supplementary material for€¦ · 7/9/2016 · 3 15n-leu amino acid defined diet: 50%...

www.sciencemag.org/content/353/6304/1161/suppl/DC1

Supplementary Material for

Tissue of origin dictates branched-chain amino acid metabolism in mutant Kras-driven cancers

Jared R. Mayers, Margaret E. Torrence, Laura V. Danai, Thales Papagiannakopoulos, Shawn M. Davidson, Matthew R. Bauer, Allison N. Lau, Brian W. Ji, Purushottam D. Dixit, Aaron M. Hosios, Alexander Muir, Christopher R. Chin, Elizaveta Freinkman,

Tyler Jacks, Brian M. Wolpin, Dennis Vitkup, Matthew G. Vander Heiden*

*Corresponding author. Email: [email protected]

Published 9 September 2016, Science 353, 1161 (2016) DOI: 10.1126/science.aaf5171

This PDF file includes:

Materials and Methods Figs. S1 to S10 Tables S1 to S8 Full Reference List

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Materials and Methods Experimental Mice

All studies were approved by the MIT committee on animal care (IACUC). All experimental groups were assigned based on genotype. All animals were numbered and experiments conducted blinded. After data collection genotypes were revealed and animals assigned to groups for analysis.

KPC: Experimental KPC mice were male mice on a pure C57BL/6J background. Experimental mice were heterozygous for the conditional lox–stop–lox KrasG12D allele, heterozygous for the conditional lox–stop–lox Trp53R172H allele and expressed Cre–recombinase under control of the Pdx–1–promoter (Tg(Ipf1-cre)1Tuv) (26). Littermate controls lacked either the LSL–KrasG12D allele, the Cre allele, or both. Control mice were sacrificed at the same time as their tumor–bearing littermates.

KP–/–C (PDAC): Experimental KP–/–C mice were male mice on a pure C57BL/6J background. Experimental mice were heterozygous for the conditional lox–stop–lox KrasG12D allele, homozygous for loxP sites flanking exons 2–10 of Trp53 and expressed Cre–recombinase under control of the Pdx–1–promoter (Tg(Ipf1-cre)1Tuv) (19). Littermate control mice lacked either the Cre–recombinase allele, LSL–KrasG12D allele, or both as previously described (18), such that control pancreas samples used throughout were derived from a combination of p53 wildtype and null pancreata. Cancer cell lines derived from these mice were used for syngenic implantation studies. For FACS sorting, mixed background male mice with the KP–/–C alleles plus a LSL-tdTomato reporter gene expressed under control of the Rosa-26 promoter (Gt(ROSA)26Sor tm9(CAG-tdTomato)Hze/+) were used. Five-week old mice were used for the experiment presented in fig. S1, whereas 8-10-week-old mice were used for all other experiments presented in the study.

KP Non–small cell lung cancer: Experimental NSCLC mice were male mice on a pure C57BL/6J background. Experimental mice were heterozygous for the conditional lox–stop–lox KrasG12D allele, homozygous for loxP sites flanking exons 2–10 of Trp53 were administered 2.5x107 pfu of Cre–expressing adenovirus intratracheally as previously described (20). Control mice lacked the lox–stop–lox KrasG12D allele, but contained the floxed-Trp53 allele. High–titer adenovirus was obtained from the Gene Transfer Vector Core (University of Iowa). Cre was administered when the mice were 8-12 weeks of age. Cancer cell lines derived from these mice were used for syngenic implantation studies. Mice 8-weeks post-infection were used for the experiment presented in fig. S1, whereas mice 12-14-weeks post-infection were used for all other experiments presented in the study.

Subcutaneous and orthotopic implantation studies: Male C57BL/6J mice aged 6-12 weeks at the start of the study were used for these experiments.

Diets

Standard Chow Diet: RMH 3000 (Prolab). Amino Acid Defined Diet: Control (unlabeled) amino acid-defined diet (TD.110839) was

designed in consultation with and subsequently obtained from Harlan Teklad. 13C-BCAA Amino Acid Defined Diet: 20% 13C–leucine and 20% 13C–valine labeled diet was

based on diet TD.110839 and produced by Cambridge Isotopes and Harlan Teklad. KP–/–C PDAC mice and NSCLC mice were fed this diet for 7 days beginning at seven weeks of age (PDAC) and 12-weeks post infection with adenoviral Cre-recombinase (NSCLC).

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15N-Leu Amino Acid Defined Diet: 50% 15N–leucine labeled diet was based on diet TD.110839 and produced by Cambridge Isotopes and Harlan Teklad. KP–/–C PDAC mice were fed this diet for 7 days beginning at seven weeks of age (PDAC). NSCLC mice were fed this diet for 6 days, beginning 12-weeks post infection with adenoviral Cre-recombinase (NSCLC).

Plasma Collection

Mice were anesthetized under 2% isofluorane–oxygen mixture and retro–orbitally bled approximately 4.5 hours after the onset of the light cycle. Blood was immediately placed in EDTA-tubes and centrifuged to separate plasma. Plasma was aliquoted and frozen at -80˚C for further analysis.

LC–MS Plasma Amino Acid Measurements

Plasma amino acids were measured by LC–MS at the Koch Institute at the Massachusetts Institute of Technology (Cambridge, MA) using methods previously described (18). Raw data were analyzed as peak area tops using the open–access MAVEN software tool (40). GC–MS Assessment of Stable Isotope Labeling

Plasma polar metabolites were extracted in ice–cold 4:1 methanol:water with norvaline internal standard (5µL plasma in 200µL extraction solution). Extracts were clarified by centrifugation and the supernatant evaporated under nitrogen and frozen at –80˚C for subsequent derivitization. Dried polar metabolites were dissolved in 20µL of 2% methoxyamine hydrochloride in pyridine (Thermo) and held at 37˚C for 1.5hr. After dissolution and reaction, tert–butyldimethylsilyl derivatization was initiated by adding 25µL N–methyl–N–(tert–butyldimethylsilyl)trifluoroacetamide + 1% tert–butyldimethylchlorosilane (Sigma) and incubating at 37˚C for 1hr.

All tissues used for metabolomic analysis were snap frozen using a BioSpec Biosquezer (#1210). To extract free polar metabolites, 10-30 mg of tissue was pulverized in liquid nitrogen using at Retsch Cryomill (#20.749.0001) and Retsch Cryomill Grinding Balls (#22.455.0002) for 2x 2min cycles at 25 Hz. Powdered tissue was extracted with 5:3:5 ice-cold methanol:water (with 13.3ng/µL Norvaline internal standard):chloroform. Extract was vortexted for 10min at 4˚C and centrifuged at for 10min at 20,000g at 4˚C in a benchtop centrifuge. An equal volume of the top (aqueous) phase was removed for each sample and evaporated under nitrogen and frozen at –80˚C for subsequent derivitization. Dried polar metabolites were dissolved in 1µL/mg tissue of 2% methoxyamine hydrochloride in pyridine (Thermo) and held at 37˚C for 1.5hr. After dissolution and reaction, tert–butyldimethylsilyl derivatization was initiated by adding 1.25µL/mg tissue N–methyl–N–(tert–butyldimethylsilyl)trifluoroacetamide + 1% tert–butyldimethylchlorosilane (Sigma) and incubating at 60˚C for 1hr.

The acid hydrolysis protocol was adapted from Antoniewicz and colleagues (41). Briefly, acid hydrolysis of tissue proteins was performed on snap frozen tissues by placing 1–5mg tissue in 1mL 18% hydrochloric acid overnight at 100˚C. 50µL supernatant was evaporated under nitrogen and frozen at –80˚C for subsequent derivitization. Dried hydrolysates were re–dissolved in pyridine (10µL/1mg tissue) prior to tert–butyldimethylsilyl derivatization, which was initiated by adding N–methyl–N–(tert–butyldimethylsilyl)trifluoroacetamide + 1% tert–butyldimethylchlorosilane (12.5µL/1mg tissue, Sigma) and incubating at 60˚C for 1h.

GC/MS analysis was performed using an Agilent 7890 GC equipped with a 30m DB–35MS capillary column connected to an Agilent 5975B MS operating under electron impact ionization

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at 70eV. One microliter of sample was injected in splitless mode at 270°C, using helium as the carrier gas at a flow rate of 1mlmin−1. For measurement of amino acids, the GC oven temperature was held at 100°C for 3min and increased to 300°C at 3.5°Cmin−1. The MS source and quadrupole were held at 230°C and 150°C, respectively, and the detector was run in scanning mode, recording ion abundance in the range of 100–605 m/z. MIDs were determined by integrating the appropriate ion fragments (41) listed in table S7 and corrected for natural isotope abundance using an algorithm adapted from Ferandez and colleagues (42).

LC-MS Assessment of Stable Isotope Labeling

Tissue samples were collected and extracted as before. After drying the polar fraction, samples were resuspended in ice-cold 1:1 Acetonitrile:Water mix at 40µL/10mg tissue. LC/MS analyses were conducted on a QExactive benchtop orbitrap mass spectrometer equipped with a heated electrospray ionization (HESI) probe, which was coupled to a Dionex UltiMate 3000 UPLC system (Thermo Fisher Scientific, San Jose, CA). External mass calibration was performed using the standard calibration mixture every 7 days. For 13C-BCAA tracing in tissues, 2.5 µl of each sample was injected into the LC/MS. For 15N-BCAA tracing into nucleotides, 10 µl of each sample was injected into the LC/MS.

