Expression of the human folylpoly-γ-glutamate synthetase (FPGS) gene is controlled by tissue/lineage specific and proliferation-dependent mechanisms. Levels of FPGS mRNA, protein, and enzyme activity are 2–3 fold higher in B-precursor (Bp) ALL cell lines and primary cells when compared to T-lineage ALL. These differences correlate with intracellular accumulation of long chain methotrexate (MTX) polyglutamates (PG), and more important with clinical sensitivity to MTX. However, significant heterogeneity of FPGS expression exists within hematopoietic cells of the same lineage, suggesting additional factors influence FPGS expression in Bp- and T-ALL. cDNA microarray data was analyzed for expression of folate-related genes (DHFR, FPGS, RFC, MTHFR, and γ-GGH) in six prognostic leukemia subtypes (T-ALL, hyperdiploid (> 50 chromosomes), BCR-ABL, E2A-PBX1, TEL-AML1, and MLL) (Yeoh et al, Cancer Cell, 2002). It was found that only the FPGS gene exhibited significant heterogeneity of expression compared the others analyzed. Therefore, we investigated the effect of distinct non-random genomic translocations on FPGS gene expression to determine whether gene fusions may be responsible for the heterogeneity of FPGS expression in ALL cells. Using quantitative fluorescence real-time RT-PCR, we first detected reduced levels of FPGS mRNA expression in the RCH-ACV (Bp-ALL, t(1:19)/E2A-PBX1) and REH (Bp-ALL, t(12:21)/TEL-AML1) human leukemia cell lines expressing non-random chromosomal gene fusions when compared to control (NALM6). To determine if expression of fusion proteins encoded by the E2A-PBX1 and TEL-AML1 translocations lead to decrease FPGS promoter activity and mRNA expression, we used a FPGS-luciferase reporter gene assay. The CHO FPGS null mutant cell line AUXB1, was triple transfected with constructs expressing luciferase (pFPGS-luc), β-galactosidase (pCMVβ), and E2A-PBX1 (pCMVKJ7) or TEL-AML1 (pTEL-AML1) and assayed for luciferase and β-galactosidase activity. Expression of E2A-PBX1 and TEL-AML1, determined by quantitative RT-PCR and Western blot, resulted in 45% and 30% decrease in the level of normalized FPGS-luciferase activity, respectively. These data indicate that FPGS gene transcription is decreased by both translocations, either by direct binding to regulatory elements or indirectly via downstream pathways influenced by E2A-PBX1 and TEL-AML1. Therefore, to confirm these findings and to identify genes or pathways in Bp-ALL regulated by E2A-PBX1 and TEL-AML1 gene fusions, we constructed NALM6 stable cell lines expressing E2A-PBX1 or TEL-AML1 using vectors pCI-neo/E2A-PBX1 and pCI-neo/TEL-AML1. In both NALM6 stable cell lines the level of FPGS mRNA expression was decreased by more than 50% compared to wild type (NALM6). Total RNA was then extracted from NALM6, NALM6/E2A-PBX1, and NALM6/TEL-AML1 cells and analyzed by cDNA microarray. Gene expression profile analysis from existing databases and our NALM6/E2A-PBX1 and NALM6/TEL-AML1 stable cell lines identified genes differentially expressed compared to NALM6 control. These included transcription factors (CSDA, AKAP12, ARL2, PBX1, TEL), cell cycle regulators (CDK2P1), signal transduction (PIK3C3), proliferation and cell migration (PTPRK), zinc finger binding protein (PHF2), among others. This study is the first to demonstrate the causal relationship between the presence of E2A-PBX1 and TEL-AML1 gene fusions and altered FPGS expression and may explain the differential sensitivity to MTX exhibited by these childhood ALL phenotypes.