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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

Metabolic fingerprint of the oncometabolic mutation IDH1 R132H in MCF10A breast epithelial cellsA. Representative micrographs of non-starved IDH1 +/+ and R132H/+ cells following 48 hours culture in PM-M1 plates containing primarily carbohydrate and carboxylate substrates (see text for a detailed explanation). B. Phenetic maps of carbon utilization patterns of IDH1+/+ and R132H/+ cells obtained after evaluation of OD values in PM-M1, PM-M2, PM-M3, and PM-M4 plates (see text for a detailed explanation). Phenotypes that are lost are colored green and phenotypes that are gained are colored red; the exact relative values are given by a corresponding color as indicated at the color scales. C. Left. Representative micrographs of starved IDH1 +/+ and R132H/+ cells following 48 hours culture in PM-M1 plates containing primarily carbohydrate and carboxylate substrates (see text for a detailed explanation). Right. Phenetic maps of starved-IDH1 +/+ and R132H/+ cells were generated as shown in B.
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Figure 1: Metabolic fingerprint of the oncometabolic mutation IDH1 R132H in MCF10A breast epithelial cellsA. Representative micrographs of non-starved IDH1 +/+ and R132H/+ cells following 48 hours culture in PM-M1 plates containing primarily carbohydrate and carboxylate substrates (see text for a detailed explanation). B. Phenetic maps of carbon utilization patterns of IDH1+/+ and R132H/+ cells obtained after evaluation of OD values in PM-M1, PM-M2, PM-M3, and PM-M4 plates (see text for a detailed explanation). Phenotypes that are lost are colored green and phenotypes that are gained are colored red; the exact relative values are given by a corresponding color as indicated at the color scales. C. Left. Representative micrographs of starved IDH1 +/+ and R132H/+ cells following 48 hours culture in PM-M1 plates containing primarily carbohydrate and carboxylate substrates (see text for a detailed explanation). Right. Phenetic maps of starved-IDH1 +/+ and R132H/+ cells were generated as shown in B.

Mentions: Because the color formed from each substrate reflected the energy-producing activity of the associated catabolic pathway, as expected R132H/+ and WT cells exhibited very similar and strong reductive responses in wells containing D-glucose (Figure 1A, green boxes [positive controls]), with little or no response in wells lacking any carbon source (Figure 1A, red boxes [negative controls]). Before qualitative and quantitative analyses (see below), all wells visually resembling the negative control wells were scored as “negative”, and all wells with a noticeable purple color (greater than negative control wells) were scored as “positive”. Wells with an extremely faint color, or with small purple flecks or clumps, were scored as “borderline”. To quantitatively compare each condition rapidly and systematically, we followed the same scoring procedure as we recently employed to delineate the nutritional phenome of epithelial-to-mesenchymal (EMT)-induced cancer stem-like cellular states [57]. Briefly, we calculated the fold change in the optical density of each “positive” substrate at 590 nm that resulted from the accumulation of reduced dye over a 6-hour period after normalization of the values to those of the negative-control wells included in each of the PM-M plates (Figure 1B). To quantify these evaluations, we also calculated a comparison score from the absolute ratio between the metabolic flows of R132H/+ and WT cells occurring at the same time point (6 h) (Figure 1B).


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)

Metabolic fingerprint of the oncometabolic mutation IDH1 R132H in MCF10A breast epithelial cellsA. Representative micrographs of non-starved IDH1 +/+ and R132H/+ cells following 48 hours culture in PM-M1 plates containing primarily carbohydrate and carboxylate substrates (see text for a detailed explanation). B. Phenetic maps of carbon utilization patterns of IDH1+/+ and R132H/+ cells obtained after evaluation of OD values in PM-M1, PM-M2, PM-M3, and PM-M4 plates (see text for a detailed explanation). Phenotypes that are lost are colored green and phenotypes that are gained are colored red; the exact relative values are given by a corresponding color as indicated at the color scales. C. Left. Representative micrographs of starved IDH1 +/+ and R132H/+ cells following 48 hours culture in PM-M1 plates containing primarily carbohydrate and carboxylate substrates (see text for a detailed explanation). Right. Phenetic maps of starved-IDH1 +/+ and R132H/+ cells were generated as shown in B.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 1: Metabolic fingerprint of the oncometabolic mutation IDH1 R132H in MCF10A breast epithelial cellsA. Representative micrographs of non-starved IDH1 +/+ and R132H/+ cells following 48 hours culture in PM-M1 plates containing primarily carbohydrate and carboxylate substrates (see text for a detailed explanation). B. Phenetic maps of carbon utilization patterns of IDH1+/+ and R132H/+ cells obtained after evaluation of OD values in PM-M1, PM-M2, PM-M3, and PM-M4 plates (see text for a detailed explanation). Phenotypes that are lost are colored green and phenotypes that are gained are colored red; the exact relative values are given by a corresponding color as indicated at the color scales. C. Left. Representative micrographs of starved IDH1 +/+ and R132H/+ cells following 48 hours culture in PM-M1 plates containing primarily carbohydrate and carboxylate substrates (see text for a detailed explanation). Right. Phenetic maps of starved-IDH1 +/+ and R132H/+ cells were generated as shown in B.
Mentions: Because the color formed from each substrate reflected the energy-producing activity of the associated catabolic pathway, as expected R132H/+ and WT cells exhibited very similar and strong reductive responses in wells containing D-glucose (Figure 1A, green boxes [positive controls]), with little or no response in wells lacking any carbon source (Figure 1A, red boxes [negative controls]). Before qualitative and quantitative analyses (see below), all wells visually resembling the negative control wells were scored as “negative”, and all wells with a noticeable purple color (greater than negative control wells) were scored as “positive”. Wells with an extremely faint color, or with small purple flecks or clumps, were scored as “borderline”. To quantitatively compare each condition rapidly and systematically, we followed the same scoring procedure as we recently employed to delineate the nutritional phenome of epithelial-to-mesenchymal (EMT)-induced cancer stem-like cellular states [57]. Briefly, we calculated the fold change in the optical density of each “positive” substrate at 590 nm that resulted from the accumulation of reduced dye over a 6-hour period after normalization of the values to those of the negative-control wells included in each of the PM-M plates (Figure 1B). To quantify these evaluations, we also calculated a comparison score from the absolute ratio between the metabolic flows of R132H/+ and WT cells occurring at the same time point (6 h) (Figure 1B).

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