Systematic approaches to study cancer cell metabolism
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1 Systematic approaches to study cancer cell metabolism Kivanc Birsoy Laboratory of Metabolic Regulation and Genetics The Rockefeller University, NY
2 Cellular metabolism is complex ~3,000 metabolic genes enzymes and transporters ~20,000 human genes
3 Cells require metabolic rewiring to Metabolic pathways contribute to cellular functions proliferate Metabolic Pathways fatty acids cholesterol nucleic acids Cellular Functions Energy Building blocks proliferation Post-translational Modifications Signaling glucose amino acids vitamins ATP differentiation response to the environment
4 Understand which metabolic pathways are required for cancer cell proliferation Determine the metabolic diversity among cancer cells
5 Cancer cells have different metabolism than normal cells Differentiated Cells Proliferating Cells
6 Most cancer cells respire 25 Oxgen consumption rate / ug (protein) Raji NCI-H82 NCI-H524 SNU-16 U-937 MC116 NCI-H929 KMS-26 rho cell
7 Cells require electron metabolic transport rewiring chain to activity proliferate. for proliferation fatty acids cholesterol nucleic acids O 2 CO 2 glucose amino acids vitamins ATP ETC inhibitors
8 The mitochondrial electron transport chain is involved in multiple biological processes I II III IV H + H + cyt c H + Q NADH Succinate H2O ADP ATP ATP synthesis Uncoupling/ Thermogenesis Redox balance ROS production Signaling
9 Functional Genomics Screens in mammalian cells shrna based screens Birsoy et. al., Nature (2014)
10 A CRISPR based genetic screen synthetic interactions with phenformin 30,000 sgrnas ~3000 metabolic genes sgrna library construction UNTREATED Jurkat Cell Line Pool Infection Pool of Knock out cells Compare sgrna abundance PHENFORMIN
11 Metabolic genes that enable growth under ETC inhibition Differential gene score (Phenformin-Untreated) Phenformin Aspartate -4 a-kg OAA GOT1-2 PLP 0 0 PDXK -2 Pyridoxal -4 untreated 2 Glutamate GOT1 NDUFA11 CAD NDUFB2 DAD1 PDXK COQ4 GSS SCO2 GPI SLC25A1 PMM2 MCAT GUK1 UROD PDE12 ADSL ACAD9 CTPS1 TRPC1 ACADL NDUFA10 ATP8B3 NDUFB9 TYMS GOT1 related ETC Nucleotide syn. not categorized
12 ETC inhibition kills cells lacking GOT1 Fold change in cell number in 5 days (log 2 ) Wild Type GOT1_KO1 GOT1_KO2 GOT1_KO1 + GOT1 cdna GOT1_KO2 + GOT1 cdna Phenformin (μm) Wild Type GOT1_KO1 GOT1_KO2 Phen (10 μm) Phen (10 μm) Phen (10 μm)
13 GOT1? Aspartate a-kg GOT1 PLP PDXK Pyridoxal OAA Glutamate ndance phenylalanine leucine Wild Type isoleucine methionine tryptophan valine proline tyrosine threonine alanine taurine glutamine asparagine Untreated Phenformin (10 μm) glycine histidine serine glutamate lysine arginine aspartate
14 GOT reaction reverses under mitochondrial respiration inhibition Wild Type GOT1 null Purine and Pyrmidine synthesis Protein synthesis NADH NAD OAA Asp GOT1 MDH1 Malate Purine and Pyrmidine synthesis Asp GOT2 MDH2 Malate Protein synthesis OAA NADH NAD ETC NADH NAD Relative abundance compared to control 6 5 Asp 4 3 OAA Wild type Cellular Aspartate levels GOT1 MDH1 Malate GOT1_KO1 GOT1_KO2 Wild type GOT1_KO1 GOT1_KO2 + Phenformin Asp GOT2 MDH2 Malate OAA Purine and Pyrmidine synthesis NADH NAD TCA Protein synthesis NADH CYTOSOL NAD ETC NADH NAD OAA Asp GOT1 MDH1 Malate OAA Asp GOT1 MDH1 Malate Asp GOT2 OAA MDH2 Malate NADH ETC NAD TCA MITOCHONDRIA Asp GOT2 OAA MDH2 Malate NADH ETC NAD TCA CYTOSOL MITOCHONDRIA TCA CYTOSOL MITOCHONDRIA CYTOSOL MITOCHONDRIA
15 Aspartate addition enables cell proliferation and viability under ETC inhibition Fold change in cell number in 5 days (log 2 ) ** -2 Phenformin (μm): Aspartate: Jurkat clones: Wild Type GOT1_KO2 GOT1_KO2 + Vector + Vector + GOT1 cdna ** ** GOT1_KO2 Untreated Aspartate ` Phen. (10 μm) Phen. (10 μm) Aspartate: 10 mm
16 Pyruvate supplementation can rescue proliferation defects under ETC inhibition Pyruvate O 2 CO 2 ETC inhibitors
17 Pyruvate supplementation provides NAD+ under ETC inhibition NAD+ / NADH ** 1 0 Phenformin: Pyruvate: Aspartate: - - -
18 Pyruvate-mediated rescue of ETC inhibition is dependent on GOT1 Pyruvate GOT1 Aspartate ETC inhibitors Fold change in cell number in 5 days (log 2 ) Relative Fold change in cell number aspartate in 5 days (log 2 ) abundance Fold change in cell number in 5 days (log 2 ) Jurkat clones: -2-4 Phenformin: Antimycin: Piericidin: PBS GOT1_KO1 GOT1_KO2 Wild Type ** ** ** 4 0 Phenformin: Pyruvate: - - e ** Wild Type Pyruvate ns GOT1_KO2
19 Studying mtdna mutations in culture EtBr treatment Fusion How do we grow these cells with mtdna mutations?
20 Aspartate is sufficient to enable proliferation of cybrids with patient-derived mtdna mutations Myoclonic Epilepsy with Ragged Red Fibers Parkinsonism/ MELAS
21 Strategies to increase aspartate in the serum Aspartate used in our experiments: 10 mm
22 Aspartate is sufficient to enable proliferation of cybrids with patient-derived mtdna mutations RPMI (150 um Asp) RPMI w/o Asp RPMI w/ (Aspartate Pyruvate (10(1 mm) SLC1A3 Aspartate Fold change in cell number CYTB days Fold change in cell number CYTB + SLC1A days
23 Aspartate levels decrease upon prolonged hypoxia Fold%change%compared%to%normoxic% 2$ 1$ 0.5$ aspartate$ glutamate$ asparagine$ glutamine$ cis/trans$hydroxyproline$ alanine$ proline$ serine$ threonine$ valine$ citrulline$ glycine$ phenylalanine$ isoleucine$ his<dine$ tyrosine$ serotonin$ leucine$ methionine$ 5=HIAA$ 5=hydroxytryptophan$ tryptophan$ arginine$ ornithine$ lysine$
24 Acknowledgements Ka+e Crielli Konnor La David M. Saba+ni Richard Possemato (Nutrostats) Tim Wang (CRISPR Screening) Walter Chen (ATPIF1) Broad Ins*tute Clary Clish (Metabolite Profiling) Levi Garraway Netherlands Cancer Ins*tute Thijn Brummelkamp (Haploid GeneKc Screens)
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