Identification of Genes Differentially Expressed after HDACi Treatment in TEL/AML1-Positive Leukaemic Cells.

The role of the TEL/AML1 fusion gene in leukaemogenesis is still not understood despite its frequent occurrence in children with acute lymphoblastic leukaemia (ALL; 25%). Both partner genes are important for the development and maintenance of normal haematopoiesis. The TEL part of the fusion protein contains domains interacting with the mSin3, N-CoR and HDAC-3 corepressors. A part of the AML1 gene involved in the fusion carries a DNA binding domain that controls the interaction with the cis-regulatory elements of the target genes. The TEL/AML1 protein seems to behave like a repressor blocking the expression of the genes originally transactivated by AML1. Target genes of AML1 protein are described in myeloid differentiation, but not in lymphopoiesis. We aim to identify these target genes. In our previous experiments we monitored the changes of immunophenotype after histone deacetylase inhibitors (HDACi; valproate (VPA), Trichostatin A (TSA)) administration of TEL/AML1[+] cells. Surviving TEL/AML1[+] cells showed drift in expression of CD10 and CD20 surface antigens and in intracellular level of RAG-1 and TdT proteins, thus reflecting a change in the differentiation stage. The protein expressions of RAG1 and TdT were confirmed to RNA level by RQ-RT-PCR (control(C) vs. TSA p<0.0001, C vs. VPA p=0.0002). We performed an expression profiling of treated vs. untreated TEL/AML1[+] cells and TEL/AML1[+] vs. other ALL patients. First we calculated a cut-off expression for each gene in ALL patients. According to the analysis of these cut-offs we selected genes specifically over- or under-expressed in TEL/AML1[+] patients. These genes were compared to genes which expression changed significantly after HDACi. Finally, we analysed interdigitated genes for their divergence between TEL/AML1[+] and other subgroups of ALL from publicly available expression profiling data. By this combined multi-step approach, we chose 5 genes which expression is i) specific for TEL/AML1[+] patients and ii) significantly changed after HDACi administration. Microarray data of chosen genes showed downregulation of JunD, ACK1, PAK1 and PDGFRB in TEL/AML1[+] patients as well as in our cell-line model with increased expression after HDACi treatment. TCF4 gene was upregulated in the studied group and the administration of HDACi lead to its downregulation. We confirmed changes of chosen genes expression levels by RQ-RT-PCR: JunD - C vs. TSA p=0.013, C vs. VPA p= 0.0008; PDGFRB - C vs. TSA p< 0.0001, C vs. VPA p=0.016; TCF4 - C vs. TSA p< 0.0001, C vs. VPA p=0.0002; ACK1 - C vs. VPA p=0.07. These genes have a fundamental role in cell cycle progression and proliferation, therefore their role in leukaemogenesis is presumptive. These data also support the hypothesis that the effect of HDACi on TEL/AML1[+] cells is directly related to the function of TEL/AML1 protein, and the treatment with HDACi is able to release cells from differentiation block caused by TEL/AML1 aberrant transcription factor. Supported by Zentiva,Inc.