Haem Oxygenase Is Synthetically Lethal with the Tumour Suppressor Fumarate Hydratase

LETTER doi:10.1038/nature10363 Haem oxygenase is synthetically lethal with the tumour suppressor fumarate hydratase Christian Frezza1, Liang Zheng1, Ori Folger2, Kartik N. Rajagopalan3, Elaine D. MacKenzie1, Livnat Jerby2, Massimo Micaroni4, Barbara Chaneton1, Julie Adam5, Ann Hedley1, Gabriela Kalna1, Ian P. M. Tomlinson6, Patrick J. Pollard5, Dave G. Watson7, Ralph J. Deberardinis3, Tomer Shlomi8*, Eytan Ruppin2,9* & Eyal Gottlieb1 Fumarate hydratase (FH) is an enzyme of the tricarboxylic acid majority of fumarate was unlabelled (m10), indicating that glucose is cycle (TCA cycle) that catalyses the hydration of fumarate into a minor source of carbon for the TCA cycle in both Fh1fl/fl and Fh12/2 malate. Germline mutations of FH are responsible for hereditary cells (Fig. 1e). On the other hand, when cells were incubated with 13C- leiomyomatosis and renal-cell cancer (HLRCC)1. It has previously glutamine most of the fumarate was labelled (Fig. 1f). In Fh12/2 cells been demonstrated that the absence of FH leads to the accumula- cultured with uniformly labelled glutamine (Fig. 1f), the vast majority of tion of fumarate, which activates hypoxia-inducible factors (HIFs) the labelled fumarate contained all four carbon atoms derived from at normal oxygen tensions2–4. However, so far no mechanism that glutamine (m14). By contrast, in Fh1fl/fl cells, the fumarate pool con- explains the ability of cells to survive without a functional TCA tained substantial fractions of molecules with fewer than four 13C atoms cycle has been provided. Here we use newly characterized genetically due to processing of fumarate beyond the Fh1 step. The lack of these modified kidney mouse cells in which Fh1 has been deleted, and products in Fh12/2 cells confirms that no accessory pathway exists in apply a newly developed computer model of the metabolism of these these cells to circumvent the loss of Fh1 enzymatic activity, indicating a cells to predict and experimentally validate a linear metabolic path- true blockade of the cycle. Notably, the 13C-enrichment profiles of way beginning with glutamine uptake and ending with bilirubin e +Cr – CL1–/– CL19 excretion from Fh1-deficient cells. This pathway, which involves the a +/+ +/+ fl/fl –/ b 9 Cre L1 h1 + fl biosynthesis and degradation of haem, enables Fh1-deficient cells to Fh1 Fh1 F Fh1 Fh1 +/+ +/+ fl/ –/– C –/– CL1 1,000 Fh1 Fh1 Fh1 Fh1 Fh1 800 use the accumulated TCA cycle metabolites and permits partial 600 FH 400 Fh1fl/fl mitochondrial NADH production. We predicted and confirmed –/– 200 Fh1 Fh1+/+ Actin that targeting this pathway would render Fh1-deficient cells non- 100 viable, while sparing wild-type Fh1-containing cells. This work goes c d beyond identifying a metabolic pathway that is induced in Fh1- 12 12 ) Fh1–/– fl/fl –/– fl/fl 4 Fh1 Fh1 Fh1–/– 10 10 deficient cells to demonstrate that inhibition of haem oxygenation Fh1 2 is synthetically lethal when combined with Fh1 deficiency, provid- 8 8 Fumarate ing a new potential target for treating HLRCC patients. 6 6 0 4 4 Succinate –2 Standard Standard Fumarate To study metabolic adaptation of FH-deficient cells we first gener- Succinate Malate 2 2 , normalized to Relative abundance Metabolite intensity Malate ated immortalized kidney cells from mice homozygous for a con- 10 –4 (log te 10 11 13 14 19 10 11 13 14 19 ate 3 n ditionally targeted Fh1 allele . These cells contain LoxP sites flanking Retention time (min) Retention time (min) Citra Malate fl/fl Fumarate ucci exons 3 and 4 (Fh1 ) (Fig. 1a and Supplementary Fig. 2a) and express ef gS 2/2 normal levels of Fh1 protein (Fig. 1b). To generate Fh1 cells the Fh1fl/fl Fh1fl/fl Fh1fl/fl 100 Fh1–/– CL1 100 Fh1–/– CL1 Fh1–/– CL1 fl/fl Fh1–/– CL19 Fh1–/– CL19 800 Fh1–/– CL19 Fh1 cells were infected with recombinant adenovirus expressing Cre 80 80 600 recombinase and two clones (CL1 and CL19) were selected from the 60 13C-glucose 60 13C-glutamine infected pools. Both clones were genetically confirmed to contain 40 40 400 homozygous Fh1-deleted (knockout) alleles (Fig. 1a) and did not 20 20 200 Fumarate (% of pool) 0 Fumarate (% of pool) 0 0 express Fh1 protein (Fig. 1b). Glutamine consumption +2 +3 fl/fl +1 per h) (nmol per mg protein CL1 m+0 m+1 m m m+4 m+0 m m+2 m+3 m+4 –/– CL19 To assess the biochemical consequences of Fh1 deletion on TCA Fh1 –/– Fh1 cycle function, the intracellular levels of several TCA cycle metabolites Fh1 were measured by gas chromatography–mass spectrometry (GC–MS) Figure 1 | Characterization of Fh1-deficient cells. a, PCR analysis of genomic (Fig. 1c). Fh12/2 cells accumulate substantial amounts of fumarate, DNA of epithelial kidney cells of the indicated genotype. Numbers indicate base which reached levels more than a 100-fold that of Fh1fl/fl cells. In addi- pairs. b, Fh1 protein levels of the indicated cells were detected by western blot tion, a sevenfold increase in succinate levels and a marked decrease of analysis. c, Representative