De Paepe, Anne

De Weer, An

Mestdagh, Pieter

Poppe, Bruce

Speleman, Franki

Van Roy, Nadine

Van Vlierberghe, Pieter

Vandesompele, Jo

Verhasselt, Bruno

English

Ghent University Academic Bibliography

13TH CONGRESS OF THE EUROPEAN HEMATOLOGYASSOCIATIONCOPENHAGEN, DEMNARK,JUNE 12 - 15, 2008ABSTRACT BOOKcence demonstrated the de-localization of the protein within the nucle-ous in CBF AML but it is completely cytoplasmatic in leukemic cells withnormal karyotype and in MDS. Transfection experiments with PR3 plas-mid demonstrate that overexpression of PR3 results into a significantincreasing of the proliferation rate and reduced apoptosis. By contrasttransfection with shRNA triggers apoptosis and cell growth inhibition.Conclusion: PR3 gene expression and protein are significantly increasedin AML and MDS, particularly in CBF leukemias in which the proteinis completed delocalized within the nucleous. PR3 overepression is asso-ciated with CEBP/α downmodulation. Ectopic expression of PR3induces increased proliferation and apoptosis arrest. The mechanismunderlying the nuclear localization of PR3 in CBF leukemias and itspathogenetic role remains unexplored.0026CXCR-4 POSITIVE ACUTE MYELOID LEUKEMIAS (AGE < 60 YEARS):IMMUNOPHENOTYPIC, CYTOMORPHOLOGIC, CYTOGENETIC, ANDMOLECULAR CHARACTERISTICSU. Oelschlaegel, B. Mohr, U. Schaekel, F. Kroschinsky, M. Schaich,G. Ehninger, C. ThiedeUniversity Hospital Dresden, DRESDEN, GermanyBackground. The interaction of the CXCR-4 receptor with its ligandSDF-1 plays a key role in the process of homing and mobilization ofstem cells and might be involved in the trafficking of leukemic blasts,too. Aims. The aim of our study was to correlate the CXCR-4 expressionwith the main diagnostic features of AML. Methods. 682 AML patients(other than AML M3) under the age of 60 were included in our investi-gation. CXCR-4 expression was measured in whole bone marrow orperipheral blood performing multi-color flow cytometry (CD45FITC/CXCR-4 PE/CD34 PerCP5.5). The cut-off was set at 20% CXCR-4 positive cells. Cytomorphologic, cytogenetic, and molecular analyseshave been done in parallel. Results. Ninety percent of all patients expressCXCR-4 (616/682 patients). These leukemias were significantly moreassigned to a monocytic phenotype (33% vs 19%, p=0.0228). The per-centage of CD34+, HLA DR+, aberrant CD19+, or CD7+ patients wasclearly lower in the CXCR-4 positive vs negative group (51% vs 83%,p<0.0001; 91% vs 100%, p=0.0330; 4% vs 11%, p=0.0236; 9% vs 17%,p=0.0802). Furthermore, in CXCR-4 positive patients low risk cytoge-netics was present less frequently (10% vs 27%, p=0.0002), whereaspatients with high risk cytogenetics were equally distributed in bothgroups (22% vs 27%). There were significantly more NPM1 mutated orFLT3-ITD mutated cases in the CXCR-4 positive group (38% vs 8%,p<0.0001; 24% vs 6%, p=0.0005). In addition, the median expression lev-el of CXCR-4 was higher on blasts of FLT3-ITD positive vs negativepatients (81% vs 71%, p=0.0115). Interestingly, patients with NPM1mutations expressed signifcantly more CXCR-4 compared to non mutat-ed cases (87.9% vs 62.7%; p<0.0001), independent from the FLT3 sta-tus. Conclusions. CXCR-4 positive vs negative acute myeloid leukemiasdisplayed distinct immunophenotypic and molecular characteristics.Especially the NPM1 mutation status seems to be correlated withCXCR-4 expression. 0027FLT3 INTERNAL TANDEM DUPLICATION INVOLVING ITS UBIQUITINDEPENDENT ENDOCYTOSIS MOTIF SUSPEND MODULATION BY HDM2AND ARE ASSOCIATED WITH INFERIOR SURVIVAL IN AMLL. Wergeland,1 E. Oveland,2 S. Gry,3 S.L. Bedringaas,4 R. Hovland,5Bruserud,4 B.T. Gjertsen61University of Bergen, BERGEN; 2Department of Biomedicine, University ofBergen, BERGEN; 3Bergen University College, BERGEN; 4Institute of Medi-cine, University of Bergen, BERGEN; 5Center for Medical Genetics, Hauke-land University Hospital, BERGEN; 6Department of Medicine, HaukelandUniversity Hospital, BERGEN, NorwayBackground. Internal tandem duplications in the juxtamembrane regionof the receptor tyrosine kinase Flt3 (Flt3-ITD) and elevated expressionof the oncogenic E3 ubiquitin ligase Hdm2 are frequent features of acutemyeloid leukemia (AML). Hdm2 is a well known suppressor of p53, butis also associated with endocytosis of cell surface receptors. Recently wehave shown that Hdm2 and Flt3 are reciprocally modulated upon DNA-damage therapy of AML cells in vitro and in vivo. Aims. To elucidate themechanism behind the Flt3-Hdm2 modulation and the impact of vari-ous Flt3-ITDs. Furthermore, determine if the Flt3-Hdm2 modulationmay