LC separation was performed on a ZIC-pHILIC 2.1 x 150 mm (5 µm particle size) column (EMD). Buffer A was 20 mM ammonium carbonate, 0.1% ammonium hydroxide; buffer B was acetonitrile. The chromatographic gradient was run at a flow rate of 0.150 ml/min as follows: 0-20 min.: linear gradient from 80% to 20% B; 20-20.5 min.: linear gradient from 20% to 80% B; 20.5-28 min.: hold at 80% B.

The mass spectrometer was operated with the spray voltage set to 3.0 kV, the heated capillary held at 275°C, and the HESI probe held at 350°C. The sheath gas flow was set to 40 units, the auxiliary gas flow was set to 15 units, and the sweep gas flow was set to 1 unit. For 13C-BCAA tracing in tissues, the MS data acquisition was performed in full-scan, polarity switching mode in a range of 70-1000 m/z, with the resolution set at 70,000, the AGC target at 106, and the maximum injection time at 20 msec. For 15N-BCAA tracing into nucleotides, the MS data were acquired in either positive or negative ion mode in a range of 285-385 m/z, with the resolution set at 140,000, the AGC target at 106, and the maximum injection time at 250 msec.

Relative quantitation of polar metabolites was performed with XCalibur QuanBrowser 2.2 (Thermo Fisher Scientific) using a 5 ppm mass tolerance and referencing an in-house library of chemical standards.

Tracer enrichment measurements were corrected for natural abundance for either 13C or 15N tracers as described by Yuan et al. (43).

Relative Abundance of Amino Acids in Tissue Samples

To determine the relative abundance of amino acids in the samples, protein hydrolysis with 18% hydrochloric acid was modified to include an amino acid internal standard mixture of U-13C and U-15N species (Cambridge Isotopes, # MSK-A2-1.2) at a final concentration of 100µM. The ratio of ion counts of unlabeled and fully labeled isotopomers of each amino acid was calculated. The fully labeled standards contain each amino acid at equal concentrations, so these calculated ratios represent their relative abundances. We therefore normalized the sum of these ratios to equal one to calculate the fraction of protein amino acids represented by each individual amino

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acid. Cysteine and tryptophan residues are acid labile and not recoverable from the hydrolyzed samples.

DNA Isolation and Enzymatic DNA Digest

DNA was extracted using Phenol/Chloroform/Isoamyl extract mix (25:24:1 pH 8.0, Sigma P3803). DNA was precipitated with 0.1 volumes of 3M Sodium Acetate pH 5.2 and 3 volumes isopropanol overnight at -20°C. After pelleting and washing, DNA was resuspended in water. 20µL DNA was mixed with 6µL 100mM Succinic Acid-NaOH 50mM CaCl2 pH5.9, 0.1µL 0.02U/µL Bovine Spleen Phosphodiesterase II (Sigma P9041), 0.1µL 2000GelUnits/µL Micrococcal Nuclease (NEB 0247) and incubated at 37°C for 2.5 hrs. Reaction was then extracted with 90µL 2:1 Chloroform:Methanol and the resulting methanol fraction was evaporated under nitrogen. Sample was resuspended in 50:50 Acetonitrile:Water at 100ng/µL for Q-Exactive Mass Spectroscopic Analysis.

RNA Extraction and qPCR

Approximately 10-20mg of frozen tissue was homogenized (ProScientific Pro200) in Trizol (Life Technologies) and RNA extracted according to the manufacturer’s instructions. RNA concentration was determined using a Nanodrop (#ND1000) and 1µg of cDNA synthesized using iScript cDNA synthesis kit (BioRad). Quantitative-RT PCR was carried out on 5ng of cDNA using a final concentration of 2µM each of forward and reverse primers (table S8) with LuminoCt SYBR Green qPCR ReadyMix on a Roche Lightcycler II qPCR machine. Samples were normalized to the geometric mean of a panel of endogenous control genes (table S8) as previously described (44).

FACS Sorting and RNA isolation from Sorted Cells

Tumors were digested with 3mg/mL Dispase II (Roche), 1 mg/mL Collagenase I (Sigma), and 0.1mg/mL DNAse I (Sigma) in PBS for 30 mins at 37°C. To halt digestion, EDTA was then added to a final concentration of 10mM EDTA. Cells were strained through 70uM strainers and resuspended in flow cytometry staining buffer (eBioscience). Sytox Red (Life Technologies) was used for live/dead staining. Cell sorting was performed with a BD FACSAria III. RNA was isolated using the Ambion RNAqueous-Micro Total RNA Isolation Kit.

Protein Immunoblots

Approximately 20-30mg of frozen tissue was homogenized in RIPA buffer containing Protease Inhibitor Cocktail Tablets (Roche). Lysates were clarified by centrifugation in a benchtop centrifuge (Eppendorf Centrifugre 5145R) at 4˚C. Following clarification, protein concentration was determined using Bradford Protein Assay Dye (BioRad). All samples were run on SDS-acrylamide gels. After transfer to PVDF membrane and blocking in 5% BSA, blotting was performed using primary antibodies against Slc7a5 (Bioss, #bs-10125R), Bcat1 (Abcam, #ab110761), Bcat2 (Pierce Biotechnology, #PA5-21549), Bckdha (Novus Biologicals, #NBP1-79616), Hsp90 (Cell Signaling, #4877) and Vinculin (Sigma, # V9131). Secondary antibodies were goat anti-rabbit-HRP and goat-anti mouse (EMG Millipore). Western blot quantification was performing using ImageJ Image Analysis Software (NIH). Levels of BCAA catabolic enzymes were normalized to levels of vinculin and Hsp90.

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Microarray expression data sets Microarray expression data sets for pancreatic ductal adenocarcinoma, non-small cell lung

cancer, and normal tissue were obtained from the NCBI GEO database (30) (accession numbers GSE15471, GSE16515, GSE71989 for pancreas samples and GSE18842 for lung samples) and were restricted to those using Affymetrix U133 Plus 2.0 GeneChips. The affyQCReport package from Bioconductor was used to search for poor quality chips. GeneChip arrays that passed quality control checks were normalized using the GCRMA (45) algorithm from Bioconductor. For each cancer type, tumor and normal samples from the same study were processed together.

The limma (46) method from Bioconductor was used to calculate differential expression of metabolic genes with results reported as ratios on the log2 scale. P-values for differential expression were corrected for multiple hypothesis testing using the Benjamini and Hochberg method (47), controlling for false discovery rate at 5%. Expression changes of all human genes assigned to metabolic pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) (48) database were analyzed in addition to those assigned to the BCAA catabolic pathway.

TCGA RNA-seq data sets

Raw read counts were obtained from the Broad Firehose (https://gdac.broadinstitute.org/) LUAD and LUSQ January 28, 2016 data runs. Differential expression analysis was performed using the voom-limma (48) method from Bioconductor with results reported as ratios on the log2 scale. The Benjamini and Hochberg method was use to control the false discovery rate at 5%. Expression changes of metabolic genes in the KEGG database were analyzed in addition to those assigned to the BCAA catabolic pathway. Principal Component Analysis and Hierarchical Cluster Analysis

Principal component analysis (PCA) was performed on qPCR expression data from mouse tumors and normal control tissues. The principal components were obtained by first mean-centering each variable and then scaling by the respective standard deviation. The correlation matrix was then calculated, along with the corresponding eigenvalues and eigenvectors.

Hierarchical cluster analysis was performed on log-transformed qPCR expression data from mouse tumors and normal control tissues. The complete linkage method and Euclidean distance metric were adopted for clustering. Cell Lines

C57BL/6J KP NSCLC and PDAC (KP–/–C) cell lines were derived as previously described (18). Briefly, end-stage tumors were dissected from indicated mice and mechanically chopped before trypsin disaggregation. Tumor cells were propagated in DMEM with 10% FBS, 4 mM glutamine and penicillin/streptomycin. Cell lines were tested and determined to be negative for mycoplasma.

Cell Culture

Isotope Tracing Experiments. For labeled-glutamine tracing, DMEM lacking glutamine was supplemented with [α-15N]-glutamine (Cambridge Isotopes) to the standard DMEM concentration and with 10% dialyzed FBS. For leucine tracing, DMEM lacking leucine and containing either 4mM, 400µM or 40µM glutamine was supplemented with 15N-leucine (Cambridge Isotopes) to the standard DMEM concentration and with 10% dialyzed FBS. For 3-

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and 24-hr tracing experiment, 1x105 cells were plated six hours prior to the start of the experiment.

Proliferation Assay. 1x104 cells/well were plated and grown for four days in standard DMEM (without pyruvate) conditions with 10% FBS. Growth rate (in doublings/day) was calculated by the following equation:

Doublings/day = [log2(#cells day 4/#cells day0)]/4days

Tracing Experiments for Bcat KO lines. DMEM media lacking leucine was supplemented

with 15N-leucine (Cambridge Isotopes) to the standard DMEM concentration and with 10% dialyzed FBS. For 24-hr tracing experiment, 1.5x105 cells were plated six hours prior to the start of the experiment.

