Histone Deacetylase Inhibitors Induce FPGS mRNA Expression and Intracellular Accumulation of Long-Chain Methotrexate Polyglutamates in Childhood Acute Lymphoblastic Leukemia: Implications for Combination Therapy

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2923-2923
Author(s):  
Guy J Leclerc ◽  
Caihong Mou ◽  
Gilles M Leclerc ◽  
Abdul M Mian ◽  
Julio C Barredo
Keyword(s):  
Transcription Factors ◽  
Mrna Expression ◽  
Histone Deacetylase ◽  
Cell Lines ◽  
Chromatin Remodeling ◽  
Histone Deacetylase Inhibitors ◽  
Lymphoblastic Leukemia ◽  
Intracellular Accumulation ◽  
Essential Components ◽  
Long Chain

Abstract Folate cofactors are essential components of one carbon metabolism and are required for the biosynthesis of purines, pyrimidines, serine and methionine. The classical folate antagonist methotrexate (MTX) continues to be a universal component of most ALL treatment regimens. 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 thymidylate synthetase (TS). The FPGS gene is regulated by the transcription factors NFY and Sp1. Using DNaseI assays we identified a hypersensitive site mapping closely upstream of exon 1, suggesting that chromatin remodeling may contribute to FPGS gene regulation. To investigate the role of histone modifications and chromatin remodeling on FPGS expression and uncover interactions between NFY, Sp1 and HDAC1, we performed co-immunoprecipitation and Western blotting. Our results demonstrate that HDAC1 complexes with NFY and Sp1 transcription factors in both B- and T-ALL cells. DNA affinity precipitation assays (DAPA) revealed that HDAC1 is recruited by NFY and Sp1 to the FPGS promoter. These findings suggest that transcription of the FPGS gene may be regulated by NFY and Sp1 factors interacting with HDAC1, and leading to chromatin remodeling. We then examined the effect of the histone deacetylase inhibitors (HDACIs) sodium butyrate (NaBu) and suberoylanilide hydroxamic acid (SAHA) on the expression of FPGS and other folate-related genes in NALM6 (Bp-ALL), REH (TEL/AML1+, Bp-ALL), SupB15 (BCR/ABL+, Bp-ALL), and CCRF-CEM (T-ALL) cells using qRT-PCR. In all cell lines examined, treatment with HDACIs increased FPGS mRNA expression by 2- to 5-fold, whereas the level of DHFR and TS mRNA expression were decreased. On this basis, we hypothesized that induction of FPGS expression by HDACIs, results in higher accumulation of MTX-PG and enhanced MTX cytotoxicity in ALL cells. Further, the concomitant decrease in the expression of the MTX-PG target enzymes DHFR and TS, would enhance the cytotoxicity of the combination of HDACIs plus MTX in ALL cells. To test this hypothesis, NALM6, REH, and SupB15 cells were treated with MTX (4h) + SAHA (24h), and cell viability assessed. We determined that SAHA increased the intracellular accumulation of long chain MTX-PGs (n ≥3 Glu) in ALL cells, correlating with the upregulation of FPGS expression in SAHA-treated cells. Treatment with MTX + SAHA increased cytotoxicity by ~30% with a calculated combination index of ≤ 0.8 indicating synergy. Analysis of apoptosis using AnnexinV/PI staining revealed a 2 to 3-fold increase in apoptotic cell death in all cell lines treated with this combination. Our data suggest HDACIs enhance MTX cytotoxicity by upregulation of FPGS expression, increased accumulation of MTXPG and downregulation of DHFR and TS. The synergism exhibited by the combination of MTX and SAHA suggests it should be tested in ALL patients, in particular those who exhibit phenotypes with de novo or acquired resistance to MTX.

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.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 864-864 ◽  
Author(s):  
Guy J. Leclerc ◽  
Julio C. Barredo
Keyword(s):  
Transcription Factors ◽  
Acute Lymphoblastic Leukemia ◽  
Mrna Expression ◽  
Histone Deacetylase ◽  
Chromatin Remodeling ◽  
Binding Sites ◽  
Histone Deacetylase Inhibitor ◽  
Lymphoblastic Leukemia ◽  
Atp Binding ◽  
Deacetylase Inhibitor

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.

scholarly journals Comparing the Effect of Multiple Histone Deacetylase Inhibitors on SSTR2 Expression and [111In]In-DOTATATE Uptake in NET Cells

Cancers ◽  
2021 ◽  
Vol 13 (19) ◽  
pp. 4905
Author(s):  
Maria J. Klomp ◽  
Simone U. Dalm ◽  
Peter M. van Koetsveld ◽  
Fadime Dogan ◽  
Marion de Jong ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Histone Deacetylase ◽  
Cell Lines ◽  
Histone Deacetylase Inhibitors ◽  
Peptide Receptor Radionuclide Therapy ◽  
Expression Levels ◽  
Beneficial Outcome ◽  
Hdaci Treatment ◽  
Mrna Expression Levels

The aim of this study was to increase somatostatin type-2 receptor (SSTR2) expression on neuroendocrine tumor (NET) cells using histone deacetylase inhibitors (HDACis), potentially increasing the uptake of SSTR2-targeted radiopharmaceuticals and subsequently improving treatment efficacy of peptide receptor radionuclide therapy (PRRT). Human NET cell lines BON-1, NCI-H727, and GOT1 were treated with HDACis (i.e., CI-994, entinostat, LMK-235, mocetinostat, panobinostat, or valproic acid (VPA); entinostat and VPA were the HDACis tested in GOT1 cells) to examine SSTR2 mRNA expression levels and uptake of SSTR2-targeting radiotracer [111In]In-DOTATATE. Reversibility of the induced effects was examined after drug-withdrawal. Finally, the effect of VPA on radiosensitivity was investigated. A strong stimulatory effect in BON-1, NCI-H727, and GOT1 cells was observed after HDACi treatment, both on SSTR2 mRNA expression levels and [111In]In-DOTATATE uptake. The effects of the HDACis were largely reversible over a period of seven days, demonstrating largest reductions within the first day. The reversibility profile of the induced effects suggests that proper timing of HDACi treatment is most likely essential for a beneficial outcome. In addition to increasing SSTR2 expression levels, VPA enhanced the radiosensitivity of all cell lines. In conclusion, HDACi treatment increased SSTR2 expression, and radiosensitivity was also enhanced upon VPA treatment.

