Mutant Kras copy number defines metabolic reprogramming and therapeutic susceptibilities

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Abstract

The RAS/MAPK (mitogen-activated protein kinase) signalling pathway is frequently deregulated in non-small-cell lung cancer, often through KRAS activating mutations1,2,3. A single endogenous mutant Kras allele is sufficient to promote lung tumour formation in mice but malignant progression requires additional genetic alterations4,5,6,7. We recently showed that advanced lung tumours from KrasG12D/+;p53-null mice frequently exhibit KrasG12D allelic enrichment (KrasG12D/Kraswild-type > 1) (ref. 7), implying that mutant Kras copy gains are positively selected during progression. Here we show, through a comprehensive analysis of mutant Kras homozygous and heterozygous mouse embryonic fibroblasts and lung cancer cells, that these genotypes are phenotypically distinct. In particular, KrasG12D/G12D cells exhibit a glycolytic switch coupled to increased channelling of glucose-derived metabolites into the tricarboxylic acid cycle and glutathione biosynthesis, resulting in enhanced glutathione-mediated detoxification. This metabolic rewiring is recapitulated in mutant KRAS homozygous non-small-cell lung cancer cells and in vivo, in spontaneous advanced murine lung tumours (which display a high frequency of KrasG12D copy gain), but not in the corresponding early tumours (KrasG12D heterozygous). Finally, we demonstrate that mutant Kras copy gain creates unique metabolic dependences that can be exploited to selectively target these aggressive mutant Kras tumours. Our data demonstrate that mutant Kras lung tumours are not a single disease but rather a heterogeneous group comprising two classes of tumours with distinct metabolic profiles, prognosis and therapeutic susceptibility, which can be discriminated on the basis of their relative mutant allelic content. We also provide the first, to our knowledge, in vivo evidence of metabolic rewiring during lung cancer malignant progression.

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Figure 1: Mutant Kras copy gain upregulates glycolysis in MEFs and lung tumour cells.
Figure 2: Mutant Kras copy gain drives glycolysis and directs glucose metabolism towards TCA cycle and glutathione synthesis.
Figure 3: Mutant Kras copy-number dictates redox state, metabolic dependencies and therapeutic susceptibilities.
Figure 4: Mutant Kras copy gain results in increased malignancy and metabolic rewiring in vivo.

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Primary accessions

Gene Expression Omnibus

Data deposits

Microarray data have been deposited in the Gene Expression Omnibus under accession number GSE75871.

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Acknowledgements

We thank T. Jacks (KrasLSL-G12D), A. Berns (p53Fx) and the National Cancer Institute Mouse Repository for mice. We also thank S. Kleeman, P. Ogger and S. Costa for assistance with redox cell profiling, cell viability assays and LC–MS, respectively. We are very grateful to Cancer Research UK Cambridge Institute Biological Resources Unit staff for support with in vivo work and all the members of the Martins laboratory for comments and advice. This work was supported by the Medical Research Council.

Author information

C.P.M. and E.M.K. designed the study and E.M.K. performed all experiments with assistance from F.K.T. (in vivo work, qPCR, Seahorse) and E.G. (Pdh activity). E.G. and C.F. contributed to the design of metabolomics experiments and performed LC–MS. All authors contributed to data analysis and discussion. C.P.M. and E.M.K. wrote the paper and all authors contributed to editing.

Correspondence to Carla P. Martins.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Enhanced glycolysis in homozygous KrasG12D cells.