Slc7a5 Overexpression

The murine Slc7a5 open reading frame was PCR amplified from a cDNA library obtained from Origene Technologies (Rockville, MD). The Slc7a5 open reading frame was subcloned into pLHCX (Clontech, Mountain View, CA) by standard laboratory methods using the HindIII and ClaI restriction sites. The entire coding sequence of this construct was confirmed by sequencing analysis. Target PDAC cells were infected with either control empty vector or Slc7a5-expressing vector. Two Slc7a5 cell lines (Lines A and B) were generated in parallel from different bacterial clones. Post-infection, cell lines were selected with hygromycin B for further experiments. Leucine Uptake Assay

1x105 cells were plated the day before the experiment. At the start of the experiment, cells were washed once in PBS before exposure to PBS containing 200µM Leucine with 1µCi/mL [1-14C]-leucine (American Radiolabeled Chemicals) for 0 min. or 2 min. at 37°C. Labeled media was aspirated and cells were washed 3x with excess ice-cold PBS before freezing in liquid nitrogen. Cells were extracted in 80% MetOH for measurement of radioactivity using scintillation counting. Macropinocytosis Assay

Procedure. Macropinocytosis experiments were conducted as previously described (49) with the following modifications. Briefly, 2.5 x 104 NSCLC or PDAC cells were plated into 24-well plates containing fibronectin coated coverslips. Cells were cultured for 12 hours in 0.5mL DMEM containing 10% FBS, then media was removed and replaced with serum free DMEM for 24 hours. After serum starvation the media was replaced with serum free DMEM containing 10 ug/mL DQ Red BSA (Thermo-Fisher, D12051) and incubated for 30 minutes at 37°C. The media was then aspirated and the plate was washed 5 times with 2 mL ice cold PBS. Cells were fixed with 4% PFA for 15 minutes. Coverslips were removed and PDAC cells stained with 1ug/mL WGA 647 (Thermo-Fisher, W32466) for 15 minutes room temperature (NSCLC cells express GFP), and then both cell lines were stained with DAPI (Thermo-Fisher, D3571) for 10 minutes and mounted in Prolong Gold mounting media (Invitrogen P36934). Cells were then imaged on an applied precision DeltaVision Spectris Imaging System with a 60X objective. Macropinocytic index was calculated by field of view as described previously (49).

Analysis. Cell area was determined based on GFP fluorescence (NSCLC) or WGA-647 staining (PDAC) imaged in the FITC channel. DQ Red BSA was imaged in the Rhodamine

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channel. Images were converted to 8-bit, Z-stacked, and thresholded using ImageJ. Cell area was read out using the “Measure” function, while DQ Red BSA area was calculated with the “Analyze Particles” function. The macropinocytic index was calculated by dividing the area of the particles by the cell area for each image.

CRISPR-Cas9 genome editing

SgRNA to Bcat1 and Bcat2 was designed by identifying exon sequence homology in Bcat1 and Bcat2 followed by an NGG PAM sequence (fig. S8A). An additional G was added to sgRNAs lacking a 5’G for U6 transcriptional initiation. U6-sgRNA-EFS-CAS9-2A-Puro vector was digested with BsmB1 and ligated with annealed sgRNAs. Lentivirus was produced by co-transfection of 293T cells with lentiviral backbone constructs and packaging vectors (delta8.2 and VSV-G) using TransIT-LT1 (Mirus Bio).

Implantation studies

Subcutaneous. For subcutaneous implantation studies, recipient mice were anesthetized with inhaled 2% isoflurane-oxygen mixture. 100µL of PBS containing 2.0 x 105 cells was delivered into the subcutaneous space on the hindflank of the animal. Volume estimates in vivo were calculated according to a modified ellipsoid volume equation:

Tumor volume = (4/3)π(length/2)(width/2)2

Lung. For orthotopic implantation studies, recipient mice were anesthetized with inhaled

2% isoflurane-oxygen mixture. A catheter was inserted into the trachea and 50µL of PBS containing 2.0 x 105 cells delivered.

Pancreas. For orthotopic implantation studies, recipient mice were anesthetized with inhaled 2% isoflurane-oxygen mixture, a vertical incision made in the abdomen at the left mid-calvicular line, the spleen mobilized, and 50µL of either PBS or PBS containing 2.0 × 105 cells was injected into the tail of the pancreas. After sacrifice, tumors were dissected and calipered to get measurements of length, width and height. Tumor volume was then calculated for ellipsoid shaped tumors:

Tumor volume = (4/3)π(length/2)(width/2)(height/2)

Statistics

Appropriate statistical tests were performed where required. Two–sided unpaired student’s t–tests were performed for all statistical analyses unless otherwise specified using Mircosoft Excel for Mac:2011 (Microsoft) or GraphPad Prism 6 (GraphPad Software). For example, two-way repeated measures ANOVA was used to assess for differences in subcutaneous allograft tumor growth over time and one-way ANOVA with Dunnett’s multiple comparisons test used for tumor weight comparisons across three groups. No statistical method was used to pre-determine sample size.

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Fig. S1. Early pancreatic cancer and lung cancer have different effects on plasma BCAA levels. (A) Plasma BCAA concentration in five-week-old control and PDAC mice. Data are presented as mean ± SEM. N = 8 control and N = 6 PDAC. (B) Plasma BCAA concentration eight weeks post-infection in control and NSCLC mice. Data are presented as mean ± SEM. N = 7 control and N = 11 NSCLC. (C) Representative H&E section from a five-week-old PDAC mouse showing areas of normal pancreas and tumor. Scale bar = 100µm. (D) Representative H&E section from an eight-weeks post-infection NSCLC mouse showing areas of normal lung and tumor. Scale bar = 100µm. (E) Plasma BCAA concentration in C57BL/6J mice four weeks after subcutaneous implantation of syngenic PDAC cells. Data are presented as mean ± SEM. N = 5 control, N = 5 PDAC. (F) Plasma BCAA concentration in C57BL/6J mice four weeks after subcutaneous implantation of syngenic NSCLC cells. Data are presented as mean ± SEM. N = 6 control, N = 7 NSCLC. Two-tailed t test was used for all comparisons between two groups. * P<0.05

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Fig. S2

11

Fig. S2. Mice with NSCLC display increased BCAA uptake and metabolism. (A-I) Mice were fed 13C-BCAA containing diet for seven days. (A) Percent plasma enrichment of fully labeled BCAAs (left panel) and relative ion counts of total BCAAs (right panel) in control and PDAC mice. Data are presented as mean ± SEM. N = 4 control and N = 4 PDAC. (B) Percent plasma enrichment of fully labeled BCAAs (left panel) and relative ion counts of total BCAAs (right panel) in control and NSCLC mice. Data are presented as mean ± SEM. N = 4 control and N = 4 NSCLC. (C) Percent enrichment of labeled free BCAAs (left panel) and relative ion counts of total free BCAAs (right panel) in control mouse pancreas tissues and PDAC mouse tumors. Data are presented as mean ± SEM. N = 4 control and N = 4 PDAC. (D) Percent enrichment of labeled free BCAAs (left panel) and relative ion counts of total free BCAAs (right panel) in control mouse lung tissues and NSCLC mouse tumors. Data are presented as mean ± SEM. N = 4 control and N = 4 NSCLC. (E) Percent enrichment of labeled BCAAs (left panel) and relative ion counts of total BCAAs (right panel) in protein hydrolysates of control mouse pancreas tissues and PDAC mouse tumors. Data are presented as mean ± SEM. N = 4 control and N = 4 PDAC. (F) Percent enrichment of labeled BCAAs (left panel) and relative ion counts of total BCAAs (right panel) in protein hydrolysates of control mouse lung tissues and NSCLC mouse tumors. Data are presented as mean ± SEM. N = 4 control and N = 4 NSCLC. (G) Labeling (%) from [U-13C]-leucine and relative total ion count of downstream leucine metabolites (Fig. 1B) in tumors from PDAC and NSCLC mice and normal tissues from their respective control mice. Data are presented as mean ± SEM. N = 4 control and N = 4 PDAC; N = 4 control and N = 4 NSCLC. (H) Relative ion counts of labeled species downstream of KIC. Data are presented as mean ± SEM. Top panel, control mouse pancreas tissues and PDAC mouse tumors (N = 4 control and N = 4 PDAC) and bottom panel, control mouse lung tissues and NSCLC mouse tumors (N = 4 control and N = 4 NSCLC). (I) Citrate labeling (%) from [U-13C]-leucine in PDAC and NSCLC. Data are presented as mean ± SEM. Top panel, N = 4 control and N = 4 PDAC. Bottom panel, N = 4 control and N = 4 NSCLC. MID = mass isotopomer distribution. Two-tailed t test was used for all comparisons between two groups. * P<0.05, ** P<0.01, *** P<0.001

12

A

B

Pancreas

0.0

5.0

10.0

15.0

Amino Acid

Perc

ent of T

ota

l P

rote

in A

min

o A

cid

Pool

Control

PDAC

Ala Arg Asp+

Asn

Glu+

Gln

Gly His Ile Leu Lys Met Phe Pro Ser Thr Tyr Val

Lung

Ala Arg Asp+

Asn

Glu+

Gln

Gly His Ile Leu Lys Met Phe Pro Ser Thr Tyr Val

Amino Acid

Perc

ent of T

ota

l P

rote

in A

min

o A

cid

Pool

Control

NSCLC

0.0

5.0

10.0

15.0

Fig. S3

Fig. S3. Amino acid composition of PDAC and NSCLC tumors compared to control normal tissue. (A) Percent of each amino acid in total tissue protein determined following acid hydrolysis of control pancreas and PDAC tumors. Data are presented as mean ± SEM. N = 7 control and N = 5 PDAC. (B) Percent of each amino acid in total tissue protein determined following acid hydrolysis of normal lung and NSCLC tumors. Data are presented as mean ± SEM. N = 6 control and N = 6 NSCLC.

