Real-time RT-PCR analysis of mRNA decay: half-life of Beta-actin mRNA in human leukemia CCRF-CEM and Nalm-6 cell lines

BACKGROUND: We describe an alternative method to determine mRNA half-life (t(1/2)) based on the Real-Time RT-PCR procedure. This approach was evaluated by using the β-actin gene as a reference molecule for measuring of mRNA stability. RESULTS: Human leukemia Nalm-6 and CCRF-CEM cells were treated with various concentrations of Actinomycin D to block transcription and aliquots were removed periodically. Total RNA was isolated and quantified using the RiboGreen(®) fluorescent dye with the VersaFluor Fluorometer System. One μg of total RNA was reverse transcribed and used as template for the amplification of a region of the β-actin gene (231 bp). To generate the standard curve, serial ten-fold dilutions of the pBactin-231 vector containing the cDNA amplified fragment were employed, β-actin mRNAs were quantified by Real-Time RT-PCR using the SYBR(®) Green I fluorogenic dye and data analyzed using the iCycle iQ system software. Using this method, the β-actin mRNA exhibited a half-life of 6.6 h and 13.5 h in Nalm-6 and CCRF-CEM cells, respectively. The t(1/2) value obtained for Nalm-6 is comparable to those estimated from Northern blot studies, using normal human leukocytes (5.5 h). CONCLUSIONS: We have developed a rapid, sensitive, and reliable method based on Real-Time RT-PCR for measuring mRNA half-life. Our results confirm that β-actin mRNA half-life can be affected by the cellular growth rate

Cytotoxic effect of 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR) on childhood acute lymphoblastic leukemia (ALL) cells: implication for targeted therapy

Acute lymphoblastic leukemia (ALL) is the most common hematological malignancy affecting children. Despite significant progress and success in the treatment of ALL, a significant number of children continue to relapse and for them, outcome remains poor. Therefore, the search for novel therapeutic approaches is warranted. The aim of this study was to investigate the AMP activated protein kinase (AMPK) as a potential target in childhood acute lymphoblastic leukemia (ALL) subtypes characterized by non-random translocation signature profiles. We evaluated the effects of the AMPK activator AICAR on cell growth, cell cycle regulators and apoptosis of various childhood ALL cells. We found that treatment with AICAR inhibited cell proliferation, induced cell cycle arrest in G1-phase, and apoptosis in CCRF-CEM (T-ALL), NALM6 (Bp-ALL), REH (Bp-ALL, TEL/AML1) and SupB15 (Bp-ALL, BCR/ABL) cells. These effects were abolished by treatment with the adenosine kinase inhibitor 5'-iodotubericidin prior to addition of AICAR indicating that AICAR's cytotoxicity is mediated through AMPK activation. Moreover, we determined that growth inhibition exerted by AICAR was associated with activation of p38-MAPK and increased expression of the cell cycle regulators p27 and p53. We also demonstrated that AICAR mediated apoptosis through the mitochondrial pathway as revealed by the release of cytochrome C and cleavage of caspase 9. Additionally, AICAR treatment resulted in phosphorylation of Akt suggesting that activation of the PI3K/Akt pathway may represent a compensatory survival mechanism in response to apoptosis and/or cell cycle arrest. Combined treatment with AICAR and the mTOR inhibitor rapamycin resulted in additive anti-proliferative activity ALL cells. AICAR-mediated AMPK activation was found to be a proficient cytotoxic agent in ALL cells and the mechanism of its anti-proliferative and apoptotic effect appear to be mediated via activation of p38-MAPK pathway, increased expression of cell cycle inhibitory proteins p27 and p53, and downstream effects on the mTOR pathway, hence exhibiting therapeutic potential as a molecular target for the treatment of childhood ALL. Therefore, activation of AMPK by AICAR represents a novel approach to targeted therapy, and suggests a role for AICAR in combination therapy with inhibitors of the PI3K/Akt/mTOR pathways for the treatment of childhood in ALL

Analysis of folylpoly-γ-glutamate synthetase gene expression in human B-precursor ALL and T-lineage ALL cells

