RNAI MEDITED SILENCING REVEALS TEL/AML1 IS DISPENSABLE FOR LEUKEMIC CLONE SURVIVAL
M Zaliova,1 J Madzo,2 G Cario,3 J Trka1
Backround. Translocation (12;21), the most frequent chromosomal aberration in childhood ALL results in TEL/AML1 fusion gene. TEL/AML1 hybrid protein most likely acts as an aberrant transcription factor that deregulates AML1-dependent transcription but its target genes, and thus also the exact role in leukemic cells remain unknown. in vivo studies showed that TEL/AML1 itself is not sufficient to cause leukemia but may induce a preleukemic state characterized by the increased numbers of multipotent or B-cell progenitors with an incomplete block of differentiation. Role of TEL/AML1 for leukemic clone establishment is being studied intensively, however, relevance of TEL/AML1 fusion gene for definitive leukemia persistance has not been approached yet. Aims. To address this question and to explore the potential of TEL/AML1-targeted therapy, we aimed to study the effect of RNAi-mediated TEL/AML1 silencing on leukemic cells. Methods. Transfection of siRNAs was used to silence TEL/AML1 and AML1 genes. HeLa cells were transfected by lipofection, REH and UOC-B6 cells (only available TEL/AML1-positive cell lines) by rectangular pulse-electroporation. TEL/AML1 level was monitored at mRNA and protein levels using qRT-PCR and western blot, respectively. Cell viability was measured using trypan blue staining followed by microscopy, apoptosis rate was monitored by staining with annexin V and propidium iodide using flow cytometry. Analysis of DNA content using staining with propidium iodide was performed to assess cell-cycle distribution. Incorporation of nucleoside analogue was measured by flow cytometry to analyse de novo DNA synthesis as an indicator of proliferation rate. GEP was performed in TEL/AML1-positive cells after TEL/AML1 silencing. Results. We designed eleven different siRNAs specifically targeting the fusion sequence to silence TEL/AML1 but prevent silencing of TEL and AML1 wild type alleles. In the first step siRNAs efficiency was measured in HeLa cells transgenic for TEL/AML1-ires2-EGFP reporter as a decrease of EGFP fluorescence by flow cytometry. In the second step five most efficient siRNAs were tested at mRNA level in TEL/AML-positive leukemic cell line. Two most efficient siRNAs were pooled and used for TEL/AML1 knock-down in REH and UOC-B6 TEL/AML1-positive cell lines. Applying two rounds of transfection within 48 hours interval we achieved 74% and 86% TEL/AML1 protein knockdown in REH and UOC-B6 cells, respectively. Seemingly counter-intuitively (based on the results from studies on other fusion oncogenes including BCR/ABL, AML1/ETO, E2A/PBX1), TEL/AML1 silencing neither decreased cell viability, nor induced apoptosis. On the contrary, TEL/AML1 depletion was accompanied by modest but significant increase in the fraction of S-phase cells and corresponding rise in the proliferation rate. Opposite effects on cell cycle and proliferation were induced by AML1 silencing, supporting our hypothesis that TEL/AML1 may block previously demonstrated AML1 role in G1/S progression through the cell cycle. In line with the lack of effect on cell viability and discreet effect on cell-cycle distribution and proliferation we found no significant changes in global gene expression pattern upon TEL/AML1 depletion. Conclusions. Our data indicate, that TEL/AML1 is dispensable for leukemia persistence, at least in studied cell lines, and, as such, would not be a suitable target for gene specific therapy.