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Inhibition of mutant IDH1 decreases D-2-HG levels without affecting tumorigenic properties of chondrosarcoma cell lines.

Suijker J, Oosting J, Koornneef A, Struys EA, Salomons GS, Schaap FG, Waaijer CJ, Wijers-Koster PM, Briaire-de Bruijn IH, Haazen L, Riester SM, Dudakovic A, Danen E, Cleton-Jansen AM, van Wijnen AJ, Bovée JV - Oncotarget (2015)

Bottom Line: Specific inhibition of mutant IDH1 using AGI-5198 decreased levels of D-2-HG in a dose dependent manner.After 72 hours of treatment one out of three mutant IDH1 cell lines showed a moderate decrease in viability , while D-2-HG levels decreased >90%.Likewise, prolonged treatment (up to 20 passages) did not affect proliferation and migration.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands.

ABSTRACT
Mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 are found in a subset of benign and malignant cartilage tumors, gliomas and leukaemias. The mutant enzyme causes the production of D-2-hydroxyglutarate (D-2-HG), affecting CpG island and histone methylation. While mutations in IDH1/2 are early events in benign cartilage tumors, we evaluated whether these mutations play a role in malignant chondrosarcomas. Compared to IDH1/2 wildtype cell lines, chondrosarcoma cell lines harboring an endogenous IDH1 (n=3) or IDH2 mutation (n=2) showed up to a 100-fold increase in intracellular and extracellular D-2-HG levels. Specific inhibition of mutant IDH1 using AGI-5198 decreased levels of D-2-HG in a dose dependent manner. After 72 hours of treatment one out of three mutant IDH1 cell lines showed a moderate decrease in viability , while D-2-HG levels decreased >90%. Likewise, prolonged treatment (up to 20 passages) did not affect proliferation and migration. Furthermore, global gene expression, CpG island methylation as well as histone H3K4, -9, and -27 trimethylation levels remained unchanged. Thus, while IDH1/2 mutations cause enchondroma, malignant progression towards central chondrosarcoma renders chondrosarcoma growth independent of these mutations. Thus, monotherapy based on inhibition of mutant IDH1 appears insufficient for treatment of inoperable or metastasized chondrosarcoma patients.

No MeSH data available.


Related in: MedlinePlus

The effect of long term mutant IDH1 inhibition on histone and DNA methylation and gene expression(A) Western blot showing levels of trimethylation of Histone H3 K4, K9 and K27 after treatment with 1.5 μM AGI5198 or 5 mM D-2-HG for 10 or 20 passages. Treatments did not have an effect on trimethylation of each of the lysines in the mutant IDH1 chondrosarcoma cell lines. (B) Global methylation levels after prolonged treatment with AGI-5198 remain unchanged, with overall mean β-values of both treated and untreated samples of approximately 0.5. (C) When analyzing the CpG islands specific probes separately, minimal demethylation of the genome is seen. (D) Differences in CpG island specific methylation levels are predominantly seen in the L835 cell line treated for 10 passages. (E) Unsupervised hierarchical clustering of all expressed genes in the RNAseq dataset showed that the expression profiles of the cell lines are not dramatically changed by the treatment, since expression profiles of the treated cell lines are more similar to their untreated counterparts than to other treated cell lines. Samples were labelled according to the cell line, followed by the treatment (AGI=treated with 1.5 μM AGI-5198; CONTROL=treated with DMSO) and the number of passages (10=10 passages of continuous treatment; 20=20 passages of continuous treatment).
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Figure 5: The effect of long term mutant IDH1 inhibition on histone and DNA methylation and gene expression(A) Western blot showing levels of trimethylation of Histone H3 K4, K9 and K27 after treatment with 1.5 μM AGI5198 or 5 mM D-2-HG for 10 or 20 passages. Treatments did not have an effect on trimethylation of each of the lysines in the mutant IDH1 chondrosarcoma cell lines. (B) Global methylation levels after prolonged treatment with AGI-5198 remain unchanged, with overall mean β-values of both treated and untreated samples of approximately 0.5. (C) When analyzing the CpG islands specific probes separately, minimal demethylation of the genome is seen. (D) Differences in CpG island specific methylation levels are predominantly seen in the L835 cell line treated for 10 passages. (E) Unsupervised hierarchical clustering of all expressed genes in the RNAseq dataset showed that the expression profiles of the cell lines are not dramatically changed by the treatment, since expression profiles of the treated cell lines are more similar to their untreated counterparts than to other treated cell lines. Samples were labelled according to the cell line, followed by the treatment (AGI=treated with 1.5 μM AGI-5198; CONTROL=treated with DMSO) and the number of passages (10=10 passages of continuous treatment; 20=20 passages of continuous treatment).

Mentions: Since mutations in IDH1 are associated with inhibition of demethylases [21, 27], we tested the effect of prolonged treatment with AGI-5198 and D-2-HG on trimethylation levels of H3K4, H3K9 and H3K27. The mutant IDH1 chondrosarcoma cell line L835, treated with 1.5 μM AGI-5198 for 10 passages, and the mutant IDH1 cell lines HT1080 and JJ012, treated for 20 passages, did not show any difference in lysine trimethylation of these specific histone marks (Figure 5A).


