a K562 cell line (K562-rn), and in vivo, in nilotinib-resistant CML patients. To characterize how Lyn mediates resistance, we performed a phosphoproteomic study using SILAC (Stable Isotope Labelling with Amino acid in Cell culture). Quantification and identification of phosphotyrosine proteins in the nilotinib resistant cells point out two tyrosine kinases, the spleen tyrosine kinase Syk and the UFO receptor Axl. The two tyrosine kinase Syk and Axl interact with Lyn as seen by coimmunopreciptation. Syk is phosphorylated on tyrosine 323 and 525/526 in Lyn dependent manner in nilotinib resistant cells. The inhibition of Syk tyrosine kinase by R406 or BAY31-6606 restores sensitivity to nilotinib in K562-rn cells. In parallel, the inhibition of Syk expression by ShRNA in K562-rn cells abolishes Lyn and Axl phosphorylation and then interaction between Lyn and Axl leading to a full restoration of nilotinib efficacy. In the opposite, the coexpression of Lyn and Syk in nilotinib sensitive K562 cells induced resistance to nilotinib whereas a Syk kinase dead mutant did not. These results highlight for the first time the critical role of Syk in resistance to tyrosine kinase inhibitors in CML disease emphasizing the therapeutic targeting of this tyrosine kinase. Moreover, Axl, which is already a target in solid tumor, will be also an interesting pathway to target in CML. Disclosures: No relevant conflicts of interest to declare.

MiR-150 Suppresses MLL-AF9 Associated Leukemia Through Simultaneously Targeting Multiple Oncogenes,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3461-3461
Author(s):  
Beiyan Zhou
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Expression Patterns ◽  
Gene Profiling ◽  
Fusion Genes ◽  
Leukemic Stem Cells ◽  
Hematopoietic Stem ◽  
Mll Gene

Abstract Abstract 3461 The mixed lineage leukemia (MLL) gene codes for an evolutionarily conserved histone methyltransferase that is crucial for early hematopoiesis. As a result of a chromosomal translocation involving locus 11q23 results in formation of chimeras composed of the 5' part of the MLL gene fused with more than 60 partner genes lead to disruption of normal function of MLL as a histone methytransferase and acquisition of transcriptional properties conferred by the partner genes. MLL fusion genes (MLL-FG) are often the causal mutations for aggressive acute myeloid and lymphoid leukemias (AML and ALL) that correlated with poor prognosis. In order to treat or even eliminate MLL-associated leukemias, extensive studies on the regulatory mechanism underlying MLL associated transformation and progression have been carried out. Leukemic stem cells (LSC) can derive from either hematopoietic stem or progenitor cells with the recruitment of MLL-fusion genes (MLL-FG) and wild type MLL protein. We report that miR-150, a key hematopoietic regulatory microRNA (miRNA) and one of the most downregulated miRNAs in MLL-associated leukemias, acts as a tumor suppressor to block the leukemogenic potency of leukemic stem cells. When expression of miR-150 was restored, a significantly suppressed leukemic stem cell potency of MLL-AF9 cells was observed both in vivo and in vitro. Gene profiling analysis demonstrated that elevated miR-150 altered various aspects of gene expression patterns in MLL-AF9 cells, including stem cell signatures, cancer pathways, and cell survival. By screening more than 30 predicted target genes, we identified multiple leukemia-associated oncogenes as bona fide miR-150 targets, and knockdown of these genes by shRNAs recapitulated the tumor suppressive effects observed after ectopically expression of miR-150 in MLL-AF9 cells. Disclosures: No relevant conflicts of interest to declare.

Cytogenetic Analysis of 1863 Chineses Patients with Chronic Myeloid Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4888-4888
Author(s):  
Qitian Mu ◽  
Qiuling Ma ◽  
Yungui Wang ◽  
Xiangmin Tong ◽  
Zhimei Chen ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Disease Progression ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Blast Crisis ◽  
Chronic Phase ◽  
Newly Diagnosed ◽  
Monocytic Differentiation ◽  
Granulocytic Differentiation ◽  
Monosomy 7

Abstract Abstract 4888 Background: Cytogenetic analyses of chronic myeloid leukemia (CML) have been performed previously in a large number of reports, but systematical research based on large sample sizes is seldom available. In order to further elucidate the cytogenetic nature of CML, we analyzed retrospectively the cytogenetic profiles of 1863 Ph/BCR-ABL-positive CML patients from a research center in China. Results: Of 1266 newly diagnosed CML patients, the median age was 41 years, which is younger than the median age of diagnosis in western populations. The incidence of additional chromosome abnormalities(ACAs) was 3.1% in newly-diagnosed chronic phase(CP), 9.1% in CP after therapy, 35.4% in accelerated phase(AP) and 52.9% in blast phase(BP), reflecting cytogenetic evolution with CML progression. 5.3% patients harbored a variant Ph translocation. A higher prevalence of ACAs was observed in variant Ph translocations than in classical t(9;22) in the disease progression, especially in BP(88.2% vs. 50%, p=0.002). Moreover, a hyperdiploid karyotype and trisomy 8 were closely correlated with myeloid blast crisis(BC) while a hypodiploid karyotype and monosomy 7 were associated with lymphoid-BC. Among subsets of myeloid-BC, compared with myeloid-BC with granulocytic differentiation or monocytic differentiation, myeloid-BC with minimal differentiation had higher ACAs rate (80% vs.46.8%, p=0.009 and 80% vs. 42.9%, p=0.006). Conclusion: CML tends to afflict younger population in China. In the disease progression, the incident of ACAs was higher in variant Ph translocations than in classical t(9;22). Among subsets of myeloid-BC, myeloid with minimal differentiation had distinct cytogenetic features. Disclosures: No relevant conflicts of interest to declare.