13

0

2

4

6

Control NSCLC

Plasma KIC

Control NSCLC0.0

0.5

1.0

1.5R

elat

ive

Ion

Cou

nts

(M+6

)

A

0.0

0.5

1.0

1.5

2.0

Control NSCLC

Rel

ativ

e Io

n C

ount

s

M+6

Lab

elin

g (%

) fro

m

[U-

C]-L

eu13

B

Liver Muscle WAT

Rel

ativ

e Io

n C

ount

s (M

+6)

Liver Muscle WAT

Rel

ativ

e Io

n C

ount

sLiver Muscle WATM

+6 L

abel

ing

(%) f

rom

[U-

C]-L

eu13

Tissue KIC

Liver Muscle WATLiver Muscle WAT0

1

2

3

Liver Muscle WAT0

1

2

3

Rel

ativ

e Io

n C

ount

s (M

+5)

Rel

ativ

e Io

n C

ount

s

0

2

4

6

8

M+5

Lab

elin

g (%

) fro

m [U

- C

]-Leu

13

Tissue C5-CarnitineC

*

**

ControlNSCLC

ControlNSCLC

0

1

2

3

4

5

0

1

2

3

0

10

20

30

40

50

Fig. S4

14

Fig. S4. Evidence for liver oxidation of BCAA-derived carbon in NSCLC tumor-bearing mice. (A-C) NSCLC mice were fed 13C-BCAA containing diet for seven days. (A) Relative labeled ion counts of M+6 isotopomer (left), M+6 labeling (%) from [U-13C]-leucine (center), and relative total ion counts (right) of KIC in the plasma of mice with NSCLC and their controls. Data are presented as mean ± SEM. N = 4 control and N = 4 NSCLC. (B) Relative labeled ion counts of M+6 isotopomer (left), M+6 labeling (%) from [U-13C]-leucine (center), and relative total ion counts (right) of KIC in the liver, muscle (gastrocnemius) and perigonadal white adipose tissue (WAT) of mice with NSCLC and their controls. Data are presented as mean ± SEM. N = 4 control and N = 4 NSCLC. (C) Relative labeled ion counts of M+5 isotopomer (left), M+5 labeling (%) from [U-13C]-leucine (center), and relative total ion counts (right) of C5-carnitine in the liver, muscle (gastrocnemius) and perigonadal white adipose tissue (WAT) of mice with NSCLC and their controls. Data are presented as mean ± SEM. N = 4 control and N = 4 NSCLC. Two-tailed t test was used for all comparisons between two groups. * P<0.05, ** P<0.01.

15

A Pancreas Free Leucine

Control PDAC0.0

0.5

1.0

1.5

2.0R

elat

ive

Ion

Cou

nts

(M+1

)

Control PDAC0.0

2.0

4.0

6.0

8.0

10.0

Control PDAC0.0

0.5

1.0

1.5

Rel

ativ

e Io

n C

ount

s

B Pancreas Tissue Protein

Ala Asp+Asn

Glu+Gln

Val Ile0.0

0.5

1.0

1.5

2.0

Rel

ativ

e Io

n C

ount

s (M

+1)

ControlPDAC

P = 0.05

0.0

0.5

1.0

1.5

Amino Acid Amino AcidC Lung Free Amino Acids

Leu

0

5

10

15

Ala Asp+Asn

Glu+Gln

Val Ile Leu

Ala Asp Glu Val Ile Leu0

2

4

6

Amino Acid

Rel

ativ

e Io

n C

ount

s (M

+1)

ControlNSCLC *** *** *** *

***

Ala Asp Glu Val Ile LeuAmino Acid

******

02468

10

****

D Lung - Plasma E Lung Tissue Protein Amino Acids

Rel

ativ

e Io

n C

ount

s

0.00.51.01.52.0


*

***

***

02468

10

0.00.51.01.52.0


*** *** ***** **

*

* *

* *

**

M+1

Lab

elin

g (%

) fro

m

[ N

]-Leu

15

M+1

Lab

elin

g (%

) fro

m [

N]-L

eu15

0

5

10

15

0

5

10

15

Rel

ativ

e Io

n C

ount

sM

+1 L

abel

ing

(%)

from

[ N

]-Leu

15

Rel

ativ

e Io

n C

ount

sM

+1 L

abel

ing

(%)

from

[ N

]-Leu

15R

elat

ive

Ion

Cou

nts

M+1

Lab

elin

g (%

) fro

m [

N]-L

eu15

Fig. S5

16

Fig. S5. BCAA-derived nitrogen supports non-essential amino acid and DNA synthesis in NSCLC tumors. (A-E) PDAC mice were fed 15N-leucine containing diet for seven days and NSCLC mice were fed 15N-leucine diet for six days. (A) Relative labeled ion counts of M+1 isotopomer (left), M+1 labeling (%) from 15N-leucine (center), and relative total ion counts (right) of free leucine in control mouse pancreas tissues and PDAC mouse tumors. Data are presented as mean ± SEM. N = 6 control and N = 6 PDAC. (B) Relative labeled ion counts of M+1 isotopomers (left), M+1 labeing (%) from 15N-leucine (top right), and relative total ion counts (bottom right) of amino acids from tissue protein hydrolysates in control mouse pancreas tissues and PDAC mouse tumors. Data are presented as mean ± SEM. N = 6 control and N = 6 PDAC. (C) Relative labeled ion counts of M+1 isotopomers (left), M+1 labeing (%) from 15N-leucine (top right), and relative total ion counts (bottom right) of free amino acids in control mouse lung tissues and NSCLC mouse tumors. Data are presented as mean ± SEM. N = 6 control and N = 5 NSCLC. (D) M+1 labeing (%) from 15N-leucine (top) and relative total ion counts (bottom) of free plasma amino acids in control and NSCLC mice. Data are presented as mean ± SEM. N = 5 control and N = 6 NSCLC. (E) M+1 labeing (%) from 15N-leucine (top) and relative total ion counts (bottom) of free tissue amino acids in control mouse lung tissues and NSCLC mouse tumors. Data are presented as mean ± SEM. N = 6 control and N = 6 NSCLC. Two-tailed t test was used for all comparisons between two groups. * P<0.05, ** P<0.01, *** P<0.001

17

A

C

Ile Leu Val0

5

10

15

Amino Acid

NSCLCPDAC

M+1

Lab

elin

g (%

) fro

m

[α-

N]-G

ln15

B

*

**

**

4mM 400µM 40µM0

5

10

15

Glutamine Concentration

Isoleucine M+1

4mM 400µM 40µM0

2

4

6

8

10


Valine M+1

M+1

Lab

elin

g (%

) fro

m

[ N

]-Leu

15

M+1

Lab

elin

g (%

) fro

m

[ N

]-Leu

15

* *

**

NSCLCPDAC

4mM 400µM 40µM0

10

20

30

40


M+1

Lab

elin

g (%

) fro

m

[ N

]-Leu

15

4mM 400µM 40µM


0

10

20

30

40

M+1

Lab

elin

g (%

) fro

m

[ N

]-Leu

15

4mM 400µM 40µM


0

10

20

30

40

M+1

Lab

elin

g (%

) fro

m

[ N

]-Leu

15

Alanine M+1 Aspartate M+1 Glutamate M+1

**

***

NSCLCPDAC

**

**

Fig. S6

Fig. S6. Relationship between BCAA transamination and glutamine level in vitro. (A) M+1 isoleucine (Ile), leucine (Leu) and valine (Val) labeling (%) from [α-15N]-glutamine

following exposure of PDAC or NSCLC cells for 24-hours to media containing [α-15N]-glutamine. Data are presented as mean ± SEM. N = 3 per group. (B) M+1 isoleucine (left panel) and valine (right panel) labeling (%) from 15N-leucine following exposure of PDAC or NSCLC cells for 3-hours to media containing 15N-leucine and the indicated concentration of glutamine. Data are presented as mean ± SEM. N = 3 per group. (C) M+1 alanine (left panel), aspartate (middle panel) and glutamate (right panel) labeling (%) from 15N-leucine following exposure of PDAC or NSCLC cells for 3-hours to media containing 15N-leucine and the indicated concentration of glutamine. Data are presented as mean ± SEM. N = 3 per group. Two-tailed t test was used for all comparisons between two groups. * P<0.05, ** P<0.01, *** P<0.001

