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The handle http://hdl.handle.net/1887/20983 holds various files of this Leiden University dissertation.

Author: Stechow, Louise von Title: Delineating the DNA damage response using systems biology approaches Issue Date: 2013-06-20 Identification of metabolic pathways in the DNA damage response in pluripotent stem cells

manuscript submitted Louise von Stechow*, Ainhoa Ruiz-Aracama*, Bob van de Water, Ad Peijnenburg, Erik HJ Danen, Arjen Lommen Identification of metabolic pathways in the DNA damage response in pluripotent stem cells

ABSTRACT

DNA damage triggers the activation of an orchestrated signaling network to arrest the cell cycle and repair the lesions or, in case of damage beyond repair, eliminate affected cells. Failure to properly balance the various aspects of this DNA damage response (DDR) in stem cells contributes to ageing and cancer. We and others have shown that treatment of pluripotent stem cells with chemotherapeutic agents, such as cisplatin causes a plethora of transcriptional and post-translational alterations that determine the outcome of the DDR. Here, we performed metabolic profiling by mass spectrometry of embryonic stem (ES) cells treated for different time periods with cisplatin. Integration of metabolomics with transcriptomics data connected cisplatin-regulated metabolites with regulated metabolic enzymes and identified enriched metabolic pathways. These included nucleotide metabolism, urea cycle and arginine and proline metabolism, and a group of metabolic pathways that clustered around the metabolite S-adenosylmethionine, which is a hub for methylation and transsulfuration reactions and polyamine metabolism. A number of the differentially regulated metabolic enzymes were identified as target genes of the transcription factor p53, pointing to p53-mediated alterations in metabolism in response to genotoxic stress. Altogether, our findings reveal interconnecting metabolic pathways that serve as signaling modules in the DDR.

129 INTRODUCTION

Metabolic changes are associated with a number of complex diseases, including cancer, diabetes and neurological disorders. Often, changes in the abundance of small metabolites are linked to changes in the expression or activity of metabolic enzymes or the complete rewiring of metabolic pathways, as seen for cancer cells, which frequently switch their energy production to aerobic glycolysis (known as Warburg effect) and develop a glutamine addiction 1; 2; 3. Indeed, mutations in a number of metabolic enzymes were recently related to inherited cancer syndromes 3. This link between metabolism and disease suggests that metabolomics may be used to identify biomarkers suitable for non-invasive methods to determine disease state, treatment and toxic responses 4. Changes in metabolism may be linked to stress responses, such as genotoxic stress. Irradiation or chemotherapeutic treatment alters the abundance of metabolites, including for example choline-containing compounds, lipids and several amino acids in cancer cell lines 5; 6. Interestingly, metabolites excreted by cancer-associated stromal cells can modulate chemosensitivity of cancer cells in a paracrine manner 7. Recently, the NCI60 panel of tumor cells lines was used to correlate treatment response to platinum drugs with baseline metabolic pathways extracted from metabolomics and transcriptomics 8. However, integrated approaches aimed at unraveling perturbation of metabolic pathways in response to therapy are currently lacking. The ability of cells to recognize and respond to DNA damage is of vital importance for the maintenance of an intact genome. The DNA crosslinking drug cisplatin is used as common treatment for various solid tumors, e.g. ovarian, non- small-cell lung, head and neck, bladder, colorectal and testicular cancer. Despite initial good responses to therapy, patients often develop resistance to cisplatin treatment and toxicity to healthy tissues (including neuro- and renal as well as gastric toxicity) limits the therapeutic window 9. Next to direct DNA damage, cisplatin also induces non-genotoxic perturbations, such as oxidative stress by shifting the redox balance through binding to nucleophilic molecules; and ER stress, which has been shown to kill enucleated cells 10; 11. Cancer cells typically have disabled crucial DDR signaling routes and often rewire metabolic pathways 3; 12. We recently unraveled the DDR signaling network in mouse embryonic stem (ES) cells through integration of functional genomics, phosphoproteomics, and transcriptomics 13; 14. ES cells show robust DNA damage- induced apoptosis, but have several features that can be extrapolated to cancer cells, such as the lack of G1/S-checkpoint after DNA damage, expression of marker genes (e.g. c-Myc), and a high proliferation rate 15; 16. Here, we integrated metabolomics and transcriptomics datasets to explore alterations in metabolic pathways in response to genotoxic stress in pluripotent stem cells.