a, Representative data (n = 3) of PCR analysis of the Kras and p53 loci in KrasWT/WT (WT/WT), KrasG12D/WT (G12D/WT) and KrasG12D/G12D (G12D/G12D); p53Fx/Fx MEFs after Cre-mediated recombination; and of unrecombined KrasLSL-G12D/WT;p53loxP/loxP control (Cre-) (*background band). b, IPA analysis of canonical pathways significantly altered in KrasG12D/G12D relative to KrasG12D/WT MEF transcriptomes (n = 3 per genotype). c, Representative qPCR data (n = 3) of glycolytic gene expression in MEFs. Fold change relative to WT/WT shown as triplicate mean ± s.d. (one-way ANOVA). d, ECAR in MEFs following exposure to glucose, oligomycin and 2DG. Representative data from three independent MEFs per genotype show mean value between triplicates ± s.d. (two-way ANOVA). e, Kras locus analysis of two lung cancer cell lines (L1212 and L1211) generated from spontaneous tumours from KrasG12D/WT;p53-deficient mice. PCR (top) and pyrosequencing (bottom) analysis shown (L1212: Kras heterozygous, G12D/WT; L1211: G12D homozygous, G12D/G12D). Recombined heterozygous MEFs shown as PCR control (CTRL). f, Representative qPCR data (n = 3) of glycolytic gene expression in L1211 and L1212 lung tumour cells. Fold change relative to heterozygous cells shown (mean of triplicates ± s.d.; ***P = <0.001; *P < 0.05; t-test). g, Left: basal glucose consumption in murine lung tumour cells determined by FACS analysis of 6-NBDG uptake (%). *P = 0.02; t-test. Right: extracellular lactate concentration (ng/dl/cell) in murine lung tumour cells. Data are triplicate mean ± s.d. *P = 0.0139; t-test.

Extended Data Figure 2 KrasG12D/WT and KrasG12D/G12D MEFs have similar biomass and mitochondrial functionality.

a, Total protein content in indicated MEFs relative to WT/WT. b, Total RNA per cell for each of the indicated genotypes. c, d, WT/WT, G12D/WT and G12D/G12D MEFs were profiled by CASY counter (Roche) and cell volume (c) and diameter (d) measured. ad, Mean value of three independent MEF triplicates per genotype ± s.d. e, Oxygen consumption rate (OCR) of MEFs in response to oligomycin, CCCP and rotenone (two-way ANOVA). f, NAO staining was used to determine mitochondrial mass in KrasWT/WT (WT/WT), KrasG12D/WT (G12D/WT) and KrasG12D/G12D (G12D/G12D) MEFs. Geometric mean of NAO fluorescence in cells was determined by FACS. Representative overlay (left panel) and geometric mean (right panel) displayed. g, Mitochondrial architecture was examined after Mitotracker green staining in WT/WT, G12D/WT and G12D/G12D MEFs (scale bar, 10 μm). h, TMRM staining was used to determine mitochondrial membrane potential in MEFs of indicated genotypes. Geometric mean of TMRM fluorescence in cells was determined by FACS. Representative overlay (left panel) and geometric mean (right panel) displayed. eh, Representative data of three independent MEFs per genotype show mean of triplicates ± s.d.; ***P < 0.001; one-way ANOVA.

Extended Data Figure 3 Glucose metabolism reprogramming in KrasG12D/G12D MEFs.

aj, Measurement of 13C-glucose-derived metabolites, calculated as a percentage of the total metabolite pool following LC–MS analysis of WT/WT, G12D/WT and G12D/G12D MEFs after 4 h culture with 13C-glucose-supplemented media. Representative data (of two independent MEFs per genotype) showing mean of triplicates ± s.d.; ***P < 0.001; **P < 0.01; *P < 0.05 (two-way ANOVA). Undetected isotopologues not shown.

Extended Data Figure 4 Glucose metabolism reprogramming in lung tumour cells with mutant Kras copy gain.

Measurement of 13C-glucose-derived metabolites, calculated as a percentage of the total metabolite pool following LC–MS analysis of murine (L1211 and L1212, ah) and human (H23, H358, H460, SW1573, ip) mutant Kras heterozygous and homozygous lung tumour cells. Cells were cultured for 4 h with 13C-glucose-supplemented media before analysis. Data show mean of triplicates ± s.d.; ***P < 0.001; **P < 0.01; *P < 0.05 (two-way ANOVA, relative to Krasmut heterozygous cells; ip, homozygous samples significantly different from both heterozygous cell lines indicated). Undetected isotopologues not shown.

Extended Data Figure 5 KrasG12D/WT and KrasG12D/G12D MEFs have distinct glutamine metabolism profiles.