BACKGROUND: Expression of folylpoly-γ-glutamate synthetase (FPGS) gene is two- to three-fold higher in B-precursor ALL (Bp- ALL) than in T-lineage ALL (T-ALL) and correlates with intracellular accumulation of methotrexate (MTX) polyglutamates and lymphoblast sensitivity to MTX. In this report, we investigated the molecular regulatory mechanisms directing FPGS gene expression in Bp-ALL and T-ALL cells. METHODS: To determine FPGS transcription rate in Bp-ALL and T-ALL we used nuclear run-on assays. 5'-RACE was used to uncover potential regulatory regions involved in the lineage differences. We developed a luciferase reporter gene assay to investigate FPGS promoter/enhancer activity. To further characterize the FPGS proximal promoter, we determined the role of the putative transcription binding sites NFY and E-box on FPGS expression using luciferase reporter gene assays with substitution mutants and EMSA. RESULTS: FPGS transcription initiation rate was 1.6-fold higher in NALM6 vs. CCRF-CEM cells indicating that differences in transcription rate led to the observed lineage differences in FPGS expression between Bp-ALL and T-ALL blasts. Two major transcripts encoding the mitochondrial/cytosolic and cytosolic isoforms were detected in Bp-ALL (NALM6 and REH) whereas in T-ALL (CCRF-CEM) cells only the mitochondrial/cytosolic transcript was detected. In all DNA fragments examined for promoter/enhancer activity, we measured significantly lower luciferase activity in NALM6 vs. CCRF-CEM cells, suggesting the need for additional yet unidentified regulatory elements in Bp-ALL. Finally, we determined that the putative transcription factor binding site NFY, but not E-box, plays a role in FPGS transcription in both Bp- and T-lineage. CONCLUSION: We demonstrated that the minimal FPGS promoter region previously described in CCRF-CEM is not sufficient to effectively drive FPGS transcription in NALM6 cells, suggesting that different regulatory elements are required for FPGS gene expression in Bp-cells. Our data indicate that the control of FPGS expression in human hematopoietic cells is complex and involves lineage-specific differences in regulatory elements, transcription initiation rates, and mRNA processing. Understanding the lineage-specific mechanisms of FPGS expression should lead to improved therapeutic strategies aimed at overcoming MTX resistance or inducing apoptosis in leukemic cells

Abstract 4359: Folylpolyglutamate synthetase expression is transcriptionally regulated by chromatin remodeling and recruitment of a multiprotein corepressor complex associated to non-random fusions in ALL

Abstract Non-random translocations in acute lymphoblastic leukemia (ALL) are known to alter gene transcription and molecular signaling pathways. Understanding the effects of these “altered pathways” on drug metabolism will provide the biological rationale to design combination strategies for ALL using proven antileukemic drugs, such as methotrexate (MTX), and novel targeted agents. The TEL-AML1 gene fusion is the most common translocation in childhood ALL (25%) and the E2A-PBX1 occurs in ∼5% of B-precursor (Bp) ALL. Both T-ALL and B-p ALL expressing TEL-AML1 and E2A-PBX1 fusions were shown to accumulate lower level of long-chain MTX-PGs when compared to other Bp- ALL phenotypes. Folylpolyglutamate synthetase (FPGS) expression, responsible for the synthesis of MTX-PG, is both lineage-specific and proliferation dependent in ALL cells. Using real-time qRT-PCR, we determined that primary cells and cell lines expressing TEL-AML1 and E2A-PBX1 exhibit significantly decreased FPGS mRNA expression. Using an FPGS-luciferase reporter gene assay, we determined that both TEL-AML1 and E2A-PBX1 fusions lead to down regulation of the FPGS promoter activity. Co-immunoprecipitation and ChIP assays demonstrated that TEL-AML1 decreased FPGS transcription by recruiting co-repressors (mSin3A, Rb) and HDAC1 to the native chromatin structure of the FPGS promoter region. These data suggest that TEL-AML1 associates with mSin3A and HDAC1 to repress FPGS transcription, leading to lower intracellular MTX-PGs accumulation. We also investigated the cell cycle dependence of FPGS expression and determined whether TEL-AML1 and E2A-PBX1 influence its expression. When CCRF-CEM (T-ALL) and NALM6 (Bp-ALL) cells were synchronized to G1 and S-phase, we found that their relative level of FPGS mRNA expression was respectively 1.4 and 2.9-fold higher in G1-phase than in S-phase, indicating FPGS mRNA expression is upregulated in G1-phase prior to progress into S-phase. Using ChIP assays, we demonstrated that NFYB, Sp1, E2F and Rb proteins interact with the native chromatin structure of the FPGS promoter region, suggesting they may associate with non-random fusions to regulate FPGS expression during cell cycle progression. Consistent with this observation, REH cells expressing TEL-AML1, exhibited a greater percentage of cells arrested in G0/G1 compared to NALM6 control cells (51% vs. 40%). These data suggest that TEL-AML1, and possibly other fusions, alter FPGS mRNA levels by determining the cell cycle dependence of FPGS gene expression. Our data demonstrate for the first time the molecular mechanism leading to lower MTX-PGs accumulation mediated by lower FPGS expression in TEL-AML1 lymphoblasts, and suggest that recruitment of this multiprotein corepressor complex may also regulate FPGS expression during cell cycle progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4359

