Pediatr Blood Cancer 2006;47:757–764 ABCB1 Over-Expression and Drug-Efflux in Acute Lymphoblastic Leukemia Cell Lines With t(17;19) and E2A-HLF Expression Michael Baudis, MD, 1{ Victor Prima, PhD, 1{ Yoon Han Tung, BS, 2 and Stephen P. Hunger, MD 1 * INTRODUCTION Genes encoding sequence-specific DNA binding tran- scription factors are common targets of somatic mutations in human cancer. Chromosome translocations in acute leuke- mias frequently involve transcription factor genes, often creating chimeric transcription factors with dominant gain- of-function oncogenic properties [1]. In general, oncogenic transcription factors are hypothesized to contribute to malignant transformation by directly activating or repressing transcription of specific target genes, or by interfering, in a dominant negative manner, with the transcriptional activity of endogenous proteins. In addition to playing a critical pathogenetic role during neoplastic transformation, chimeric transcription factors may also modulate the clinical pheno- type. The 19p13.3 E2A gene (also termed TCF3) is a major target of chromosome translocations in acute lympho- blastic leukemia (ALL) [2]. In about 5% of ALLs, a t(1;19)(q23;p13) disrupts E2A and fuses it to the chromo- some 1 gene PBX1, leading to expression of E2A-PBX1 chimeric proteins [3,4]. Analogous E2A-HLF fusion proteins are produced by a t(17;19)(q21;p13), present in 0.5% – 1% of ALLs [5,6]. E2A-HLF expression is associated with disseminated intravascular coagulation [7], and confers a dismal prognosis [2,8]. E2A-HLF and E2A-PBX1 consist of the amino terminal two-thirds of E2A, including two separate transcriptional activation domains (TADs), fused to portions of HLF and PBX1 containing distinct DNA binding domains and protein interaction motifs. HLF contains a basic leucine zipper (bZIP) domain that mediates site-specific DNA binding and protein dimerization [6,9]. E2A-PBX1 includes the PBX1 homeobox and an adjacent region responsible for interaction with a subset of HOX proteins and binds DNA in conjunction with MEIS proteins [3,4,10]. HLF and PBX1 are not normally expressed in lym- phocytes. Thus, these translocations have two important consequences: they ectopically express HLF/PBX1 DNA binding domains and fuse them to portions of E2A that contain TADs with potent activity in lymphoid cells, suggesting that aberrant regulation of specific target gene expression plays an important role in leukemogenesis mediated by E2A fusion proteins and may also dictate unique clinical properties of this subtype of ALL. Candidate E2A-PBX1 and E2A-HLF target genes have been identified, but none have been shown to directly mediate transformation to date. Other data suggest that a critical activity of E2A fusion proteins might be to interfere with the normal function of wild type E2A. Structure–function analyses of E2A-PBX1 revealed that the PBX1 DNA binding domain is not required for transformation of NIH-3T3 cells Background. The t(17;19)(q21;p13), which occurs in a small subset of acute lymphoblastic leukemias (ALLs) and is associated with a dismal prognosis, creates a chimeric E2A-HLF transcription factor with transforming properties. Procedure. We used representa- tional difference analysis to identify candidate E2A-HLF target genes. Transient transfection assays and an inducible expression model system were then used to evaluate the ability of E2A-HLF to modulate target gene expression. Results. We identified ABCB1 (MDR1, P-glycoprotein) as a gene differentially expressed in ALL cell lines with and without E2A-HLF expression and demonstrated that t(17;19)þ ALL cell lines expressed high levels of ABCB1 protein and had a drug efflux-positive phenotype. Although ABCB1 transcription is regulated by C/EBPb via interaction with a DNA response element that shares significant homology with the optimal E2A-HLF binding site, E2A-HLF did not directly activate transcription of reporter genes under control of ABCB1 promoter elements in transient transfection assays. However, ABCB1 expression was induced in a DNA-binding independent manner by E2A-HLF, E2A-PBX1, and truncated E2A polypeptides consisting of those portions of E2A present in leukemic fusion proteins. Conclusions. E2A-HLF-mediated over-expression of ABCB1 may play a critical role in defining the clinical phenotype of ALLs with a t(17;19), suggesting pharmacologic modulation of ABCB1 activity as a rational therapeutic strategy for this chemother- apy resistant subtype of ALL. Pediatr Blood Cancer 2006;47:757– 764. ß 2005 Wiley-Liss, Inc. Key words: ABCB1; E2A-HLF; lymphoblastic leukemia; MDR1; transcription factor; translocation ß 2005 Wiley-Liss, Inc. DOI 10.1002/pbc.20635 —————— 1 Department of Pediatrics, University of Florida College of Medicine and the University of Florida Shands Cancer Center, Gainesville, Florida; 2 Department of Pediatrics, University of Colorado Health Sciences Center, Denver, Colorado { Michael Baudis and Victor Prima contributed equally to this study. Grant sponsor: Pediatric Cancer Foundation (to SPH); Grant sponsor: American Cancer Society (to SPH); Grant number: LBC 97-463. *Correspondence to: Stephen P. Hunger, Pediatric Hematology- Oncology, University of Florida, College of Medicine, P.O. Box 100296, Gainesville, FL 32610-0296. E-mail: hungesp@peds.ufl.edu Received 22 May 2005; Accepted 24 August 2005