18

A

C D

B

Lung

Control

PDAC

Pancreas

Acadsb Auh Hadhb Pcca

0.0

0.5

1.0

1.5

2.0

Re

lative

Expre

ssio

n

NSCLC

Control

Acadsb Auh Hadhb Pcca

0.0

0.5

1.0

1.5

2.0

Re

lative

Expre

ssio

n

Control

KPC

Slc7a5 Bcat1 Bcat2 Bckdha Bckdhb Acadsb Auh Hadhb Pcca

0.0

0.5

1.0

1.5

2.0R

ela

tive

Expre

ssio

nPancreas

Re

lative

Expre

ssio

n

Slc7a5 Bcat1 Bcat2 Bckdha Bckdhb

0

1

2

3

Whole Tumor

Cancer Cells

PDAC

Glut1 Eno1

0

5

10

15

20

25

Glut1 Eno1

Re

lative

Expre

ssio

n

0

1

2

3

4

E

Re

lative

Expre

ssio

n

FLung Pancreas

* * * ***

*

*

*

**

*******

PC1 (40.6%)

PC

2 (

29.6

%)

−2 0 2 4 6

−3−2

−10

12

3

●

●●

●

●● ●

●●●●●

●●

●

●

●●

●

●● ●●●●●

●●●

●●

Control

PDAC

Pancreas Lung

Control

NSCLC

G H I

−0.05

0.0

6

0.1

6

0.2

7

0.3

7

0.4

8

0.5

8

0.6

8

0.7

90.91

GS

E15471

GS

E16515

GS

E71989

GS

E18842

LU

AD

_T

CG

A

LU

SQ

_T

CG

A

GSE15471

GSE16515

GSE71989

GSE18842

LUAD_TCGA

LUSQ_TCGA

Bck

dhb

Bca

t2

Hadhb

Auh

Aca

dsb

Pcc

a

Slc7a5

Bck

dha

Bca

t1−6 −4 −2log(expr)

Fig. S7

19

Fig. S7. Gene expression in both mouse and human tumors reflects tumor tissue-specific BCAA metabolism. (A) Relative expression of BCAA metabolic pathway genes in KPC PDAC tumors and normal pancreas. Data are presented as mean ± SEM. N = 3 control and N = 4 KPC. (B) Relative expression of BCAA metabolic pathway genes in FACS sorted tumor cells compared with parental whole tumor from KP-/-C PDAC mice. Data are presented as mean ± SEM. N = 5 tumors. Two-tailed paired-sample t test used for comparison. (C) Relative expression of downstream BCAA metabolic pathway genes in normal lung and NSCLC tumors from KP mice. Data are presented as mean ± SEM. N = 6 control and N = 6 NSCLC. (D) Relative expression of downstream BCAA metabolic pathway genes in normal pancreas and PDAC tumors from KP-/-C mice. Data are presented as mean ± SEM. N = 7 control and N = 5 PDAC. (E) Relative expression of glycolytic genes in normal lung and NSCLC tumors from KP mice. Data are presented as mean ± SEM. N = 6 control and N = 6 NSCLC. (F) Relative expression of glycolytic genes in normal pancreas and PDAC tumors from KP-/-C mice. Data are presented as mean ± SEM. N = 7 control and N = 5 PDAC. (G) Principal component analysis (PCA) of mouse BCAA catabolic gene expression in normal lung, NSCLC tumors, normal pancreas and PDAC tumors from Figs. 3, A and B and figs. S7, C and D. The first two principal components explained ~41% and ~30% of the variance respectively. (H) Hierarchical cluster analysis of mouse BCAA catabolic gene expression data. Colors on the side bar correspond to tissue samples given in fig. S7G. (I) Correlation of tumor expression fold-changes for branched-chain amino acid degradation pathway genes in human studies involving pancreas (GSE15471, GSE16515, GSE71989) and lung (GSE18842, LUAD_TCGA, LUSQ_TCGA). Average Spearman R was 0.84 for pancreas data. Spearman R with GSE18842 was 0.61 for lung adenocarcinoma and 0.86 for squamous cell carcinoma. Two-tailed t test was used for all comparisons unless otherwise stated. * P<0.05, ** P<0.01, *** P<0.001

20

A

E

B

C

Bcat1 CDS

Bcat2 CDS

sgBcat total

G C T G G T C T T T G G A G C T A C G T T T A C T G A C C A C A T G C T G A C G G T G G A G T G G T C C T C T G C G T C T G G A T G

A C T G C T G T T T G G G A A G A C C T T C A C A G A C C A C A T G C T G A T G G T G G A G T G G A A T A A C A A G G C T G G A T G

- - - - - - - - - - - - - - - - - - - - - - - - - - - C C A C A T G C T G A C G G T G G A G - - - - - - - - - - - - - - - - - - - -

Allele 1 Allele 2

NSCLC Clone A 1bp insertion 8bp deletion 8bp deletion 5bp deletion

NSCLC Clone B 1bp deletion 19bp deletion 25bp deletion 8bp deletion

PDAC 8bp deletion 8bp deletion 1bp deletion 2bp insertion

Allele 1 Allele 2

Bcat1 Bcat2

Bcat1 Bcat20.0

0.5

1.0

1.5

Gene

Re

lative

Expre

ssio

n

pLenti Control

Bcat Clone A

Bcat Clone B

Bcat1 Bcat20.0

0.5

1.0

1.5

Gene

Re

lative

Expre

ssio

n

D

pLenti Control

Bcat null

F

pLenti

Control

Bcat null

0

200

400

600

800

1000

Tum

or

Volu

me

(m

m3)

PDAC Pancreas

Orthotopic Allografts

**

I

pLenti

Control

Bcat null

Clone A

pLenti

Control

Bcat null

Clone B

***

0

5

10

15

20

***

pLenti

Control

Bcat null

M+1 G

lu L

abelin

g (%

)

from

[ N

]-Leu

15

0

2

4

6

8 ***M

+1 G

lu L

abelin

g (%

)

from

[ N

]-Leu

15

NSCLC Subcutaneous

Allografts

PDAC Subcutaneous

Allografts

pLenti

Control

Bcat null

Clone A

Bcat null

Clone B

0

200

400

600

Tum

or

We

ight (m

g)

Cell Line Cell Line

G

***

***

Cell Line Cell Line

pLenti

Control

Bcat null

0

100

200

300

Tum

or

We

ight (m

g)

H

Cell Line

Fig. S8

0

100

200

300

400

pLenti

Control

Bcat null

Cell Line

21

Fig. S8. Branched-chain amino acid transaminase (Bcat) activity is required for NSCLC tumor growth. (A) Schematic of common exon sequence of Bcat1 and Bcat2 targeted with sgRNA for CRISPR-Cas9 genome editing. Red “T” highlights a 1bp sequence difference between the two isoforms. (B) Summary of sequence analyses of Bcat1 and Bcat2 in Bcat null clones derived from syngenic NSCLC and PDAC cell lines following sgRNA targeting with CRISPR-Cas9. (C) Normalized expression measured by qPCR of Bcat1 and Bcat2 in NSCLC Bcat null Clones A and B confirming decreased expression of both genes. (D) Normalized expression measured by qPCR of Bcat1 and Bcat2 in PDAC Bcat null cells confirming decreased expression of both genes. (E) M+1 Glu labeling (%) from 15N-leucine following 24-hour 15N-leucine tracing in control infected compared with NSCLC Bcat null Clone A and B cells. Data are presented as mean ± SEM. N = 3 per group. Representative experiment from ≥2 repeats. (F) M+1 Glu labeling (%) from 15N-leucine following 24-hour 15N-leucine tracing in control infected compared with PDAC Bcat null cells. Data are presented as mean ± SEM. N = 3 per group. (G) Tumor weight of subcutaneous allograft tumors generated from control infected and Bcat null syngenic NSCLC cell lines in C57BL/6J mice. Data are presented as mean ± SEM. N = 6 per group. (H) Two independent experiments determining tumor weight of subcutaneous allograft tumors derived from control infected and Bcat null syngenic PDAC cells in C57BL/6J mice. Data are presented as mean ± SEM. Left panel corresponds to mice in Fig. 4D. N = 5 pLenti control and N = 6 Bcat null. Right panel, N = 3 pLenti control and N = 3 Bcat null. (I) Calculated tumor volumes of orthotopically implanted syngenic PDAC pLenti control and Bcat null tumors. Data are presented as mean ± SEM. N = 8 per group. Two-tailed t test was used for all comparisons between two groups. * P<0.05, ** P<0.01, *** P<0.001

22

A B

Fig. S9

***

NSCLC PDAC

Cell Type

Mac

ropi

nocy

tic In

dex

0.0

0.2

0.4

0.6

0.8

1.0NSCLC PDACDAPIDQBSA

Fig. S9. NSCLC cells exhibit decreased macropinocytosis relative to PDAC cells. (A) Representative images used to quantify macropinocytic uptake in NSCLC and PDAC cells. Cells were exposed to DQ Red BSA (DQBSA) and stained with DAPI. Scale bar = 10µm. (B) Quantified macropinocytic index based on fluorescence from DQBSA. Data were obtained from N = 7 fields-of-view (29 cells) for NSCLC and N = 6 fields-of-view (49 cells) for PDAC.

