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Oncometabolic mutation IDH1 R132H confers a metformin-hypersensitive phenotype.

Cuyàs E, Fernández-Arroyo S, Corominas-Faja B, Rodríguez-Gallego E, Bosch-Barrera J, Martin-Castillo B, De Llorens R, Joven J, Menendez JA - Oncotarget (2015)

Bottom Line: A mitochondria toxicity-screening modality confirmed a severe inability of IDH1-mutated cells to use various carbon substrates that are fed into the electron transport chain at different points.Additionally, metabolic reprogramming of IDH1-mutant cells increased their sensitivity to metformin in assays of cell proliferation, clonogenic potential, and mammosphere formation.Moreover, synergistic interactions occurred when metformin treatment was combined with the selective R132H-IDH1 inhibitor AGI-5198.

View Article: PubMed Central - PubMed

Affiliation: Metabolism and Cancer Group, Translational Research Laboratory, Catalan Institute of Oncology (ICO), Girona, Catalonia, Spain.

ABSTRACT
Metabolic flexibility might be particularly constrained in tumors bearing mutations in isocitrate dehydrogenase 1 (IDH1) leading to the production of the oncometabolite 2-hydroxygluratate (2HG). To test the hypothesis that IDH1 mutations could generate metabolic vulnerabilities for therapeutic intervention, we utilized an MCF10A cell line engineered with an arginine-to-histidine conversion at position 132 (R132H) in the catalytic site of IDH1, which equips the enzyme with a neomorphic α-ketoglutarate to 2HG reducing activity in an otherwise isogenic background. IDH1 R132H/+ and isogenic IDH1 +/+ parental cells were screened for their ability to generate energy-rich NADH when cultured in a standardized high-throughput Phenotype MicroArrayplatform comprising >300 nutrients. A radical remodeling of the metabotype occurred in cells carrying the R132H mutation since they presented a markedly altered ability to utilize numerous carbon catabolic fuels. A mitochondria toxicity-screening modality confirmed a severe inability of IDH1-mutated cells to use various carbon substrates that are fed into the electron transport chain at different points. The mitochondrial biguanide poisons, metformin and phenformin, further impaired the intrinsic weakness of IDH1-mutant cells to use certain carbon-energy sources. Additionally, metabolic reprogramming of IDH1-mutant cells increased their sensitivity to metformin in assays of cell proliferation, clonogenic potential, and mammosphere formation. Targeted metabolomics studies revealed that the ability of metformin to interfere with the anaplerotic entry of glutamine into the tricarboxylic acid cycle could explain the hypersensitivity of IDH1-mutant cells to biguanides. Moreover, synergistic interactions occurred when metformin treatment was combined with the selective R132H-IDH1 inhibitor AGI-5198. Together, these results suggest that therapy involving the simultaneous targeting of metabolic vulnerabilities with metformin, and 2HG overproduction with mutant-selective inhibitors (AGI-5198-related AG-120 [Agios]), might represent a worthwhile avenue of exploration in the treatment of IDH1-mutated tumors.

No MeSH data available.