130 Identification of metabolic pathways in the DNA damage response in pluripotent stem cells

RESULTS

Cisplatin-induced changes on the metabolome of embryonic stem cells General considerations - To explore intracellular metabolic changes in response to genotoxic stress in pluripotent stem cells, ES cells were treated with a sub-lethal dose of 5 μM cisplatin for 4 h and 8 h and lysates were prepared for metabolomics analysis (Fig S1A). At 8 h cells began to accumulate in the S/G2 phase of the cell cycle but viability was not significantly affected at these early timepoints while analysis of parallel control plates at later time points confirmed induction of apoptosis by this concentration of cisplatin (Fig Sfig1B-D). Apolar and polar fractions were collected and used for 1H-NMR and U-HPLC-Orbitrap-MS analysis, respectively. To assess whether data normalization was required to correct for potential differences in cell numbers 1H-NMR data on the apolar fraction of control and cisplatin treated ES cells at both time points were analyzed. Since no significant differences (ANOVA p<0.01) in phospholipid content or other apolar metabolites were detected (examples of overlaid spectra are given in Fig S2), normalization of metabolite data to correct for cell numbers was not required. Finally, PCA demonstrated excellent reproducibility for all 5 biological replicates within each treatment and exposure time group (Fig S3).

Identification of metabolites significantly affected by cisplatin - Polar fractions of control- and cisplatin-treated samples were compared for each time point independently. Masses were identified that significantly contributed to the observed separation between treatments at the different time points (Table S1). At 4 h, metabolites that were differentially regulated between control and cisplatin treated samples were mainly involved in methionine degradation pathways (including transmethylation, transsulfuration/glutathione synthesis), as well as polyamine synthesis and catabolism, urea cycle, proline and arginine metabolism, and nucleotide metabolism (Fig 1A). Furthermore, we detected increased levels of the metabolite N-acetyl-aspartyl- glutamic acid (NAAG), a common neuropeptide and its precursor N-acetyl-L-aspartate (NAA) 23 (Fig 1A). After 8 h of cisplatin treatment, levels of reduced glutathione and proline remained increased, while other differentially regulated metabolites were mainly involved in nucleotide metabolism (Fig 1 B, Table S1).

Expression of metabolic enzymes significantly affected by cisplatin In parallel to metabolomics, transcriptomics analysis was performed to determine cisplatin-induced changes in metabolic enzymes at 8 h. Cytoscape and IPA-based pathway analysis led to the identification of a list of 144 metabolism-related enzymes (Table S2, Fig 2A). A large proportion of these metabolic enzymes were involved in lipid metabolism; inositol phosphate metabolism (mostly myo-Inositol), glycerophospholipid and sphingolipid metabolism. We furthermore detected changes in the mRNA levels of metabolic enzymes that are involved in sugar and fatty acid metabolism (Fig 2A). Next to those, a number of differentially-regulated metabolic enzymes correlated with the

131 A cisplatin 4 h vs. control 4 h N-Acetylputrescine Carnitine

Methylation Proline Urea 4’MC 3’MC Putrescine Adenine production Polyamine Ornithine breakdown Arginine Methionine S-Adenosylmethionine pathway Carbamoyl phosphate Methylation Pyrimidine Remethylation S-Adenosylhomocysteine Methylthio synthesis THF pathway adenosine Spermine UMP Homocysteine hydrolysis GSH red/ox

Cystathione Adenosine CMP dCMP AMP Adenosine hydrolysis GTP Adenine Inosine dTMP Purine Recycling synthesis dUMP Cysteine bases Guanine Guanosine Remethylation Homoserine Hypoxanthine IMP THF pathway Uracil Uridine ATP GSH-ox GSH-red Cytosine Cytidine GMP B cisplatin 8 h vs. control 8 h N-Acetylputrescine Carnitine

Methylation Proline Urea 4’MC 3’MC Putrescine Adenine production Polyamine Ornithine breakdown Arginine Methionine S-Adenosylmethionine pathway Carbamoyl phosphate Methylation Pyrimidine Remethylation S-Adenosylhomocysteine Methylthio synthesis THF pathway adenosine Spermine UMP Homocysteine hydrolysis GSH red/ox

