Up-regulation of Survivin by the E2A-HLF Chimera Is Indispensable for the Survival of t(17;19)-positive Leukemia Cells

Mayuko Okuya,Hidemitsu Kurosawa,Jiro Kikuchi,Yusuke Furukawa,Hirotaka Matsui,Daisuke Aki,Takayuki Matsunaga,Takeshi Inukai,Hiroaki Goto,Rachel A Altura,Kenich Sugita,Osamu Arisaka,A.Thomas Look,Toshiya Inaba

doi:10.1074/jbc.m109.023762

Mayuko Okuya, Hidemitsu Kurosawa + Show 12 more

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https://doi.org/10.1074/jbc.m109.023762

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The E2A-HLF fusion transcription factor generated by t(17;19)(q22;p13) translocation is found in a small subset of pro-B cell acute lymphoblastic leukemias (ALLs) and promotes leukemogenesis by substituting for the antiapoptotic function of cytokines. Here we show that t(17;19)+ ALL cells express Survivin at high levels and that a dominant negative mutant of E2A-HLF suppresses Survivin expression. Forced expression of E2A-HLF in t(17;19)(-) leukemia cells up-regulated Survivin expression, suggesting that Survivin is a downstream target of E2A-HLF. Analysis using a counterflow centrifugal elutriator revealed that t(17;19)+ ALL cells express Survivin throughout the cell cycle. Reporter assays revealed that E2A-HLF induces survivin expression at the transcriptional level likely through indirect down-regulation of a cell cycle-dependent cis element in the promoter region. Down-regulation of Survivin function by a dominant negative mutant of Survivin or reduction of Survivin expression induced massive apoptosis throughout the cell cycle in t(17;19)+ cells mainly through caspase-independent pathways involving translocation of apoptosis-inducing factor (AIF) from mitochondria to the nucleus. AIF knockdown conferred resistance to apoptosis caused by down-regulation of Survivin function. These data indicated that reversal of AIF translocation by Survivin, which is induced by E2A-HLF throughout the cell cycle, is one of the key mechanisms in the protection of t(17;19)+ leukemia cells from apoptosis.

Highlights

The E2A-HLF fusion transcription factor, which is generated by the t(17;19)(q22;p13) translocation, is found in a small subset of pro-B cell acute lymphoblastic leukemias (ALLs)2 that occurs
E2A-HLF blocks apoptosis normally induced by cytokine deprivation in murine interleukin (IL)-3-dependent B precursor lines such as Baf-3 or FL5.12 cells, suggesting that this fusion protein contributes to leukemogenesis through modification of apoptosis regulatory pathways normally controlled by cytokines [6, 7]
The survivin gene may be a good candidate for a target gene of E2A-HLF involved in the inhibition of apoptosis in t(17;19)ϩ saline; FITC, fluorescein isothiocyanate; shRNA, short hairpin RNA; siRNA, short interfering RNA; GFP, green fluorescent protein; Electrophoretic Mobility Shift Assays (EMSA), electrophoretic mobility shift assay; BrdUrd, bromodeoxyuridine; TdT, terminal deoxynucleotidyltransferase; PARP, poly(ADP-ribose) polymerase; Terminal deoxynucleotidyltransferase-mediated dUTP nick-end-labeling (TUNEL), terminal deoxynucleotidyltransferase-mediated dUTP nick-end-labeling; PI, propidium iodide; dn, dominant negative; nt, nucleotide; 7-AAD, 7-amino-actinomycin D; PLL, plenti-Lox3.7

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Introduction

The E2A-HLF fusion transcription factor, which is generated by the t(17;19)(q22;p13) translocation, is found in a small subset of pro-B cell acute lymphoblastic leukemias (ALLs)2 that occurs. Four t(17;19)ϩ ALL cell lines (UOC-B1, YCUB-2, HAL-O1, and Endo-kun) expressed the E2A-HLF chimeric protein on immunoblot analysis (Fig. 1A, 4th panel, lanes 1– 4) either as a slower (lanes 1 and 3) or a faster migration band (lanes 2 and 4) corresponding to difference in the fusion junction, as described previously [3].

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