23

Control Vector

Slc7a5 Line A

Slc7a5 Line B

Cell Line

Dou

blin

gs/2

4hrs

in vitro proliferation

0

10

20

30

Rel

ativ

e Ex

pres

sion

Slc7a5 Expression

Control Vector

Slc7a5 Line A

Slc7a5 Line B

Cell Line

A B C

Fig. S10

Dfm

ol /

(cel

l * m

in)

Leucine Uptake

Control Vector

Slc7a5 Line A

Slc7a5 Line B

Cell Line

0.0

0.5

1.0

1.5

2.0

2.5***

***

E

0.0

0.2

0.4

0.6

0.8

1.0

1.2

0 5 10 15 200

200

400

600

800

1000

Time (days)

Tum

or V

olum

e (m

m )3

PDAC Subcutaneous Allografts

Control VectorSlc7a5 Line ASlc7a5 Line B

0

200

400

600

Tum

or W

eigh

t (m

g)

PDAC Subcutaneous Allografts

Control Vector

Slc7a5 Line A

Slc7a5 Line B

Cell Line

P = 0.06

Fig. S10. Overexpression of Slc7a5 in PDAC cells does not affect proliferation. (A) Relative expression of Slc7a5 in control infected PDAC cells and two PDAC cells lines overexpressing Slc7a5. (B) Uptake rate of leucine in vector control infected PDAC cells and PDAC cells overexpressing Slc7a5. Data are presented as mean ± SEM. N = 3 per group. One-way ordinary ANOVA with Dunnett’s multiple comparisons for control vector versus each overexpression vector. (C) Doubling time in vitro of control infected and Slc7a5 overexpressing PDAC cells. Data are presented as mean ± SEM. N = 3 per group. (D) Estimated tumor volume (mm3) of subcutaneous allografts derived from control vector infected and Slc7a5 overexpressing syngenic PDAC cell lines in C57BL/6J mice. Data are presented as mean ± SEM. N = 6 control vector and N = 5 each Slc7a5 Vectors A and B. Two-way repeated measures ANOVA used for comparison between groups. (E) Tumor weight of subcutaneous allografts derived from control vector and Slc7a5 overexpressing syngenic PDAC cells in C57BL/6J mice. Data are presented as mean ± SEM. N = 6 control vector and N = 5 each Slc7a5 Vectors A and B. One-way ordinary ANOVA (P = 0.0137) with Dunnett’s multiple comparisons for control vector versus each overexpression vector. * P<0.05, ** P<0.01, *** P<0.001

24

Table S1. Human expression data comparing PDAC to adjacent normal pancreatic tissue (GSE15471). Gene ID Gene Symbol Gene Name log2 fold

change p-value

8140 SLC7A5 solute carrier family 7 (amino acid transporter light chain, L system), member 5

0.879502861 0.00122176

587 BCAT2 branched chain amino-acid transaminase 2, mitochondrial -0.90281329 7.65E-05

586 BCAT1 branched chain amino-acid transaminase 1, cytosolic -1.684103972 0.00477334

259307 IL4I1 interleukin 4 induced 1 0.232279522 0.034091294

593 BCKDHA branched chain keto acid dehydrogenase E1, alpha polypeptide -0.613720584 0.000701634

594 BCKDHB branched chain keto acid dehydrogenase E1, beta polypeptide -0.735076396 2.48E-06

1629 DBT dihydrolipoamide branched chain transacylase E2 -0.587540291 0.000331659

1738 DLD dihydrolipoamide dehydrogenase -0.469580173 4.03E-08

35 ACADS acyl-CoA dehydrogenase, C-2 to C-3 short chain -0.877723342 1.46E-06

34 ACADM acyl-CoA dehydrogenase, C-4 to C-12 straight chain -0.928974891 9.48E-10

3712 IVD isovaleryl-CoA dehydrogenase -0.367787174 0.000388408

36 ACADSB acyl-CoA dehydrogenase, short/branched chain -1.373555807 4.44E-08

27034 ACAD8 acyl-CoA dehydrogenase family, member 8 -0.519219647 0.000294208

3030 HADHA hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase (trifunctional protein), alpha subunit

-0.584595254 0.001003697

1962 EHHADH enoyl-CoA, hydratase/3-hydroxyacyl CoA dehydrogenase -0.658770531 0.003790345

1892 ECHS1 enoyl CoA hydratase, short chain, 1, mitochondrial -0.048319001 0.745579334

3033 HADH hydroxyacyl-CoA dehydrogenase -0.547844188 0.001603

3028 HSD17B10 hydroxysteroid (17-beta) dehydrogenase 10 0.160472939 0.07307885

30 ACAA1 acetyl-CoA acyltransferase 1 -0.334432109 0.015971732

10449 ACAA2 acetyl-CoA acyltransferase 2 -0.566448642 4.78E-06

3032 HADHB hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase (trifunctional protein), beta subunit

-0.246013864 0.009645609

5095 PCCA propionyl CoA carboxylase, alpha polypeptide -0.482286092 0.00036024

5096 PCCB propionyl CoA carboxylase, beta polypeptide -0.553229461 0.000186867

84693 MCEE methylmalonyl CoA epimerase -0.769948801 6.22E-05

4594 MUT methylmalonyl CoA mutase -0.454059405 7.59E-05

26275 HIBCH 3-hydroxyisobutyryl-CoA hydrolase -0.725551273 2.74E-05

11112 HIBADH 3-hydroxyisobutyrate dehydrogenase -0.346119019 0.005247236

4329 ALDH6A1 aldehyde dehydrogenase 6 family, member A1 -1.531300679 7.64E-09

217 ALDH2 aldehyde dehydrogenase 2 family (mitochondrial) 0.017059544 0.914989371

224 ALDH3A2 aldehyde dehydrogenase 3 family, member A2 -0.830408387 0.000415913

219 ALDH1B1 aldehyde dehydrogenase 1 family, member B1 -0.160216402 0.171935365


223 ALDH9A1 aldehyde dehydrogenase 9 family, member A1 -0.38849919 7.85E-06

316 AOX1 aldehyde oxidase 1 -1.714514255 0.001243926

18 ABAT 4-aminobutyrate aminotransferase -1.913909607 3.47E-05

56922 MCCC1 methylcrotonoyl-CoA carboxylase 1 (alpha) -0.807017307 2.65E-05

64087 MCCC2 methylcrotonoyl-CoA carboxylase 2 (beta) -0.272774313 0.049069198

25

549 AUH AU RNA binding protein/enoyl-CoA hydratase -0.295488655 0.052464302

3155 HMGCL 3-hydroxymethyl-3-methylglutaryl-CoA lyase -0.337595694 0.004458408

5019 OXCT1 3-oxoacid CoA transferase 1 0.537053005 0.003720935

64064 OXCT2 3-oxoacid CoA transferase 2 -0.533796167 4.00E-06

39 ACAT2 acetyl-CoA acetyltransferase 2 0.574014232 0.000803098

38 ACAT1 acetyl-CoA acetyltransferase 1 -1.309218369 5.31E-09

3157 HMGCS1 3-hydroxy-3-methylglutaryl-CoA synthase 1 (soluble) 0.08709899 0.513755372

3158 HMGCS2 3-hydroxy-3-methylglutaryl-CoA synthase 2 (mitochondrial) -2.009941821 0.006996665

26

Table S2. Human expression data comparing lung adenocarcinoma to adjacent normal lung tissue (GSE18842) Gene ID Gene