Cystathione Adenosine CMP dCMP AMP Adenosine hydrolysis Inosine GTP Adenine dTMP Purine Recycling Cysteine synthesis dUMP bases Guanine Guanosine Remethylation Homoserine Hypoxanthine IMP THF pathway Uracil Uridine ATP GSH-ox GSH-red Cytosine Cytidine GMP

Figure 1. Network of differentially regulated metabolites. Red, Blue and Grey boxes indicate metabolites (mean of 5 replicates) upregulated, downregulated, and unchanged in cisplatin treatment, respectively (ANOVA p<0.01 and fold change 1.1); metabolites without a box were not detected. (A) Results after 4 h. (B) Results after 8 h.

metabolic pathways, which had been identified based on the changes in metabolite levels, including urea cycle and arginine/ proline metabolism, polyamine metabolism and nucleotide metabolism (Fig 2A). Interestingly, several of the cisplatin-regulated metabolic enzymes were identified as target genes of the transcription factor p53 (Fig 2B). p53 target genes implicated in lipid metabolism were commonly suppressed while p53 target genes encoding enzymes functioning in amino acid or nucleotide metabolism were mostly enhanced.

132 Identification of metabolic pathways in the DNA damage response in pluripotent stem cells

A B Nucleotide Fatty acid -5 0 5 -5 0 5 metabolism metabolism Lipid Vitamin K metabolism VKO R C 1 BD H 2 metabolism Biopterin metabolism SPR H SD 17B12 PTS AC AD S Porphyrin metabolism FEC H SLC 27A2 Oxidative phosphorylation N D U FB2 FAR S2 ITPK1 FAR SB IP6K1 FAH PLC B4 G R H PR PTPN 13 C ER C AM Amino acid PLC G 2 PLO D 3 metabolism M D P1 W H SC 1L1 PLC B1 SM YD 3 PTEN M LL3 Inositol Ppp3ca SEPH S1 metabolism Methyltransferases PLC H 1 IVD Sugar metabolism PIP4K2A PC C A PPM 1F H M G C L AG PAT5 PC M T1 ABH D 5 TPM T Lclat1 C BS Transsulfuration pathway PIK3C B G STO 1 LPIN 1 ASN S PPAP2B PR O D H ELO VL2 PYC R 2 ELO VL6 ALD H 4A1 Urea cycle M BO AT2 G PT Arginine/ Proline PAFAH 2 Glycerophospholipid G LS metabolism metabolism D G KZ PAR S2 D G KA G ATM Sphingolipid D G KH AD C metabolism PAFAH 1B1 SM S Polyamine metabolism Amino acid C D S2 SM O X metabolism PC YT1B M TH FD 1L G PD 2 FPG S Folate metabolism SG PP1 D H FR SM PD 4 ATP2B1 G BA ATP11C B4G ALT5 AD K G ALN T7 PD E3B N-glycan ST6G AL1 ATP10A metabolism FU T8 N PR 2 O-glycan STT3B APR T metabolism D PM 2 AM PD 2 G ALN T1 AK1 Propanoate metabolism M C EE AD A M LYC D PO LA1 Proteoglycan biosynthesis D SE PO LR 3A EXT1 PO LR 3B Purine metabolism D EC R 2 PO LB C PT1C PO LR 3B AC AT2 PO LR 3K M EC R PO LE3 M C AT PO LL EPH X1 Fatty acid PO LR 2D metabolism C BR 3 PO LR 1A O XC T1 PO LR 2G EC H D C 2 R R M 2 AAC S C AN T1 Pyrimidine metabolism PM VK N M E3 Bile acid AC AA2 EN TPD 5 metabolism C YP27A1 EN TPD 6 N AN S N M E4 G N E N T5C SO R D EN PP3 PM M 1 U C K2 FU K TK1 Sugar metabolism PFKP U M PS FBP2 TPK1 Pentose phosphate G AA G R K5 pathway PR PS2 Apobec1 PG LS Prkcd

Figure 2. Differentially regulated metabolic enzymes. (A) Heatmap indicating metabolic enzymes obtained from Cytoscape metabolic signaling network (FigS 4, highlighted in blue; TableS 2), differentially regulated after 8 h of cisplatin treatment and enriched metabolic pathways within the dataset. (B) Regulation of metabolic enzymes by the transcription factor p53 obtained with Ingenuity pathway analysis.