Histone Deacetylase Inhibitor Induces FPGS mRNA Expression in Childhood B-Precursor and T Acute Lymphoblastic Leukemia: Implication of Combination Therapy with Methotrexate To Enhance Cytotoxicity

Abstract Methotrexate (MTX) is an antifolate widely used to treat childhood acute lymphoblastic leukemia (ALL). MTX is retained within cells as long-chain polyglutamates (MTX-PGs), after metabolism by the enzyme folylpoly-γ-glutamate synthetase (FPGS). Intracellular retention of MTX-PGs results in enhanced cytotoxicity due to prolonged inhibition of dihydrofolate reductase (DHFR), and the additional inhibition of thymidylate synthetase (TS). The FPGS gene was shown to be regulated by the transcription factors Sp1 and NFY. We performed DNaseI hypersensitive assays and identified a hypersensitive site mapping closely upstream of exon 1 suggesting that chromatin remodeling may contribute to FPGS gene regulation. Using co-immunoprecipitation and Western blotting we investigated the role of histone modifications and chromatin remodeling on the expression of FPGS and uncovered interactions between NFY, Sp1 and HDAC1. Our results demonstrate that HDAC1 complexes with NFY and Sp1 transcription factors in both B- and T-ALL cells. DNA affinity precipitation assay (DAPA) revealed that the HDAC1-NFY and HDAC1-Sp1 complex binds to the NFY and Sp1 binding sites in the FPGS promoter. These findings suggest that transcription of the FPGS gene may be regulated by acetylation of NFY and Sp1 factors and interaction with HDAC1, and/or chromatin remodeling. We then examined the effect of the histone deacetylase inhibitor (HDACi) sodium butyrate (NaBu) on the expression of FPGS and other folate-related genes. The level of FPGS, ATP-binding cassette subfamily C (ABCC1), ATP-binding cassette subfamily G (ABCG2), DHFR, γ-glutamyl hydrolase (GGH), solute carrier family 19/folate transporter (SLC19A1), and TS mRNA gene expression was determined by qRT-PCR in NALM6 (Bp-ALL), REH (Bp-ALL, t(12,21)/TEL-AML1), SupB15 (Bp-ALL, t(9,22)/BCR-ABL), and CCRF-CEM (T-ALL) cells treated with NaBu [2mM-5mM]. In all cell lines examined, treatment with NaBu induced 2- to 5-fold the level of FPGS and ABCC1 mRNA expression whereas the level of DHFR, SLC19A1, and TS mRNA expression was decreased. Expression of GGH and ABCG2 mRNAs was increased 2-fold in CCRF-CEM but remained unaltered in Bp-ALL NaBu treated cells. Promoters of butyrate-responsive genes have been shown to contain genetic elements such as Sp1/Sp3 binding sites which interact with HDAC1 to mediate the action of NaBu. On this basis, we hypothesized that pre-treatment of ALL cells with NaBu should lead to induction of FPGS expression and subsequently, higher synthesis of MTX-PG and enhanced MTX cytotoxicity in ALL cells. To test this hypothesis, CCRF-CEM, NALM6, REH, and SupB15 cells were treated sequentially with NaBu (24h) and MTX (4h), and assessed for cell viability. Treatment of NaBu and MTX increased cell death by ∼40% in NALM6, REH, and SupB15 Bp-cells, and ∼60% in CCRF-CCEM cells when compared to treatment with each drug alone. These data suggest that combination of HDACi and MTX may represent a novel therapeutic strategy for treatment of ALL. This strategy may be particularly useful to overcome MTX resistance in patients diagnosed with phenotypes that accumulate low levels of MTX-PGs and for patients after relapse

Mechanisms of Sensitivity of B-Precursor, T-Lineage and BCR/ABL Positive Acute Lymphoblastic Leukemia (ALL) to the Glycolytic Inhibitor 2-Deoxy-D-Glucose (2-DG)

