Histone deacetylase inhibitors induce FPGS mRNA expression and intracellular accumulation of long-chain methotrexate polyglutamates in childhood acute lymphoblastic leukemia: implications for combination therapy

Abstract

Children with acute lymphoblastic leukemia (ALL) diagnosed with resistant phenotypes, and those who relapse, have a dismal prognosis for cure. The antifolate methotrexate (MTX), a universal component of ALL therapies, is metabolized by folylpoly-c-glutamate synthetase (FPGS) into long-chain polyglutamates (MTX-PG 3À7 ), resulting in enhanced cytotoxicity from prolonged inhibition of dihydrofolate reductase (DHFR) and thymidylate synthetase (TS). Using DNaseI assays, we identified a hypersensitive site upstream from exon-1, suggesting chromatin remodeling could alter FPGS expression. We demonstrated that histone deacetylase-1 (HDAC1) is recruited by NFY and Sp1 transcription factors to the FPGS promoter in ALL cell lines. We examined the effect of histone deacetylase inhibitors (HDACIs) sodium butyrate and suberoylanilide hydroxamic acid (SAHA) on the expression of FPGS and other folate-related genes. HDACIs increased FPGS mRNA expression by 2-to 5-fold, whereas DHFR and TS mRNA expression was decreased. Combination treatment with MTX plus SAHA significantly increased cytotoxicity and apoptosis in B-and T-ALL cell lines as compared with each drug alone (CIp0.8). SAHA increased the intracellular accumulation of long-chain MTX-PG 3À7 . Therefore, HDACI-induced FPGS expression increases the accumulation of MTX-PG 3À7 and cytotoxicity in ALL cell lines, which is potentiated by DHFR and TS downregulation. The synergism exhibited by the combination of MTX and SAHA warrants clinical testing in ALL patients.

The sequence of application of methotrexate and histone deacetylase inhibitors determines either a synergistic or an antagonistic response in childhood acute lymphoblastic leukemia cells

“…Incubation time with each compound was extended to 48 h and the absolute count of vital cells was assessed by flow cytometry. As shown in Figure 1b, sequential treatment with MTX followed by VPA decreased the amount of vital Reh cells significantly compared with administration of single drugs MTX and VPA (Po0.001), confirming the results reported by Leclerc et al 1 The sequential combination of both drugs in reversed order (VPA followed by MTX) was significantly less effective in reducing vital cell counts compared with administration of each drug alone (Po0.01). Taken together, altering the application schedule reversed the described synergistic effect 1 and actually had a potent antagonistic effect on treatment responsiveness of BCP-ALL cells.…”

“…We have acknowledged with great attention the recent article of Leclerc et al 1 reporting a synergistic cytotoxicity of methotrexate (MTX) and histone deacetylase inhibitors (HDACis) combination treatment in childhood acute lymphoblastic leukemia (ALL) cell lines. Both the group of Leclerc 1 and ours 2 have suggested that the introduction of HDACis into current ALL therapy regimes may provide a promising alternative.…”

“…Both the group of Leclerc 1 and ours 2 have suggested that the introduction of HDACis into current ALL therapy regimes may provide a promising alternative. Leclerc et al 1 have observed synergism after sequential treatment with MTX followed by the HDACi suberoylanilide hydroxamic acid (SAHA). However, when we changed the sequence of application by treating cells primarily with different HDACis, such as SAHA and valproic acid (VPA), and afterwards with MTX or combined MTX and HDACi concomitantly, a strong antagonistic effect was observed.…”

“…Leclerc et al 1 suggest that the synergism observed for the combination of MTX followed by HDACis warrants further testing in clinical studies. In the in vitro setting, elimination of a drug was achieved by replacement of culture medium, which allows a clearly defined sequence of exposure to each compound without overlap.…”

“…We used experimental settings similar to those presented by Leclerc et al 1 Briefly, cells of the B-cell precursor (BCP) ALL cell line Nalm6 were exposed to MTX (300 nM) or SAHA (1 mM) alone, or a sequence of SAHA followed by MTX. As shown in Figure 1a, treatment with SAHA followed by MTX significantly decreased the proportion of apoptotic Nalm6 cells compared with MTX alone (Po0.001), showing that the sequence of application has a significant effect on drug responsiveness.…”