Identification of affected metabolic pathways through integration of metabolomics and transcriptomics Identified changes in metabolites and metabolic enzymes were combined to derive integrated signaling networks. For this, 144 regulated enzymes and 35 regulated metabolites were imported in IPA and Metscape to form an integrated metabolic signaling network (Fig 3; Fig S4). Clusters of significantly enriched metabolic pathways were identified based on the criteria [>3 affected molecules including at least1 affected metabolite and 1 affected enzyme]. Lipid metabolism, despite the observed changes in expression of several enzymes in this process (Fig 2A; Table S2), was not selected due to the absence of information on affected metabolites. The sample

133 A Nucleotide metabolism Metabolomics data Polyamine metabolism

Identification of SAMe Integrated metabolic common canonical pathways Transcriptomics data signaling network pathways Urea cycle, Prol. & Arg. metabolism

B

Pyrimidine metabolism

Polyamine metabolism Glyc Cys/Meth Degradation metabolism

Urea cycle Arg/Prol

tRNA Purine charging metabolism

Figure 3. Integrated signaling network. (A) Schematic representation of metabolomics and transcriptomics data integration leading to identification of common signaling networks related to nucleotide metabolism, SAMe pathways, polyamine pathways and urea cycle, and arginine & proline metabolism. (B) Integrated signaling network of metabolic enzymes and metabolites obtained with Ingenuity pathway analysis. Clusters of significantly enriched canonical pathways and related enzymes and metabolites are highlighted. Upregulated enzymes in green, downregulated enzymes in red. Upregulated metabolites in dark red, downregulated metabolites in blue.

134 Identification of metabolic pathways in the DNA damage response in pluripotent stem cells analysis methodology (NMR) used here for the apolar (lipid) fraction does not have the required resolution for detection of metabolites on the individual species level (such as for instance PIP3 etc). Networks included a purine and a pyrimidine metabolism cluster, a cluster of S-adenosylmethionine (SAMe)-related pathways, a polyamine synthesis cluster, and a urea cycle cluster, featuring pathways related to the metabolism of proline, arginine and citrulline (Fig 3, 4, 5, Fig S5, 6). At 4 h of cisplatin treatment many metabolites linked to SAMe-related pathways were increased (Fig 1,3,4). SAMe is the methyl donor in methylation of DNA, RNA and methylation of lysine during biosynthesis of carnitine 24. We detected cisplatin-induced changes in the levels of the RNA nucleosides 3-methylcytidine, 4-methylcytidine, and of carnitine, as well as changes in mRNA levels of the methyltransferases MLL3, PCMT1 and TPMT (Fig 2A, Fig 3). We reinvestigated the list of differentially regulated genes and identified regulation of several other methylation enzymes that had been missed by IPA and Metscape analysis, including histone methyltransferases and demethlyases as well as RNA methyltransferases (Fig S7A). This additional list included the RNA methyltransferase METLL6, which has been correlated to cisplatin sensitivity in lung cancer patients 25. SAMe is a critical hub between trans-sulfuration, polyamine synthesis, and the folate cycle 24. Multiple enzymes and metabolites in these pathways were significantly regulated by cisplatin (Fig 4). This included an increase in cystathionine- beta-synthase (CBS), an enzyme that is critical for the conversion of homocysteine to

POLYAMINE SYNTHESIS

Glutamate Methylthioadenosine 7,8 DHP THF L-Glutamate Spermidine MATs FPGS FPGS Putrescine Methionine DHF DHFR THF

SMS dTMP S-adenosylmethionine Methylthioadenosine

MS methylated substrate Methyltransferases see dUMP e.g. DNA/ RNA Spermine M THFD1L FigS 7 Methyl 5,10-CH2- Fig 2A transferase SMOX THF POLYAMINE CATABOLISM methyl acceptor 5-MTHF TRANSMETHYLATION

10-CHO-THF Homocysteine S-adenosylhomocysteine FOLATE CYCLE CBS PURINE SYNTHESIS TRANSSULFURATION Purine metabolism see Cystathione FigS 5 Fig 2A Homoserine GSH ox Cysteine GSH GSTO

GSH red

Figure 4. SAMe centered pathways. Upregulated enzymes in green, downregulated enzymes in red. Upregulated metabolites in dark red, downregulated metabolites in blue.