Abstract Abstract 3081 Poster Board III-18 Novel treatment strategies are needed for patients with ALL diagnosed with resistant phenotypes or after relapse. The glucose analogue 2-deoxy-D-glucose (2-DG) is a glycolytic inhibitor that induces growth arrest and cell death by inhibiting the key glycolytic enzymes phosphoglucose isomerase (PGI) and hexokinase (HK). Cancer cells and hypoxic cells are more sensitive to 2-DG due to their reliance on glycolysis for ATP generation. In this study, we evaluated the antileukemic activity of 2-DG in T- and Bp-ALL subtypes characterized by non-random translocations. The cytotoxicity of 2-DG was determined on CCRF-CEM (PTEN mutant T-ALL), NALM6 (Bp-ALL), REH (TEL/AML1+ Bp-ALL), and the BCR/ABL+ ALL cell lines SupB15 and TOM1. Cells were treated with 4mM 2-DG for 72 h under normoxic vs. hypoxic (0.5% O2) conditions using a hypoxia box chamber (BioSpherix), and growth arrest and cell death were assessed. 2-DG induced 2-13 fold higher cell death in all ALL cells tested under normal O2 conditions compared to hypoxia. Similarly, growth inhibition was greater under normoxia. Both BCR/ABL+ ALL cell lines TOM1 and SupB15 were among the most sensitive, while the PTEN mutant CCRF-CEM (T-ALL) cell model was the least. To analyze the mechanisms of higher sensitivity of ALL cells under normoxia, we first used mannose to assess the role of glycosylation in 2-DG induced ALL cell death. Mannose reversed 2-DG induced cell death in CCRF-CEM cells but only partially in NALM6 cells, indicating inhibition of glycosylation mainly mediated death in CCRF-CEM cells, while inhibition of both glycolysis and glycosylation mediated cell death in NALM6 cells. To further assess other mechanisms leading to cell death in ALL, we evaluated mitochondrial integrity in representative Bp- and T-ALL models by determining cellular respiration with a Clark electrode (Hansatech Instruments). Our data show that cellular respiration in NALM6 was 50% lower compared to CCRF-CEM cells, suggesting that an intrinsic mitochondrial dysfunction was also responsible for 2-DG induced cell death in NALM6 cells. We then evaluated the observed higher sensitivity of BCR/ABL+ ALL cells to 2-DG. The AMPK and PI3K/Akt/mTOR pathways regulate protein, fatty acid and glucose metabolism, and key glycolytic enzymes are known to be upregulated in BCR/ABL+ cells. Therefore, we determined changes on AMPK and PI3K/Akt/mTOR signaling following exposure to 2-DG in SupB15 (BCR/ABL+) and in CCRF-CEM cells, the least sensitive ALL model examined. Western immunoblotting showed that 2-DG led to upregulation of p-AMPK (Thr172) and downregulation of p-mTOR (S2448) and p-p70S6K (Thr389). In both cell lines, 2-DG also led to cell death by triggering an unfolded protein response (UPR) evidenced by CHOP expression and PARP cleavage. We previously demonstrated that activation of AMPK leads to upregulation of Akt as a compensatory survival mechanism in ALL cells (Mol Cancer 6: 46, 2007). On this basis, we then tested induction of cell death by the combination of 2-DG (4 mM) plus Akt inhibitor X (AIX) (12 μM) in CCRF-CEM vs. SupB15 cells under normoxic conditions. Again, SupB15 cells were more sensitive to 2-DG, while AIX alone at low dose (12 μM) had minimal effect on either cell line. When both drugs were used in combination at these same concentrations, significant synergism was seen in CCRF-CEM cells but not in SupB15 cells. Analysis of AMPK, mTOR and Akt in CCRF-CEM cells treated with the combination of 2-DG plus AIX showed higher p-AMPK activation and mTOR downregulation compared to 2-DG alone, while these signaling changes were lower in SupB15 cells. Consistent with these findings, this combination led to higher induction of UPR and PARP cleavage in CCRF-CEM vs. SupB15 cells. We conclude that the greater sensitivity of ALL cells to 2-DG under normoxia is due to concomitant inhibition of glycolysis and/or glycosylation, an inherent mitochondrial dysfunction in some phenotypes, and 2-DG induced changes in AMPK and PI3K/Akt/mTOR signaling leading to the induction of UPR. Concomitant Akt inhibition sensitizes the relatively 2-DG resistant CCRF-CEM cells to death with 2-DG. Chemotherapy resistant BCR/ABL+ ALL cells exhibit significant sensitivity to 2-DG, likely due to their increased reliance on glycolysis for ATP generation. On this basis, we propose that glycolytic inhibitors represent a promising novel therapeutic strategy for ALL, particularly for BCR/ABL+ ALL. Disclosures No relevant conflicts of interest to declare