have a clinical impact in AML. Methods. Primary AML cells and celllines (NB4 and MV4-11) with wild type Flt3 (Flt3-wt) or mutated Flt3(Flt3-ITD) were used with Flt3 ligand (FL), small molecular inhibitors andsmall interfering RNA (siRNA) to elucidate the relation between Flt3and Hdm2 on protein level, mRNA expression and modulation of apop-tosis. Results. Kinase inhibition of Flt3 increase Hdm2 in Flt3-wt cells, butnot in Flt3-ITD cells. Modulation of FLt3 and Hdm2 by siRNA indicatesthat their protein levels are mutually dependent. Cell lines with Flt3-ITDhave a more rapid cycling of receptors on the cell surface than cells withFlt3-wt. A sequence alignment of the juxtamembrane region of Flt3 andother related tyrosine kinase receptors lead to the identification of aputative Ubiquitin dependent endocytosis motif (UbE) in the juxtamem-brane region of Flt3. Based on transfection studies of mutant Flt3 andHdm2, internalisation of Flt3 is dependent on both Hdm2 E3 ligase activ-ity and Flt3 UbE domain. A subset of AML patients with Flt3-ITD hasa duplication of this domain (Flt3-2xUbE). These patients have a reducedlevel of surface Flt3 which significantly correlates to an elevated level ofHdm2, and survival data suggest a negative prognostic impact in thepresence of Flt3xUbE. Conclusions. FLT3 receptor turnover involvesHdm2, and Flt3-ITD results in dysregulated receptor turnover attenuat-ed for Hdm2-induced Flt3 down-regulation. A novel type of Flt3 muta-tions (Flt3-2xUbE) is associated with the Hdm2-Flt3-signalling pathwayand may have prognostic impact within the group of Flt3-ITD positiveAML patients.0028MICRORNA PROFILING OF EVI1 DEREGULATED MYELOID LEUKEMIAA. De Weer,1 B. Poppe,1 P. Mestdagh,1 P. Van Vlierberghe,1N. Van Roy,1 A. De Paepe,1 B. Verhasselt,2 J. Vandesompele,2F. Speleman11University Hospital, Centre for Medical Genetics, GHENT; 2University Hos-pital, Dept of Chemistry, Microbiology and Immunology, GHENT, BelgiumBackground. Chromosomal rearrangements involving the EVI1 geneare a recurrent finding in malignant myeloid disorders. These transloca-tions or inversions contribute to ectopic expression of the EVI1 gene.EVI1 transcriptional activation has also been reported in approximately5 % of acute myeloid leukemia (AML) patients without chromosomaldefects affecting the EVI1 locus. Survival of patients with EVI1 overex-pressing leukemias is poor and new insights into the molecular pathol-ogy are needed as a basis for development of targeted therapies. Recent-ly, microRNA deregulation was identified as a major contributor to can-cer initiation and progression. Moreover, microRNA genes were shownto be directly regulated by activated proto-oncogenes. Aims. The aim ofthis study was to investigate which microRNA genes are implicated inthe transcriptional pathways governed by the EVI1 oncogene. Methods.SiRNA mediated EVI1 knockdown was performed in EVI1 overexpress-ing AML cell lines Kasumi-3 and UCSD-AML1 and validated by qRT-PCR and Western blotting. A total of 384 microRNAs were profiledthrough automated qRT-PCR using high-throughput quantitative stem-loop RT-PCR (Applied Biosystems). Integrated statistical analysis (SAM-analysis, delta Ct and confidence interval analysis) was performed toidentify up- and downregulated microRNAs. Results. After siRNA treat-ment, a reduction of 90% on mRNA level and a reduction of 70% onprotein level were observed for both EVI1 overexpressing cell lines Kasu-mi-3 and UCSD-AML1. Through integrated analysis several statistical-ly significant up- (miR-184 and miR-182#) and downregulated (miR-155, miR-222 and miR-210) microRNAs were identified. Conclusions. Weidentified several EVI1 regulated microRNAs after siRNA treatment ofEVI1 overexpressing cell lines which are currently under further inves-tigation in a large panel of AML patients. Furthermore, microRNA pro-filing of 3q26 rearranged EVI1 overexpressing patient samples and non-rearranged EVI1 overexpressing bone marrow samples is underway. Fur-ther studies will also include electroporation of antagomirs or microR-NA mimics for EVI1 regulated microRNAs to asses their contribution tothe leukemic phenotype. The discovery of functionally relevant microR-NAs may provide new targets for therapeutic intervention in AML. 10 | haematologica | 2008; 93(s1)13th Congress of the European Hematology Association

MICRORNA PROFILING OF EVI1 DEREGULATED MYELOID LEUKEMIA

https://biblio.ugent.be/publication/516262/file/964370

MICRORNA PROFILING OF EVI1 DEREGULATED MYELOID LEUKEMIA

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