Ezh2 Plays a Critical Role in the Progression of MLL-AF9-Induced Acute Myeloid Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 57-57
Author(s):  
Satomi Tanaka ◽  
Goro Sashida ◽  
Satoru Miyagi ◽  
Koutaro Yokote ◽  
Chiaki Nakaseko ◽  
...  
Keyword(s):  
Stem Cells ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Target Genes ◽  
Nuclear Translocation ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Histone H3 ◽  
Chip Sequencing ◽  
Acute Myeloid

Abstract Abstract 57 The polycomb group proteins function in gene silencing through histone modifications. They have been characterized as a general regulator of stem cells, but also play a critical role in cancer. EZH2 is a catalytic component of the polycomb repressive complex 2 (PRC2) and tri-methylates histone H3 at lysine 27 to transcriptionally repress the target genes. Although EZH2 is over-expressed in various cancers including hematological malignancies, it remains unknown how EZH2 contributes to the initiation and/or progression of acute myeloid leukemia (AML). To understand the role of EZH2 in AML, we transformed granulocyte macrophage progenitors (GMPs) from Cre-ERT;Ezh2+/+ and Cre-ERT;Ezh2flox/flox mice with the MLL-AF9 fusion gene. Then, Ezh2 was deleted by inducing nuclear translocation of Cre by adding tamoxifen to culture. We found that proliferation of Ezh2δ/δ transformed cells was severely compromised upon deletion of Ezh2 (Ezh2δ/δ) in liquid culture. They gave rise to a significantly reduced number of colonies in replating assays. Of note, while Ezh2+/+ cells formed compact colonies composed of immature myeloblasts, Ezh2δ/δ cells formed dispersed colonies composed of differentiated myeloid cells. We next transplanted Cre-ERT;Ezh2+/+ and Cre-ERT;Ezh2flox/flox GMPs transformed by MLL-AF9 into recipient mice. All the recipient mice developed AML by 3 weeks after transplantation. At 3 weeks after transplantation, we depleted Ezh2 by intraperitoneal injection of tamoxifen. Deletion of Ezh2 significantly prolonged the survival of the recipient mice (60 days vs. 76 days, p<0.0001), although all the mice eventually died of leukemia. Nonetheless, as was detected in vitro, Ezh2δ/δ AML cells in BM were apparently differentiated in morphology compared with the control. Ezh2δ/δ AML cells in BM gave rise to 10-fold fewer colonies in methylcellulose medium compared with Ezh2+/+ AML cells, and again showed an obvious tendency of differentiation. These observations imply that Ezh2 is critical for the progression of MLL-AF9 AML and maintains the immature state of AML cells. To elucidate the mechanism how Ezh2 promotes the progression of MLL-AF9-induced AML, we examined the genome-wide distribution of tri-methylation of histone H3 at lysine 27 (H3K27me3) by ChIP-sequencing and microarray-based expression analysis. ChIP-sequencing using Ezh2+/+ and Ezh2δ/δ BM AML cells identified 3525 and 89 genes exhibiting a ≧ 10-fold enrichment in H3K27me3 levels in Ezh2+/+ and Ezh2δ/δ AML cells, respectively, confirming a drastic reduction in the levels of global H3K27me3 in the absence of Ezh2. Microarray analysis using lineage marker (except for Mac1)−Sca-1−c-Kit+FcγRII/IIIhi BM AML cells revealed 252 upregulated and 154 downregulated genes (≧ 2-fold) in Ezh2δ/δ AML cells compared with Ezh2+/+ AML cells. Of interest, the absence of Ezh2 did not affect the transcriptional activation of the major target genes of MLL-AF9, including HoxA9 and Meis1. Because Ezh2 functions as transcriptional repressor, de-repressed genes could be direct targets of Ezh2. Based on these data, we are now engaged in further comprehensive analysis to narrow down the direct target genes of Ezh2 responsible for the progression of AML. Collectively, our findings suggest that Ezh2 is the major enzyme for H3K27me3 in AML and contributes to the progression of AML by regulating transcription a cohort of genes that are supposedly relevant to the self-renewal capacity and perturbed differentiation of AML stem cells. Disclosures: No relevant conflicts of interest to declare.

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