Stress-Induced AMPK Interactome Analysis Reveals Novel Protein-Protein Interactions Associated with Chromatin Remodeling and Transcription Machinery in Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) is a leading cause of cancer-related death among children, adolescents and young adults. Survival rates for relapsed/refractory ALL remain dismal. We have previously reported that ALL cells are vulnerable to energy/metabolic stress conditions following AMP-activated protein kinase (AMPK) activation, leading to cell death. AMPK is the master metabolic regulator, and our lab and others have reported it interacts with chromatin-associated proteins to epigenetically regulate gene expression in response to energy/metabolic stress. To identify genome-wide genes regulated by direct association of AMPK to chromatin in response to energy/metabolic stress, we previously used ChIP-seq and RNA-seq in Bp-ALL (KASUMI-2) and T-ALL (KE-37) cells treated with or without glucose or AICAR, a well-known AMPK activator, and found that (i) AMPK interacts with components of the transcription machinery including the TATA-Box Binding Protein Associated Factor (TAF) and RNA polymerase II, (ii) AMPKα2 was enriched for genes involved in histone H3-K4 methylation, protein localization, Notch signaling, and mRNA destabilization, (iii) AMPKa2 was recruited to promoter regions of the epigenetic regulators KMT2A and SETD1A, and (iv) the mRNA expression of KMT2A and SETD1A genes was upregulated in KASUMI-2 cells treated with AICAR. To further investigate AMPK's involvement in epigenetic mechanisms in response to energy/metabolic stress, we performed a comprehensive interactome analysis using TurboID-based proximity labeling proteomics. AMPK is a highly conserved heterotrimeric kinase complex composed of catalytic α-subunit (α1 or α2) and regulatory β (β1 or β2) and γ (γ1, γ2, or γ3) subunits. We generated HEK293T cells stably expressing TurboID fused to the AMPKα1, AMPKα2, AMPKβ1 or AMPKγ1 (which are the most abundant AMPK subunits expressed in ALL cells), and these stable cell lines were treated with the potent allosteric AMPK activator PF-06409577 for 1 hr, to mimic the cell's rapid response to energy/metabolic stress. Following characterization of these stable cell lines and small-scale optimization experiments, treated samples and controls were submitted to mass spectrometry analysis. Proteomics data analysis identified novel AMPK's protein-protein interactions (PPI) which were altered (enhanced or reduced) following PF-06409577 treatment. Among PPI altered by PF-06409577 treatment, 148 were enhanced and 144 reduced for AMPKα1, 169 enhanced and 172 reduced for AMPKα2, 250 enhanced and 188 reduced for AMPKβ1 and 289 enhanced and 153 reduced for AMPKγ1. For proteomics data visualization we used Cytoscape software. The GO analysis of proteins interacting with AMPKα1β1γ1 and/or AMPKα2β1γ1 heterotrimeric complexes in cells treated with PF-06409577 showed that energy/metabolic stress enhances their interaction with proteins involved in gene expression, cellular metabolic processes, chromatin organization, DNA repair, histone modifications, etc.. Using the STRING database, we performed MCL clustering analysis and uncovered a major cluster of known epigenetic regulators (e.g., histone modifiers such as KDM6A, HDAC2, SETD2, KMT2A and SETD1A etc.) and components of the transcription machinery (such as NELFE, POLR2A etc.) that interacted with AMPK under stress-induced conditions. These data support our previous findings uncovered by ChIP-seq and RNA-seq analysis. Consequently, our findings further validate an epigenetic role for AMPK via interactions with putative members of chromatin-associated and transcription machinery complexes and chromatin modifying enzymes that allow ALL cells to respond to conditions of energy/metabolic stress. To our knowledge, this is the first report describing the AMPK interactome in pediatric ALL. Further elucidation of the uncovered protein-protein interactions may lead to potential epigenetic-based targeted therapies to overcome therapeutic resistance in relapse/refractory ALL and other hematological malignancies

TEL/AML1 and E2A/PBX1 Tranlsocations Lead to Altered Folylpoly-γ-Glutamate Synthetase (FPGS) Expression in ALL

Abstract 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