Glutamine metabolism analysis in WT/WT, G12D/WT and G12D/G12D MEFs. a, Representation of carbon flux (grey circles) from uniformly labelled 13C-glutamine (13C-GLN). b, Heatmap illustrates abundance of selected labelled metabolites across triplicates of representative MEFs (two independent MEFs per genotype analysed) based on metabolomics analysis. ci, Measurement of 13C-glutamine-derived metabolites, calculated as a percentage of the total metabolite pool following LC–MS analysis of WT/WT, G12D/WT and G12D/G12D MEFs after 4 h culture with 13C-glutamine-supplemented media. Representative data (two independent MEFs per genotype) show mean of triplicates ± s.d. (two-way ANOVA). j, Oxygen consumption rate (OCR) of WT/WT, G12D/WT and G12D/G12D MEFs upon glutamine (4 mM) addition. Representative data of three independent MEFs per genotype showing mean of triplicates ± s.d. k, Relative diversion (percentage) of glutamine to TCA (aKG m + 5) or GSH (GSH m + 5) in MEFs of indicated genotypes based on metabolomics data. Representative MEF data (n = 2 MEFs per genotype) show triplicate mean ± s.d. (one-way ANOVA). ***P < 0.001; **P < 0.01; *P < 0.05.

Extended Data Figure 6 KrasG12D homozygous cells depend on glucose metabolism reprogramming for ROS management.

a, GSSG levels in G12D/WT and G12D/G12D MEFs relative to WT/WT. Mean data (n = 3 MEFs per genotype) ± s.d. shown. b, ROS levels in MEFs following 48 h of 2DG treatment. Data were normalized to vehicle treatment (CTRL). c, Percentage of AnnexinV/PI double-positive G12D/G12D MEFs following 48 h of 2DG treatment in the presence (+) or absence (−) of NAC. d, Ratio of reduced to oxidized glutathione (GSH/GSSG) determined for WT/WT, G12D/WT and G12D/G12D MEFs after incubation with 2DG, BSO or both (2DG + BSO) for 48 h, normalized to vehicle. bd, Representative data from three independent MEFs per genotype presented. Mean data for triplicates ± s.d. shown (two-way ANOVA). e, Representative data of GSH/GSSG ratio and GSH levels in murine G12D/G12D tumour cells relative to G12D/WT (t-test). f, Differential sensitivity of lung tumour cells to nutrient depletion. Lung tumour cells were cultured in normal media and low glucose conditions for 72 h and viable cells counted and normalized to CTRL (two-way ANOVA). g, Percentage of AnnexinV/PI double-positive murine tumour cells following 48 h treatment with BSO, 2DG, or both (2DG + BSO). eg, Representative data (n = 3 independent experiments) depict triplicate mean ± s.d. (***P < 0.001, two-way ANOVA). ***P < 0.001; **P < 0.01; *P < 0.05.

Extended Data Figure 7 Increased mutant Kras allelic content leads to glucose metabolism reprogramming in lung tumours in vivo.

ai, Control (no Cre) and tumour-bearing KrasG12D/+;p53Fx/Fx mice were infused with 13C-glucose 12 (early group) or 16 weeks (late group) after adenoviral-Cre treatment and individual lung tumours (early, n = 16; late, n = 12) or control lung (normal, n = 3) collected for LC–MS analysis (three technical replicates per sample). Selected 13C-glucose-derived metabolites shown, calculated as a percentage of the total metabolite pool. Mean abundance per cohort ± s.e.m. shown. ***P < 0.001; *P < 0.05 (two-way ANOVA).

Extended Data Table 1 Mutant heterozygous and homozygous KRAS NSCLC cell lines
Extended Data Table 2 KRAS status in panel of human lung adenocarcinomas

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This file contains Supplementary Figures 1-2, which show the uncropped gels from the main and Extended Data figures. (PDF 634 kb)

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Kerr, E., Gaude, E., Turrell, F. et al. Mutant Kras copy number defines metabolic reprogramming and therapeutic susceptibilities. Nature 531, 110–113 (2016) doi:10.1038/nature16967

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