135 cystathione 24. Cystathione is a precursor for glutathione and levels of oxidized and reduced glutathione were increased after 4 h of cisplatin treatment, while after 8 h only the reduced form persisted (Fig 1, Table S1). Enzymes related to tetrahydrofolate (THF) synthesis, which is a crucial part of the folate cycle were also affected. THF is not only involved in transmethylation, but is also crucial to pyrimidine and purine synthesis. This included upregulation of FPGS and DHFR, which are directly involved in THF synthesis, and downregulation of MTHFD1L, which is involved in the ATP/ADP-dependent interconversion of 10-formyl-THF (needed for purine synthesis) (Fig 4). A cluster of reactions related to polyamine synthesis and catabolism were identified based on cisplatin-regulation of enzymes and metabolites (Fig 4,5). After 4 h of cisplatin treatment we identified increased levels of the polyamine metabolites spermine and its precursor putrescine. At 8 h the concentrations were normalized again, while at the same time spermine synthase (SMS) mRNA levels were reduced and spermine oxidase (SMOX) mRNA levels were increased. In addition, changes in metabolites and enzymes related to urea cycle and proline/ arginine metabolism were identified (Fig 1,5). Arginine catabolism appeared to be enhanced even though we did not observe transcriptional changes in superoxide dismutase or NO-synthase. ADC was found to be strongly increased, potentially resulting in a higher conversion of arginine to agmatine, which itself is an inhibitor of NO-synthase. Moreover, expression of GATM, which forms a creatine precursor from arginine and glycine was enhanced (Fig 5) 26. Enhanced levels

Prolyl tRNA PARS2 biosynthesis Proline PRODH

PYCR2 P5C ALDH4A1 P5C Glutamate

Ornithine Ornithine Carbamoyl Carbamoyl phosphate phosphate Urea

Citrulline Mitochondrion Nucleus Putrescine POLYAMINE SYNTHESIS Polyamine metabolism see Fig 4 Cytoplasm ADC

Glycine Arginine Argininesuccinate UMP

GATM CITRIC ACID CYCLE PYRIMIDINE SYNTHESIS Pyrimidine metabolism see Creatine FigS 6 Fig 2A

Figure 5. Urea cycle and the metabolism of arginine and proline. Upregulated enzymes in green, downregulated enzymes in red. Upregulated metabolites in dark red, downregulated metabolites in blue.

136 Identification of metabolic pathways in the DNA damage response in pluripotent stem cells of metabolic products of arginine catabolism such as urea, putrescine (and spermine) and proline were also detected 26 (Fig 5). Next to increased proline levels at 4 h and 8 h of treatment enzymes involved in reduction of P5C to proline (PYCR2), as well as proline breakdown (PRODH, Aldh4a1) were up-regulated 27. Notably, IPA analysis identified PRODH, Aldh4a1, and GATM as known p53 target genes 28; 29 (Fig 2A). Since many of the enzymes and metabolites have been previously implicated in anti- or prooxidant function (e.g. glutathione, methionine, proline, PRODH, SMOX) we tested whether cisplatin treatment, at the time points of our analysis, led to an increase in reactive oxygen species (ROS) formation. However, while hydrogen peroxide led to a strong increase in ROS levels, no significant increase in intracellular ROS levels were detected by cisplatin treatment, at any of the studied time points (FigS 7B). Lastly, several metabolites and metabolic enzymes involved in purine/pyrimidine metabolism were significantly regulated by cisplatin treatment (Fig 1,2,3; Fig S5,6). These were implicated in de novo synthesis and salvage pathways (Fig S5,6) and included a group of enzymes encoded by genes that were identified in IPA as p53 targets. For instance, DNA polymerase β and RRM2 were upregulated while the catalytic subunit of the replicative B-family DNA polymerase α was downregulated 30; 31 (Fig 2B).