Symbol Gene Name log2 fold

change p-value

8140 SLC7A5 solute carrier family 7 (amino acid transporter light chain, L system), member 5 2.526891751 6.23E-13

587 BCAT2 branched chain amino-acid transaminase 2, mitochondrial 0.453635541 0.003881953

586 BCAT1 branched chain amino-acid transaminase 1, cytosolic 0.807841298 0.013203348

259307 IL4I1 interleukin 4 induced 1 1.554637499 1.11E-06

593 BCKDHA branched chain keto acid dehydrogenase E1, alpha polypeptide 0.226473838 0.073768274

594 BCKDHB branched chain keto acid dehydrogenase E1, beta polypeptide 0.405589777 0.10000218

1629 DBT dihydrolipoamide branched chain transacylase E2 0.711942322 0.000365403

1738 DLD dihydrolipoamide dehydrogenase 0.322748437 0.005217065

35 ACADS acyl-CoA dehydrogenase, C-2 to C-3 short chain -0.372841503 0.000793325

34 ACADM acyl-CoA dehydrogenase, C-4 to C-12 straight chain -0.446473763 0.000226808

3712 IVD isovaleryl-CoA dehydrogenase -0.628354316 0.000721463

36 ACADSB acyl-CoA dehydrogenase, short/branched chain -1.695405701 2.78E-08



-0.135673055 0.071471145

1962 EHHADH enoyl-CoA, hydratase/3-hydroxyacyl CoA dehydrogenase 0.78441437 0.000216506

1892 ECHS1 enoyl CoA hydratase, short chain, 1, mitochondrial 0.485018276 2.15E-05

3033 HADH hydroxyacyl-CoA dehydrogenase 0.219528794 0.207298092

3028 HSD17B10 hydroxysteroid (17-beta) dehydrogenase 10 0.670151503 4.40E-08




-0.120032456 0.109495435


5096 PCCB propionyl CoA carboxylase, beta polypeptide 1.421420616 2.85E-11

84693 MCEE methylmalonyl CoA epimerase 0.19575875 0.231716118

4594 MUT methylmalonyl CoA mutase -0.565339317 0.000718517

26275 HIBCH 3-hydroxyisobutyryl-CoA hydrolase 0.106494012 0.632709265

11112 HIBADH 3-hydroxyisobutyrate dehydrogenase 0.445133888 0.00299894


217 ALDH2 aldehyde dehydrogenase 2 family (mitochondrial) -1.742763109 1.15E-12


219 ALDH1B1 aldehyde dehydrogenase 1 family, member B1 0.960176481 5.93E-06



316 AOX1 aldehyde oxidase 1 -3.32817822 3.40E-20

18 ABAT 4-aminobutyrate aminotransferase -0.64764613 0.125137447

56922 MCCC1 methylcrotonoyl-CoA carboxylase 1 (alpha) 0.388764727 0.051035278

27

64087 MCCC2 methylcrotonoyl-CoA carboxylase 2 (beta) 1.190292844 1.38E-09




64064 OXCT2 3-oxoacid CoA transferase 2 -0.087938141 0.196592754





28


change p-value


0.695729277 0.055891107

587 BCAT2 branched chain amino-acid transaminase 2, mitochondrial -0.587343204 0.014782749




594 BCKDHB branched chain keto acid dehydrogenase E1, beta polypeptide -0.29063685 0.075005553

1629 DBT dihydrolipoamide branched chain transacylase E2 -0.55337088 0.010313609

1738 DLD dihydrolipoamide dehydrogenase -0.321787985 0.021112408


34 ACADM acyl-CoA dehydrogenase, C-4 to C-12 straight chain -0.823919118 3.92E-05

3712 IVD isovaleryl-CoA dehydrogenase 0.109736254 0.497366466

36 ACADSB acyl-CoA dehydrogenase, short/branched chain -1.120427046 0.001225893



-0.396494941 0.019975795


1892 ECHS1 enoyl CoA hydratase, short chain, 1, mitochondrial 0.278663082 0.111478307


3028 HSD17B10 hydroxysteroid (17-beta) dehydrogenase 10 0.181845127 0.27170365

30 ACAA1 acetyl-CoA acyltransferase 1 0.092593182 0.690004538



-0.152380334 0.298839704



84693 MCEE methylmalonyl CoA epimerase -0.605450867 0.005927251


26275 HIBCH 3-hydroxyisobutyryl-CoA hydrolase -0.230225489 0.292635698

11112 HIBADH 3-hydroxyisobutyrate dehydrogenase 0.193265533 0.361841563




219 ALDH1B1 aldehyde dehydrogenase 1 family, member B1 0.278048635 0.328069203



316 AOX1 aldehyde oxidase 1 -3.327431917 1.24E-05


56922 MCCC1 methylcrotonoyl-CoA carboxylase 1 (alpha) -0.761894863 0.001967284

64087 MCCC2 methylcrotonoyl-CoA carboxylase 2 (beta) 0.369865204 0.069242708

29









30


change p-value


1.834506135 0.048832563

587 BCAT2 branched chain amino-acid transaminase 2, mitochondrial -0.915125942 0.005263568




594 BCKDHB branched chain keto acid dehydrogenase E1, beta polypeptide -0.687946873 0.156787101

1629 DBT dihydrolipoamide branched chain transacylase E2 -2.138467417 4.04E-05

1738 DLD dihydrolipoamide dehydrogenase -0.309437161 0.194182069


34 ACADM acyl-CoA dehydrogenase, C-4 to C-12 straight chain -0.227530954 0.339717236

3712 IVD isovaleryl-CoA dehydrogenase 0.364308007 0.136514652

36 ACADSB acyl-CoA dehydrogenase, short/branched chain -1.370334091 0.001696882



-1.199004767 0.000134058


1892 ECHS1 enoyl CoA hydratase, short chain, 1, mitochondrial 0.104757048 0.61074395


3028 HSD17B10 hydroxysteroid (17-beta) dehydrogenase 10 -0.218220037 0.082112071




-0.176206766 0.336134576



84693 MCEE methylmalonyl CoA epimerase -1.089729015 0.008120642


26275 HIBCH 3-hydroxyisobutyryl-CoA hydrolase -0.604614912 0.049045949

11112 HIBADH 3-hydroxyisobutyrate dehydrogenase -0.618580292 0.001237186




219 ALDH1B1 aldehyde dehydrogenase 1 family, member B1 -0.067959408 0.879445206



316 AOX1 aldehyde oxidase 1 -4.611602293 0.000559186


56922 MCCC1 methylcrotonoyl-CoA carboxylase 1 (alpha) -1.16890529 0.001695464

64087 MCCC2 methylcrotonoyl-CoA carboxylase 2 (beta) -0.275297083 0.213689093

31






38 ACAT1 acetyl-CoA acetyltransferase 1 -1.624761864 0.001229512



32

Table S5. Human expression data comparing lung adenocarcinoma to normal lung tissue (LUAD_TCGA) Gene ID Gene Symbol logFC AveExpr t P.Value adj.P.Val B

8140 SLC7A5 2.301489817 6.358231189 10.42262512 2.03E-23 1.99E-22 41.91341697

587 BCAT2 -0.022329943 5.405346113 -0.238943734 0.811234173 0.837543421 -7.962364892

586 BCAT1 -0.082200745 5.812571896 -0.390719929 0.696148682 0.734856037 -7.949366128

259307 IL4I1 1.802053865 3.662717891 7.146453383 2.70E-12 1.13E-11 16.96043713

593 BCKDHA -0.064986379 5.742374666 -0.805448303 0.420893276 0.471526877 -7.695807869

594 BCKDHB -0.089449287 3.848210819 -1.05811492 0.290446434 0.338490079 -7.215969219

1629 DBT -0.165486395 4.901371519 -2.308007721 0.021351989 0.030447339 -5.296980892

1738 DLD 0.037319195 6.153408635 0.518807164 0.604094408 0.648584069 -7.903129538

35 ACADS -0.396142929 4.654196909 -3.721228554 0.000217685 0.000404804 -1.096055568

34 ACADM -0.149175994 5.561180575 -1.893454362 0.058798186 0.078016723 -6.224218221

3712 IVD -0.454413704 6.120665928 -4.234544074 2.67E-05 5.56E-05 0.78537962

36 ACADSB -0.656685952 5.224157391 -5.221457536 2.48E-07 6.60E-07 5.310708694

27034 ACAD8 0.952248253 5.346434799 6.865131266 1.72E-11 6.71E-11 14.77617986

3030 HADHA -0.16085849 7.678322593 -3.398825913 0.000723518 0.001259949 -2.325968325

1962 EHHADH 0.029588819 3.912137766 0.267564907 0.789129991 0.818635495 -7.734194387

1892 ECHS1 0.496639082 6.83135147 6.780040815 2.98E-11 1.13E-10 14.08150294

3033 HADH -0.073366638 5.661570217 -0.888721427 0.374523663 0.425039729 -7.619794388

3028 HSD17B10 0.506258189 5.855151605 6.499293438 1.75E-10 6.21E-10 12.40300803

30 ACAA1 -0.407743414 5.772675264 -5.017906592 6.97E-07 1.77E-06 4.293828537

10449 ACAA2 -0.837258146 5.703806931 -8.277737019 8.78E-16 4.84E-15 24.34694946

3032 HADHB -0.021523161 6.805987063 -0.408996065 0.682694521 0.722433188 -7.971203158

5095 PCCA -0.246456074 3.753144879 -2.016385373 0.044223175 0.059868769 -5.753673006

5096 PCCB 0.343767432 5.710113172 4.699194071 3.27E-06 7.66E-06 2.850605682

84693 MCEE 0.132703208 2.801890502 1.308484783 0.191230609 0.232045672 -6.694952278

4594 MUT 0.11151478 5.342882293 1.519870588 0.129092016 0.161790721 -6.820741756

26275 HIBCH 0.207361423 4.500464409 2.463148936 0.014063513 0.020557426 -4.826842489

11112 HIBADH 0.546603418 5.672095469 6.273912746 6.92E-10 2.33E-09 11.07907501

4329 ALDH6A1 -0.143182697 3.97983577 -1.19603012 0.232176743 0.276353846 -7.092230071

217 ALDH2 -1.376449884 8.081884479 -10.37850121 2.99E-23 2.89E-22 41.38215198

224 ALDH3A2 -0.455354534 7.29385346 -3.207001887 0.001415631 0.002381613 -2.950344994

219 ALDH1B1 0.947103168 5.207573479 7.848908012 2.05E-14 1.02E-13 21.41181195

501 ALDH7A1 0.165878163 5.557560458 1.785293215 0.074739652 0.097505234 -6.400096771

223 ALDH9A1 -0.23186742 6.512121218 -3.239786141 0.00126511 0.002143239 -2.849056789

316 AOX1 -2.15049672 2.471108098 -12.67670724 1.18E-32 2.09E-31 63.05942585

18 ABAT -0.270201151 3.825653793 -1.616717801 0.106486521 0.135456773 -6.492303448

56922 MCCC1 -0.310875957 5.036842343 -3.089395627 0.002102324 0.003454369 -3.242611828

64087 MCCC2 0.60856239 6.249504932 7.598211993 1.22E-13 5.69E-13 19.51128747

549 AUH -0.307891419 3.26732002 -4.000960494 7.13E-05 0.000141411 0.193214559

33

3155 HMGCL 0.094091401 5.573266149 1.277615691 0.20189947 0.243699726 -7.181453788

5019 OXCT1 0.035856218 4.423932006 0.217854779 0.827619389 0.851982284 -7.835471736

64064 OXCT2 0.753014278 -1.341418463 3.932433184 9.43E-05 0.00018367 0.41322003

39 ACAT2 -0.435561364 4.614165503 -4.402822034 1.27E-05 2.77E-05 1.596491751

38 ACAT1 -0.637666221 5.665301524 -6.551388268 1.27E-10 4.55E-10 12.67279193

3157 HMGCS1 -1.093236292 6.104568228 -9.63348088 1.82E-20 1.43E-19 34.99752964

3158 HMGCS2 -3.494020182 -1.784574072 -8.586127586 8.40E-17 5.02E-16 27.27033633

34

Table S6. Human expression data comparing lung squamous cell carcinoma to normal lung tissue (LUSQ_TCGA) Gene ID Gene Symbol logFC AveExpr t P.Value adj.P.Val B

