Abstract
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.