Related in: MedlinePlus

Differential mitochondrial fuel of IDH1 mutant cellsA. Representative micrograph of the utilization patterns of 8 different mitochondrial substrates in IDH1+/+ and R132H/+ cells cultured in the absence or presence of graded concentrations of metformin for 48 h. The hexose D-galactose is metabolized to NADH via mitochondrial activity, whereas α-D-glucose can bypass these mitochondrial functions. Glucose-1-phosphate is metabolized differently from glucose and galactose, whereas ribose-containing inosine and xylitol are both metabolized (albeit differently) via the pentose phosphate pathway. αKG directly enters the TCA cycle, whereas the ketones β-hydroxybutyrate and pyruvic acid enter the TCA cycle upon metabolism and linkage to coenzyme A. B. Representative micrographs of the utilization patterns of 8 different mitochondrial substrates in IDH1+/+ and R132H/+ cells cultured in the absence or presence of graded concentrations of phenformin (top) or AICAR (bottom) for 48 h.
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Figure 2: Differential mitochondrial fuel of IDH1 mutant cellsA. Representative micrograph of the utilization patterns of 8 different mitochondrial substrates in IDH1+/+ and R132H/+ cells cultured in the absence or presence of graded concentrations of metformin for 48 h. The hexose D-galactose is metabolized to NADH via mitochondrial activity, whereas α-D-glucose can bypass these mitochondrial functions. Glucose-1-phosphate is metabolized differently from glucose and galactose, whereas ribose-containing inosine and xylitol are both metabolized (albeit differently) via the pentose phosphate pathway. αKG directly enters the TCA cycle, whereas the ketones β-hydroxybutyrate and pyruvic acid enter the TCA cycle upon metabolism and linkage to coenzyme A. B. Representative micrographs of the utilization patterns of 8 different mitochondrial substrates in IDH1+/+ and R132H/+ cells cultured in the absence or presence of graded concentrations of phenformin (top) or AICAR (bottom) for 48 h.

Mentions: Because the above results indicated that significant remodeling of the metabotype occurs in R132H/+ cells, we employed a mitochondria toxicity-screening (PM-M TOX1) assay to evaluate the differential mitochondrial utilization of eight-carbon substrate that are fed into the ETC at different points. In the absence of exogenously-added glucose (the so-called “substrate metabolism assay”; Figure 2A), WT cells could obtain significant amounts of energy from α-D-glucose and inosine (absolute rate of dye reduction > 0.2) and very reduced amounts from substrates such as D-galactose, D-glucose-1-phosphatye and xylitol (absolute rates of dye reduction ~ 0.1). When exogenous glucose was omitted, WT cells were unable to obtain energy from α-keto-glutaric acid, DL-hydroxy-butyric acid and pyruvic acid (absolute rates of dye reduction < 0.1). In contrast, R132H/+ cells exhibited a notably reduced ability to generate energy-rich NADH from α-D-glucose and inosine and were unable to obtain significant amounts of energy from all other mitochondrial substrates (Figure 2A).


Oncometabolic mutation IDH1 R132H confers a metformin-hypersensitive phenotype.

Cuyàs E, Fernández-Arroyo S, Corominas-Faja B, Rodríguez-Gallego E, Bosch-Barrera J, Martin-Castillo B, De Llorens R, Joven J, Menendez JA - Oncotarget (2015)

Differential mitochondrial fuel of IDH1 mutant cellsA. Representative micrograph of the utilization patterns of 8 different mitochondrial substrates in IDH1+/+ and R132H/+ cells cultured in the absence or presence of graded concentrations of metformin for 48 h. The hexose D-galactose is metabolized to NADH via mitochondrial activity, whereas α-D-glucose can bypass these mitochondrial functions. Glucose-1-phosphate is metabolized differently from glucose and galactose, whereas ribose-containing inosine and xylitol are both metabolized (albeit differently) via the pentose phosphate pathway. αKG directly enters the TCA cycle, whereas the ketones β-hydroxybutyrate and pyruvic acid enter the TCA cycle upon metabolism and linkage to coenzyme A. B. Representative micrographs of the utilization patterns of 8 different mitochondrial substrates in IDH1+/+ and R132H/+ cells cultured in the absence or presence of graded concentrations of phenformin (top) or AICAR (bottom) for 48 h.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4494938&req=5

