Abstract 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 representational 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)+ ALL cell lines expressed high levels of ABCB1 protein and had a drug efflux-positive phenotype. Although ABCB1 transcription is regulated by C/EBPbeta 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 chemotherapy resistant subtype of ALL.