8140 SLC7A5 3.43564825 7.707835006 16.8420549 1.06E-51 1.70E-50 106.4644738

587 BCAT2 0.07009414 5.318307538 0.789526849 0.430141792 0.462844102 -7.897165607

586 BCAT1 -0.688161797 5.359877887 -3.719734352 0.000219746 0.000334717 -1.397369172

259307 IL4I1 1.296770374 3.453601085 6.269297405 7.30E-10 1.73E-09 11.21977782

593 BCKDHA 0.079824619 5.779109943 0.928442663 0.353582178 0.386300753 -7.802215369

594 BCKDHB -0.07226825 3.684809521 -0.840701212 0.400877901 0.433848221 -7.671861771

1629 DBT 0.164670241 5.022387251 2.257039152 0.024394191 0.030722493 -5.64125545

1738 DLD 0.472083711 6.473703967 5.931205524 5.30E-09 1.19E-08 8.827576396

35 ACADS -0.688906151 4.141312537 -8.68132614 4.38E-17 1.54E-16 27.21097897

34 ACADM -0.371239168 5.144530653 -4.691191107 3.43E-06 6.16E-06 2.582944004

3712 IVD -0.645242161 5.791703655 -6.78309739 3.03E-11 7.80E-11 13.8768321

36 ACADSB -0.937195974 4.796808291 -8.847962164 1.19E-17 4.34E-17 28.43082989

27034 ACAD8 -0.2502558 4.257898037 -3.025178338 0.002600091 0.003600605 -3.58739773

3030 HADHA -0.048020113 7.710428249 -0.821136923 0.411921353 0.444919058 -7.911004339

1962 EHHADH 1.034244319 4.674421838 8.219699856 1.45E-15 4.76E-15 23.86230287

1892 ECHS1 0.451173254 6.698471514 5.523701135 5.11E-08 1.08E-07 6.617537985

3033 HADH 0.009166245 5.628533204 0.10419606 0.917051457 0.925609627 -8.224064291

3028 HSD17B10 0.868935793 6.207759135 10.67223442 2.61E-24 1.33E-23 43.62216891

30 ACAA1 -0.833287959 5.297952201 -12.55693223 5.29E-32 3.90E-31 61.21244863

10449 ACAA2 -1.598338832 4.859858995 -11.46266038 1.91E-27 1.13E-26 50.77961214

3032 HADHB 0.085773364 6.782436419 1.478288742 0.1398986 0.16181226 -7.160907287

5095 PCCA -0.633654611 3.330750953 -7.456879784 3.44E-13 9.94E-13 18.47590952

5096 PCCB 0.842988114 6.090580657 9.092035539 1.72E-18 6.55E-18 30.34813146

84693 MCEE -0.141246973 2.454128328 -1.802403963 0.072025057 0.086354068 -6.183095022

4594 MUT 0.09017852 5.215313953 1.228996733 0.219594598 0.247453796 -7.445942964

26275 HIBCH 0.259467139 4.413522371 2.712491386 0.006885437 0.009149068 -4.440336234

11112 HIBADH 0.331576682 5.35150903 4.126505311 4.25E-05 6.89E-05 0.198328863

4329 ALDH6A1 -0.883081122 3.325422375 -9.281187504 3.75E-19 1.48E-18 31.99751141

217 ALDH2 -2.311539127 7.018829786 -17.05700482 9.50E-53 1.60E-51 108.8074819

224 ALDH3A2 -0.459840083 7.056178264 -3.137152311 0.001796571 0.002524887 -3.367434368

219 ALDH1B1 0.740029643 5.111153608 6.320069534 5.38E-10 1.29E-09 11.16559328

501 ALDH7A1 -0.906626359 4.440622329 -4.201445115 3.09E-05 5.08E-05 0.499215659

223 ALDH9A1 -0.10132745 6.470811855 -1.225070269 0.221069152 0.249042786 -7.501370219

316 AOX1 -3.443645528 1.534859041 -23.28947731 3.58E-84 1.74E-82 181.1221231

18 ABAT -0.815746782 3.178580439 -5.589189087 3.58E-08 7.66E-08 7.171077491

56922 MCCC1 0.554866022 5.692969451 4.016982659 6.71E-05 0.000107042 -0.247020887

64087 MCCC2 0.456443463 6.070667953 5.888844777 6.75E-09 1.51E-08 8.60376602

35

549 AUH -0.377647125 3.065586136 -5.361194814 1.22E-07 2.51E-07 6.064739271

3155 HMGCL -0.344838377 5.090438162 -4.626389578 4.64E-06 8.23E-06 2.296688655

5019 OXCT1 0.916244765 5.228583589 6.940823736 1.09E-11 2.90E-11 14.98423096

64064 OXCT2 0.136104799 -1.86673463 0.658902471 0.510232056 0.543357378 -7.109385516

39 ACAT2 0.588374543 5.528288775 4.66015788 3.96E-06 7.08E-06 2.469036769

38 ACAT1 -0.93819422 5.172873981 -11.63615179 3.76E-28 2.31E-27 52.39608243

3157 HMGCS1 0.255430691 7.291842146 1.752511445 0.080238867 0.095618141 -6.719621068

3158 HMGCS2 -4.265592639 -3.501736693 -10.30012009 6.98E-23 3.32E-22 40.98001202

36

Table S7. Metabolite Fragments Used for Isotope Quantification in GC/MS analysis Metabolite Carbons Formula m/z Leu 123456 C14H32O2NSi2 302 Ile 123456 C14H32O2NSi2 302 Val 12345 C13H30O2NSi2 288 Ala 123 C11H26O2NSi2 260 Asp 1234 C18H40O3NSi3 418 Glu 12345 C19H42O4NSi3 432 Lys 123456 C20H47O2N2Si3 431 Citrate 123456 C26H55O7Si4 591

37

Table S8. qRT-PCR Primer Sets Gene Forward (5’ à 3’) Reverse (5’ à 3’) Slc7a5 ATATCACGCTGCTCAACGGTG CTCCAGCATGTAGGCGTAGTC Bcat1 TCGGGGAGTTGATAACAAGATCC GGTCCTCACAGCAGATCGG Bcat2 AAAGCATACAAAGGTGGAGACC CGTAGAGGCTCGTTCCGTTG Bckdha CTCCTGTTGGGACGATCTGG CATTGGGCTGGATGAACTCAA Bckdhb AGTGCCCTGGATAACTCATTAGC GCATCGGAAGACTCCACCAAA Acadsb CCCAACCTGCTTGTCTCCTTG ATCCCTGGATCACCGATTTCT Pcca TTCATACCAATGCCTAGTGGTGT GACAGCCTCATCCGCCATTTT Auh AGCTGGCTCTAGCGTGTGA GTTCTGTTCTAACACATGGCTGA Hadhb ACTACATCAAAATGGGCTCTCAG AGCAGAAATGGAATGCGGACC Glut1 CAGTTCGGCTATAACACTGGTG GCCCCCGACAGAGAAGATG Eno1 TGCGTCCACTGGCATCTAC CAGAGCAGGCGCAATAGTTTTA 18s* CGCTTCCTTACCTGGTTGAT GAGCGACCAAAGGAACCATA Rpl13a* GGGCAGGTTCTGGTATTGGAT GGCTCGGAAATGGTAGGGG Gapdh* AGGTCGGTGTGAACGGATTTG TGTAGACCATGTAGTTGAGGTCA β-actin* GGCTGTATTCCCCTCCATCG CCAGTTGGTAACAATGCCATGT Rplp0* AGATTCGGGATATGCTGTTGGC TCGGGTCCTAGACCAGTGTTC B2m* TTCTGGTGCTTGTCTCACTGA CAGTATGTTCGGCTTCCCATTC Hprt* TCAGTCAACGGGGGACATAAA GGGGCTGTACTGCTTAACCAG Pgk1* ATGTCGCTTTCCAACAAGCTG GCTCCATTGTCCAAGCAGAAT * endogenous controls

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http://dx.doi.org/10.1038/nm.3217


http://dx.doi.org/10.1016/S0149-2918(05)80001-3


http://dx.doi.org/10.1016/j.ymben.2007.01.003


http://dx.doi.org/10.1002/(SICI)1096-9888(199603)31:3%3c255::AID-JMS290%3e3.0.CO;2-3

http://dx.doi.org/10.1002/(SICI)1096-9888(199603)31:3%3c255::AID-JMS290%3e3.0.CO;2-3




http://dx.doi.org/10.1186/gb-2002-3-7-research0034


http://dx.doi.org/10.1038/nbt0604-656b


http://dx.doi.org/10.2202/1544-6115.1027


http://dx.doi.org/10.1093/nar/gkp896



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