Figure 2: Differential mitochondrial fuel of IDH1 mutant cellsA. Representative micrograph of the utilization patterns of 8 different mitochondrial substrates in IDH1+/+ and R132H/+ cells cultured in the absence or presence of graded concentrations of metformin for 48 h. The hexose D-galactose is metabolized to NADH via mitochondrial activity, whereas α-D-glucose can bypass these mitochondrial functions. Glucose-1-phosphate is metabolized differently from glucose and galactose, whereas ribose-containing inosine and xylitol are both metabolized (albeit differently) via the pentose phosphate pathway. αKG directly enters the TCA cycle, whereas the ketones β-hydroxybutyrate and pyruvic acid enter the TCA cycle upon metabolism and linkage to coenzyme A. B. Representative micrographs of the utilization patterns of 8 different mitochondrial substrates in IDH1+/+ and R132H/+ cells cultured in the absence or presence of graded concentrations of phenformin (top) or AICAR (bottom) for 48 h.
Mentions: Because the above results indicated that significant remodeling of the metabotype occurs in R132H/+ cells, we employed a mitochondria toxicity-screening (PM-M TOX1) assay to evaluate the differential mitochondrial utilization of eight-carbon substrate that are fed into the ETC at different points. In the absence of exogenously-added glucose (the so-called “substrate metabolism assay”; Figure 2A), WT cells could obtain significant amounts of energy from α-D-glucose and inosine (absolute rate of dye reduction > 0.2) and very reduced amounts from substrates such as D-galactose, D-glucose-1-phosphatye and xylitol (absolute rates of dye reduction ~ 0.1). When exogenous glucose was omitted, WT cells were unable to obtain energy from α-keto-glutaric acid, DL-hydroxy-butyric acid and pyruvic acid (absolute rates of dye reduction < 0.1). In contrast, R132H/+ cells exhibited a notably reduced ability to generate energy-rich NADH from α-D-glucose and inosine and were unable to obtain significant amounts of energy from all other mitochondrial substrates (Figure 2A).

Bottom Line: A mitochondria toxicity-screening modality confirmed a severe inability of IDH1-mutated cells to use various carbon substrates that are fed into the electron transport chain at different points.Additionally, metabolic reprogramming of IDH1-mutant cells increased their sensitivity to metformin in assays of cell proliferation, clonogenic potential, and mammosphere formation.Moreover, synergistic interactions occurred when metformin treatment was combined with the selective R132H-IDH1 inhibitor AGI-5198.

View Article: PubMed Central - PubMed

Affiliation: Metabolism and Cancer Group, Translational Research Laboratory, Catalan Institute of Oncology (ICO), Girona, Catalonia, Spain.

ABSTRACT
Metabolic flexibility might be particularly constrained in tumors bearing mutations in isocitrate dehydrogenase 1 (IDH1) leading to the production of the oncometabolite 2-hydroxygluratate (2HG). To test the hypothesis that IDH1 mutations could generate metabolic vulnerabilities for therapeutic intervention, we utilized an MCF10A cell line engineered with an arginine-to-histidine conversion at position 132 (R132H) in the catalytic site of IDH1, which equips the enzyme with a neomorphic α-ketoglutarate to 2HG reducing activity in an otherwise isogenic background. IDH1 R132H/+ and isogenic IDH1 +/+ parental cells were screened for their ability to generate energy-rich NADH when cultured in a standardized high-throughput Phenotype MicroArrayplatform comprising >300 nutrients. A radical remodeling of the metabotype occurred in cells carrying the R132H mutation since they presented a markedly altered ability to utilize numerous carbon catabolic fuels. A mitochondria toxicity-screening modality confirmed a severe inability of IDH1-mutated cells to use various carbon substrates that are fed into the electron transport chain at different points. The mitochondrial biguanide poisons, metformin and phenformin, further impaired the intrinsic weakness of IDH1-mutant cells to use certain carbon-energy sources. Additionally, metabolic reprogramming of IDH1-mutant cells increased their sensitivity to metformin in assays of cell proliferation, clonogenic potential, and mammosphere formation. Targeted metabolomics studies revealed that the ability of metformin to interfere with the anaplerotic entry of glutamine into the tricarboxylic acid cycle could explain the hypersensitivity of IDH1-mutant cells to biguanides. Moreover, synergistic interactions occurred when metformin treatment was combined with the selective R132H-IDH1 inhibitor AGI-5198. Together, these results suggest that therapy involving the simultaneous targeting of metabolic vulnerabilities with metformin, and 2HG overproduction with mutant-selective inhibitors (AGI-5198-related AG-120 [Agios]), might represent a worthwhile avenue of exploration in the treatment of IDH1-mutated tumors.

No MeSH data available.


Related in: MedlinePlus