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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

Prolonged treatment of IDH1/2 wildtype and IDH1 mutant cell lines with mutant IDH1 inhibitor AGI-5198 and metabolite D-2-HGProlonged treatment with 1.5 μM IDH1 mutant inhibitor AGI-5198 did not affect intracellular D-2-HG levels of the IDH1/2 wildtype cell line CH2879 after 10 and 20 passages, while treatment with D2HG resulted in increased levels of L2HG and D2HG intracellularly (A). Chondrosarcoma cells harboring an IDH1 mutation (L835 (B), HT1080 (C), JJ012 (D)) show decreased levels of intracellular D-2-HG compared to untreated cells after continuous treatment with 1.5 μM AGI-5198 for 10 and 20 passages, respectively. For L835, due to slower growth rate, only 10 passages were evaluated. The xCelligence assay demonstrated no effect of prolonged inhibition of mutant IDH1 on proliferation (E) or migration (F). Graphs show one representative experiment out of three experiments.
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Figure 4: Prolonged treatment of IDH1/2 wildtype and IDH1 mutant cell lines with mutant IDH1 inhibitor AGI-5198 and metabolite D-2-HGProlonged treatment with 1.5 μM IDH1 mutant inhibitor AGI-5198 did not affect intracellular D-2-HG levels of the IDH1/2 wildtype cell line CH2879 after 10 and 20 passages, while treatment with D2HG resulted in increased levels of L2HG and D2HG intracellularly (A). Chondrosarcoma cells harboring an IDH1 mutation (L835 (B), HT1080 (C), JJ012 (D)) show decreased levels of intracellular D-2-HG compared to untreated cells after continuous treatment with 1.5 μM AGI-5198 for 10 and 20 passages, respectively. For L835, due to slower growth rate, only 10 passages were evaluated. The xCelligence assay demonstrated no effect of prolonged inhibition of mutant IDH1 on proliferation (E) or migration (F). Graphs show one representative experiment out of three experiments.

Mentions: To assess the role of mutant IDH1 in chondrosarcoma cell proliferation, we incubated the cell lines with the IDH1 mutant inhibitor AGI-5198 for 72 hours. Indeed the levels of D-2-HG in culture media decreased in a dose-dependent manner in the mutant IDH1 cell lines JJ012 (R132G), HT1080 (R132C) and L835 (R132C) with IC50s of 0.7 μM, 0.5 μM and 0.35 μM, respectively (Figure 3A). As expected, D-2-HG levels of the mutant IDH2 SW1353 cell line were not affected (Figure 3A). Interestingly, even at doses that led to >90% decrease in D-2-HG (20 μM AGI-5198 for 72 hours), only a minor effect on cell viability using a WST-1 assay was seen (approximately 65% viability left in JJ012 after 72 hours)(Figure 3B). Furthermore, mutant IDH1 inhibition did not influence the colony forming capacity of the mutant IDH1 cell lines HT1080 and JJ012 and the mutant IDH2 cell line SW1353 after 10 days of treatment, whereas L835 was not able to form colonies in both the control and the AGI-5198 conditions (Figure 3C). Since glioma cells transfected with mutant IDH1 need 20 passages to show an effect on methylation [33], we treated the mutant IDH1 cell lines JJ012, HT1080 and the IDH1/2 wildtype cell line CH2879 for up to 20 passages. Due to its slower growth rate, L835 was treated for up to 10 passages. As expected, treatment of CH2879 with 5mM D-2-HG for 10 passages showed a slight increase of intracellular levels of D-2-HG (10 times compared to control) (Figure 4A). We confirmed that after treatment with 1.5 μM AGI-5198 for 10 and 20 passages intracellular levels of D-2-HG were decreased in mutant IDH1 cell lines L835, HT1080 and JJ012 compared to untreated cells (Figure 4B-4D). However, the xCelligence assay did not show any effect of AGI-5198 treatment on proliferation or migration (Figure 4E-4F). Moreover, the ability of IDH1 mutant cells to migrate in 3D extracellular matrix scaffolds was not affected by treatment with 1.5 μM AGI-5198 (Supplementary Figure 2). Indeed, migration was not dependent on the IDH1/2 mutation status, since mutant IDH1 and mutant IDH2 cell lines as well as the IDH1/2 wildtype cell line showed migration in the timeframe of this experiment.