Insights into histone deacetylase inhibitors-induced apoptosis in cancer cell lines

2008 ◽  
Vol 6 (9) ◽  
pp. 28
Author(s):  
P. Ruiz-Rico ◽  
M.P. Menéndez-Gutiérrez ◽  
E. Carrasco-García ◽  
R. Martínez-Mira ◽  
L. Rocamora-Reverte ◽  
...  
Keyword(s):  
Histone Deacetylase ◽  
Cell Lines ◽  
Cancer Cell ◽  
Histone Deacetylase Inhibitors ◽  
Cancer Cell Lines ◽  
Induced Apoptosis

scholarly journals Development of Coumarin-Based Hydroxamates as Histone Deacetylase Inhibitors with Antitumor Activities

Molecules ◽  
2020 ◽  
Vol 25 (3) ◽  
pp. 717 ◽  
Author(s):  
Na Zhao ◽  
Feifei Yang ◽  
Lina Han ◽  
Yuhua Qu ◽  
Di Ge ◽  
...  
Keyword(s):  
Molecular Docking ◽  
Histone Deacetylase ◽  
Cell Lines ◽  
Histone Deacetylases ◽  
Histone Deacetylase Inhibitors ◽  
Human Cancer ◽  
Immunoblot Analysis ◽  
Docking Study ◽  
Molecular Docking Study ◽  
Human Cancer Cell Lines

Histone deacetylases (HDACs) have been proved to be promising targets for the treatment of cancer, and five histone deacetylase inhibitors (HDACis) have been approved on the market for the treatment of different lymphomas. In our previous work, we designed a series of novel coumarin-containing hydroxamate HDACis, among which compounds 6 and 7 displayed promising activities against tumor growth. Based on a molecular docking study, we further developed 26 additional analogues with the aim to improve activity of designed compounds. Several of these new derivatives not only showed excellent HDAC1 inhibitory effects, but also displayed significant growth inhibitory activities against four human cancer cell lines. Representative compounds, 13a and 13c, showed potent anti-proliferative activities against solid tumor cell lines with IC50 values of 0.36–2.91 µM and low cytotoxicity against Beas-2B and L-02 normal cells. Immunoblot analysis revealed that 13a and 13c dose-dependently increased the acetylation of histone H3 and H4. Importantly, the two compounds displayed much better anti-metastatic effects than SAHA against the MDA-MB-231 cell line. Moreover, 13a and 13c arrested MDA-MB-231 cells at G2/M phase and induced MDA-MB-231 cell apoptosis. Finally, the molecular docking study rationalized the high potency of compound 13c.

Up-regulation of costimulatory/adhesion molecules by histone deacetylase inhibitors in acute myeloid leukemia cells

Blood ◽  
2000 ◽  
Vol 96 (12) ◽  
pp. 3847-3856 ◽  
Author(s):  
Takahiro Maeda ◽  
Masayuki Towatari ◽  
Hiroshi Kosugi ◽  
Hidehiko Saito
Keyword(s):  
Acute Myeloid Leukemia ◽  
Adhesion Molecules ◽  
Histone Deacetylase ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Histone Deacetylase Inhibitors ◽  
Trichostatin A ◽  
Hl60 Cells ◽  
Hla Dr ◽  
Acute Myeloid

Abstract Histone deacetylase inhibitors (HDACIs) have been used to focus on the effects of inducing gene expression through the acetylation of histones which results in chromatin remodeling. The study explored whether HDACIs could induce the expression of costimulatory/adhesion molecules on acute myeloid leukemia (AML) cells, thereby effectively inducing tumor immunity. The expression of CD80, CD86, human leukocyte antigen (HLA)-DR, HLA-ABC, and intracellular adhesion molecule–1 (ICAM-1) was tested in human AML cell lines after the addition of HDACI, sodium butyrate (SB). Generally, increased expression of CD86 was observed by SB treatment in a majority of cell lines, and ICAM-1 was expressed in fewer cell lines. Essentially the same results were obtained using other HDACIs such as FR901228, trichostatin A, and trapoxin A. Quantitation of transcripts of CD86 accompanied with RNA synthesis inhibition assay and nuclear run-on assay revealed that SB up-regulates the CD86 expression transcriptionally. Furthermore, chromatin immunoprecipitation experiments showed that HDACI treatment caused remarkable acetylation on histone H3 and H4 at CD86 promoter chromatin in vivo. In 30 clinical AML samples, CD86 expression was significantly increased (P < .001) by SB treatment, and the expression of HLA-DR and ICAM-1 was moderately increased (P < .05) by SB treatment. Finally, the allogeneic mixed leukocyte reaction (allo-MLR) against HL60 cells pretreated with SB was enhanced 4-fold compared with allo-MLR obtained with non-treated HL60 cells. These results suggest that the immunotherapeutic use of HDACIs may become a novel tool for treatment of AML.

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