Abstract
Polyglutamylation of antifolates catalyzed by folylpoly-γ-glutamate synthetase (FPGS) is essential for their intracellular retention and cytotoxic activity. Hence, loss of FPGS expression and/or function results in lack of antifolate polyglutamylation and drug resistance. Members of the TGF-β/Smad signaling pathway are negative regulators of hematopoiesis and deregulation of this pathway is considered a major contributor to leukemogenesis. Here we show that FPGS gene expression is inversely correlated with the binding of a Smad4/Ets-1 complex to exon12 of FPGS in both acute lymphoblastic leukemia cells and acute myeloid leukemia blast specimens. We demonstrate that antifolate resistant leukemia cells harbor a heterozygous point mutation in exon12 of FPGS which disrupts FPGS activity by abolishing ATP binding, and alters the binding pattern of transcription factors to the genomic region of exon12. This in turn results in the near complete silencing of the wild type allele leading to a 97% loss of FPGS activity. We show that exon12 is a novel intragenic transcriptional regulator, endowed with the ability to drive transcription in vitro, and is occupied by transcription factors and chromatin remodeling agents (e.g. Smad4/Ets-1, HP-1 and Brg1) in vivo. These findings bear important implications for the rational overcoming of antifolate resistance in leukemia.
Highlights
Folates are essential vitamins which serve as cofactors in a variety of key processes such as de novo nucleotide biosynthesis, amino acid biosynthesis and DNA methylation [1, 2]
To explore the clinical relevance of our findings, which suggested that the expression of folylpoly-γ-glutamate synthetase (FPGS) is repressed by a Smad4/Ets-1 complex, we studied the binding of these transcription factors (TFs) to exon12 of FPGS in blast cells from acute myeloid leukemia (AML) patients
We found that the WT FPGS allele was selectively silenced presumably due to the binding of Smad4 to the genomic region of WT exon12
Summary
Folates are essential vitamins which serve as cofactors in a variety of key processes such as de novo nucleotide biosynthesis, amino acid biosynthesis and DNA methylation [1, 2]. FPGS plays a key role in intracellular retention and antitumor activity of polyglutamatable antifolates [5]. Antifolates including methotrexate (MTX) are a key component in ALL chemotherapy, acute myeloid leukemia (AML) was found to have intrinsic resistance to these important antimetabolites. Three naturally occurring mutations have been shown to underlie loss of FPGS function in leukemia cells: C388F decreased the affinity of FPGS for glutamate by 23-fold [11]. The transforming growth factor-β (TGF-β) signaling pathway has key roles in cell differentiation, apoptosis, development and carcinogenesis [13]. We further demonstrate that FPGS gene expression is inversely correlated with the binding of a Smad4/Ets-1 complex to exon in both ALL cells and AML blast specimens
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