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)

Prolonged treatment of IDH1/2 wildtype and IDH1 mutant cell lines with mutant IDH1 inhibitor AGI-5198 and metabolite D-2-HGProlonged treatment with 1.5 μM IDH1 mutant inhibitor AGI-5198 did not affect intracellular D-2-HG levels of the IDH1/2 wildtype cell line CH2879 after 10 and 20 passages, while treatment with D2HG resulted in increased levels of L2HG and D2HG intracellularly (A). Chondrosarcoma cells harboring an IDH1 mutation (L835 (B), HT1080 (C), JJ012 (D)) show decreased levels of intracellular D-2-HG compared to untreated cells after continuous treatment with 1.5 μM AGI-5198 for 10 and 20 passages, respectively. For L835, due to slower growth rate, only 10 passages were evaluated. The xCelligence assay demonstrated no effect of prolonged inhibition of mutant IDH1 on proliferation (E) or migration (F). Graphs show one representative experiment out of three experiments.
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Related In: Results  -  Collection

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Figure 4: Prolonged treatment of IDH1/2 wildtype and IDH1 mutant cell lines with mutant IDH1 inhibitor AGI-5198 and metabolite D-2-HGProlonged treatment with 1.5 μM IDH1 mutant inhibitor AGI-5198 did not affect intracellular D-2-HG levels of the IDH1/2 wildtype cell line CH2879 after 10 and 20 passages, while treatment with D2HG resulted in increased levels of L2HG and D2HG intracellularly (A). Chondrosarcoma cells harboring an IDH1 mutation (L835 (B), HT1080 (C), JJ012 (D)) show decreased levels of intracellular D-2-HG compared to untreated cells after continuous treatment with 1.5 μM AGI-5198 for 10 and 20 passages, respectively. For L835, due to slower growth rate, only 10 passages were evaluated. The xCelligence assay demonstrated no effect of prolonged inhibition of mutant IDH1 on proliferation (E) or migration (F). Graphs show one representative experiment out of three experiments.
Mentions: To assess the role of mutant IDH1 in chondrosarcoma cell proliferation, we incubated the cell lines with the IDH1 mutant inhibitor AGI-5198 for 72 hours. Indeed the levels of D-2-HG in culture media decreased in a dose-dependent manner in the mutant IDH1 cell lines JJ012 (R132G), HT1080 (R132C) and L835 (R132C) with IC50s of 0.7 μM, 0.5 μM and 0.35 μM, respectively (Figure 3A). As expected, D-2-HG levels of the mutant IDH2 SW1353 cell line were not affected (Figure 3A). Interestingly, even at doses that led to >90% decrease in D-2-HG (20 μM AGI-5198 for 72 hours), only a minor effect on cell viability using a WST-1 assay was seen (approximately 65% viability left in JJ012 after 72 hours)(Figure 3B). Furthermore, mutant IDH1 inhibition did not influence the colony forming capacity of the mutant IDH1 cell lines HT1080 and JJ012 and the mutant IDH2 cell line SW1353 after 10 days of treatment, whereas L835 was not able to form colonies in both the control and the AGI-5198 conditions (Figure 3C). Since glioma cells transfected with mutant IDH1 need 20 passages to show an effect on methylation [33], we treated the mutant IDH1 cell lines JJ012, HT1080 and the IDH1/2 wildtype cell line CH2879 for up to 20 passages. Due to its slower growth rate, L835 was treated for up to 10 passages. As expected, treatment of CH2879 with 5mM D-2-HG for 10 passages showed a slight increase of intracellular levels of D-2-HG (10 times compared to control) (Figure 4A). We confirmed that after treatment with 1.5 μM AGI-5198 for 10 and 20 passages intracellular levels of D-2-HG were decreased in mutant IDH1 cell lines L835, HT1080 and JJ012 compared to untreated cells (Figure 4B-4D). However, the xCelligence assay did not show any effect of AGI-5198 treatment on proliferation or migration (Figure 4E-4F). Moreover, the ability of IDH1 mutant cells to migrate in 3D extracellular matrix scaffolds was not affected by treatment with 1.5 μM AGI-5198 (Supplementary Figure 2). Indeed, migration was not dependent on the IDH1/2 mutation status, since mutant IDH1 and mutant IDH2 cell lines as well as the IDH1/2 wildtype cell line showed migration in the timeframe of this experiment.

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