Inhibition of mutant IDH1 decreases D-2-HG levels without affecting tumorigenic properties of chondrosarcoma cell lines.

Suijker J, Oosting J, Koornneef A, Struys EA, Salomons GS, Schaap FG, Waaijer CJ, Wijers-Koster PM, Briaire-de Bruijn IH, Haazen L, Riester SM, Dudakovic A, Danen E, Cleton-Jansen AM, van Wijnen AJ, Bovée JV - Oncotarget (2015)

The effect of long term mutant IDH1 inhibition on histone and DNA methylation and gene expression(A) Western blot showing levels of trimethylation of Histone H3 K4, K9 and K27 after treatment with 1.5 μM AGI5198 or 5 mM D-2-HG for 10 or 20 passages. Treatments did not have an effect on trimethylation of each of the lysines in the mutant IDH1 chondrosarcoma cell lines. (B) Global methylation levels after prolonged treatment with AGI-5198 remain unchanged, with overall mean β-values of both treated and untreated samples of approximately 0.5. (C) When analyzing the CpG islands specific probes separately, minimal demethylation of the genome is seen. (D) Differences in CpG island specific methylation levels are predominantly seen in the L835 cell line treated for 10 passages. (E) Unsupervised hierarchical clustering of all expressed genes in the RNAseq dataset showed that the expression profiles of the cell lines are not dramatically changed by the treatment, since expression profiles of the treated cell lines are more similar to their untreated counterparts than to other treated cell lines. Samples were labelled according to the cell line, followed by the treatment (AGI=treated with 1.5 μM AGI-5198; CONTROL=treated with DMSO) and the number of passages (10=10 passages of continuous treatment; 20=20 passages of continuous treatment).
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Related In: Results  -  Collection

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Figure 5: The effect of long term mutant IDH1 inhibition on histone and DNA methylation and gene expression(A) Western blot showing levels of trimethylation of Histone H3 K4, K9 and K27 after treatment with 1.5 μM AGI5198 or 5 mM D-2-HG for 10 or 20 passages. Treatments did not have an effect on trimethylation of each of the lysines in the mutant IDH1 chondrosarcoma cell lines. (B) Global methylation levels after prolonged treatment with AGI-5198 remain unchanged, with overall mean β-values of both treated and untreated samples of approximately 0.5. (C) When analyzing the CpG islands specific probes separately, minimal demethylation of the genome is seen. (D) Differences in CpG island specific methylation levels are predominantly seen in the L835 cell line treated for 10 passages. (E) Unsupervised hierarchical clustering of all expressed genes in the RNAseq dataset showed that the expression profiles of the cell lines are not dramatically changed by the treatment, since expression profiles of the treated cell lines are more similar to their untreated counterparts than to other treated cell lines. Samples were labelled according to the cell line, followed by the treatment (AGI=treated with 1.5 μM AGI-5198; CONTROL=treated with DMSO) and the number of passages (10=10 passages of continuous treatment; 20=20 passages of continuous treatment).
Mentions: Since mutations in IDH1 are associated with inhibition of demethylases [21, 27], we tested the effect of prolonged treatment with AGI-5198 and D-2-HG on trimethylation levels of H3K4, H3K9 and H3K27. The mutant IDH1 chondrosarcoma cell line L835, treated with 1.5 μM AGI-5198 for 10 passages, and the mutant IDH1 cell lines HT1080 and JJ012, treated for 20 passages, did not show any difference in lysine trimethylation of these specific histone marks (Figure 5A).

Bottom Line: Specific inhibition of mutant IDH1 using AGI-5198 decreased levels of D-2-HG in a dose dependent manner.After 72 hours of treatment one out of three mutant IDH1 cell lines showed a moderate decrease in viability , while D-2-HG levels decreased >90%.Likewise, prolonged treatment (up to 20 passages) did not affect proliferation and migration.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands.

ABSTRACT
Mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 are found in a subset of benign and malignant cartilage tumors, gliomas and leukaemias. The mutant enzyme causes the production of D-2-hydroxyglutarate (D-2-HG), affecting CpG island and histone methylation. While mutations in IDH1/2 are early events in benign cartilage tumors, we evaluated whether these mutations play a role in malignant chondrosarcomas. Compared to IDH1/2 wildtype cell lines, chondrosarcoma cell lines harboring an endogenous IDH1 (n=3) or IDH2 mutation (n=2) showed up to a 100-fold increase in intracellular and extracellular D-2-HG levels. Specific inhibition of mutant IDH1 using AGI-5198 decreased levels of D-2-HG in a dose dependent manner. After 72 hours of treatment one out of three mutant IDH1 cell lines showed a moderate decrease in viability , while D-2-HG levels decreased >90%. Likewise, prolonged treatment (up to 20 passages) did not affect proliferation and migration. Furthermore, global gene expression, CpG island methylation as well as histone H3K4, -9, and -27 trimethylation levels remained unchanged. Thus, while IDH1/2 mutations cause enchondroma, malignant progression towards central chondrosarcoma renders chondrosarcoma growth independent of these mutations. Thus, monotherapy based on inhibition of mutant IDH1 appears insufficient for treatment of inoperable or metastasized chondrosarcoma patients.

No MeSH data available.


Related in: MedlinePlus