chromatogram of a GC–MS analysis performed from the indicated cell lines. Annotated metabolites are indicated by the arrows. malate and citrate were detected (Fig. 1d). In cancer cells, the TCA cycle 2/2 5 d, Fold change of the indicated TCA cycle metabolites in Fh1 compared to is largely supplied through the metabolism of glucose and glutamine . control Fh1fl/fl cells (n 5 3). e, f, Isotopomer distribution of intracellular To investigate the effects of Fh1 deletion on carbon supply to the TCA 13 13 13 fumarate. Cells were incubated with either C-glucose (e)or C-glutamine cycle, cells were cultured in medium containing C-labelled glucose (f) and fumarate isotopomers (with the indicated mass shift) were analysed 13 and unlabelled glutamine, or vice versa, and C-enrichment of fuma- (n 5 3). g, Glutamine consumption rate of the indicated cell lines (n 5 3). All rate and succinate was analysed. When 13C-glucose was used, the data are presented as mean 6 s.e.m. 1Cancer Research UK, Beatson Institute for Cancer Research, Switchback Road, Glasgow G61 1BD, UK. 2The Blavatnik School of Computer Science, Tel Aviv University, Tel Aviv 69978, Israel. 3Department of Pediatrics and McDermott Center for Human Growth and Development, University of Texas–Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd, Dallas, Texas 75390-9063, USA. 4The University of Queensland, Institute for Molecular Bioscience, St Lucia, Brisbane, Queensland 4072, Australia. 5Henry Wellcome Building for Molecular Physiology, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK. 6Molecular and Population Genetics Laboratory, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, UK. 7Strathclyde Institute of Pharmaceutical and Biomedical Sciences, University of Strathclyde, 27 Taylor Street, Glasgow G4 0NR, UK. 8Computer Science Department, Technion, Israel Institute of Technology, Haifa, 32000, Israel. 9The Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel. *These authors contributed equally to this work. 8 SEPTEMBER 2011 | VOL 477 | NATURE | 225 ©2011 Macmillan Publishers Limited. All rights reserved RESEARCH LETTER succinate perfectly matched fumarate results, underlining the presence decrease in respiration of Fh12/2 cells was associated with an increase of a flux of carbons from glutamine to fumarate via succinate. In addi- in glycolysis as indicated initially by the increased extracellular acidifi- tion, no reverse flux of TCA cycle metabolites was detected (Sup- cation rate (Fig. 2b) and further confirmed by the increase in glucose plementary Fig. 2b, c). (See Supplementary Fig. 1 for a diagram sum- consumption and lactate production (Fig. 2c, d). marizing these results.) Consistent with the increased contribution of Metabolic networks are highly interconnected, making them resistant glutamine to the pool of TCA cycle intermediates, Fh12/2 cells used to most single gene perturbations7. To elucidate the metabolic changes significantly more glutamine than Fh1fl/fl cells (Fig. 1g). that enable the survival of Fh12/2 cells despite having a truncated TCA Because the TCA cycle is a major source for mitochondrial NADH, cycle, we took a systemic approach based on flux balance analysis (FBA) the truncation of the TCA cycle may havesevere bioenergetic outcomes. to obtain a genome-scale model of cellular metabolism, as very com- Indeed, steady-state levels of mitochondrial NADH in Fh12/2 cells monly used in modelling of microbial metabolism8. We adopted an in were lower than those of control cells (Fig. 2a; untreated and Sup- silico modelling approach for cancer metabolism9 (see Supplementary plementary Fig. 2d for quantification). However, Fh12/2 cells still Information for details) to predict genes for which elimination together 2/2 generate significant amounts of NADH and retained normal mitochon- with Fh1 would selectively affect the growth ability of Fh1 cells fl/fl drial membrane potential (Fig. 2a). In addition, depolarizing the mito- without affecting the wild-type Fh1 cells (predicted to be synthetic chondria by using the protonophore carbonyl cyanide m-chlorophenyl lethal with Fh1). This modelling involved the reconstruction of genome- fl/fl 2/2 hydrazone (CCCP) decreased mitochondrial NADH, whereas blocking scale metabolic network models of the metabolism of Fh1 and Fh1 NADH oxidation with rotenone, an inhibitor of NADH:ubiquinone cells, using a generic computational method for building metabolic 10 oxidoreductase, increased mitochondrial NADH in both control and models of specific human tissues . The reconstruction of the models began with the identification of core sets of metabolic-enzyme-coding Fh1-deficient cells. These results indicate that despite having a trun- fl/fl 2/2 cated TCA cycle, Fh1-deficient cells retain significant mitochondrial genes that are expressed in Fh1 and Fh1 cells, based on accom- bioenergetic activity and are capable of generating and oxidizing panying microarray gene expression analysis (Supplementary Fig.

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