DISCUSSION

The DDR activates, in a damage-specific fashion, the appropriate enzyme complexes dedicated to the repair of a variety of DNA lesions. It is critical that the DDR is integrated with ongoing cellular activities, including cell cycle progression, transcription, and translation. In the case of severe damage it is important to arrest the cell cycle, prevent aberrant transcription and translation, preserve energy, and, if damage is beyond repair, to activate cell death mechanisms. This is particularly relevant in stem cells where defective DDR signaling can have major impacts on ageing and cancer 32; 33. Clearly, the DDR extends into all vital cellular processes. Our current study identifies alterations in metabolic pathways that are likewise controlled by the DDR in pluripotent stem cells. It must be noted that the metabolomics profiles generated here provide a snapshot of the total dynamic regulation of metabolism under standard and stressed conditions This is for instance due to technical limitations that exclude certain metabolites from the analysis, the labile nature of some metabolites, and the absence of information on flux. Nevertheless, the strong reproducibility of the data (typically a bottleneck in metabolomics) and the connections with affected metabolic enzymes provide high- confidence data on the metabolic response of ES cells to genotoxic insult. Interestingly, a number of metabolic enzymes we find to be transcriptionally modified by cisplatin are target genes of the key DDR transcriptional regulator, p53. This is in agreement with our recent finding that p53 is a major DDR signaling hub in ES cells 13. Moreover, the integration of transcriptionally regulated metabolic enzymes and significantly affected

137 metabolites allows identification of integrated metabolic networks that are responsive to genotoxic stress. These cluster in pathways centered on purine/pyrimidine metabolism, SAMe-related pathways, polyamine synthesis, and the urea cycle.

Changes in methylation pathways Our analysis shows an increase in SAMe-related metabolites in response to cisplatin treatment. SAMe is formed from methionine and ATP with the help of methionine adenosyltransferases (MATs) and functions as a general methyl donor in almost all cellular methylation reactions 24. Methylation patterns (especially those of DNA) change during embryonic development and neoplastic transformation and can be affected by genotoxic stress 34. RNA-methylation has been associated with structural features, RNA stability and function (e.g. tRNA codon specificity) 35. Mammalian cells lack the necessary kinases to phosphorylate modified nucleosides into nucleoside triphosphate, which prevents their recycling and incorporation in mRNA 36. Instead, modified nucleosides are excreted. Enhanced urinary levels of modified RNA nucleosides, including 3-methylcytidine have been suggested as a potential biomarker for certain cancers 37; 38. We detect changes in the expression of RNA- but not DNA-methyl transferases and decreased levels of methylated RNA nucleosides in response to cisplatin treatment, including 3- and 4-methylcytidine.

Changes in transsulfuration pathways and folate cycle After methyl transfer SAMe is converted into S-adenosylhomocysteine (SAH), which in turn is hydrolyzed to form adenosine and homocysteine. Homocysteine, can be either reduced to cysteine, which functions as a crucial precursor for glutathione (Transsulfuration pathway), or be remethylated to methionine (Folate cycle) 24. Our integrated metabolic network associated with SAMe points to changes in these pathways. Glutathione - a cytoprotective compound - has been reported to chelate cisplatin and to play a role in copper transporter mediated cisplatin efflux. Furthermore, glutathione has a function in cellular redox regulation and can act as a protective agent against cisplatin-induced oxidative stress 39; 40; 41. In accordance with this, GSTO1, a member of the highly conserved omega class of glutathione transferases with dehydroascorbate reductase activity, which has been implicated in resistance to various genotoxic drugs and irradiation, shows increased levels after cisplatin treatment 42; 43. In addition, cisplatin treatment causes changes in the levels of enzymes associated with folate synthesis and the folate cycle although altered levels of relevant metabolites are not seen. Expression of DHFR and FGPS is upregulated whereas MTHFD1L is decreased, which could provide a connection to the observed changes in purine metabolism through ATP/ADP- dependent interconversion of 10-formyl-THF 44. Interestingly, antifolates, such as the DHFR interactor methotrexate are used as antineoplastic agents 45.

138 Identification of metabolic pathways in the DNA damage response in pluripotent stem cells

Changes in polyamine related pathways SAMe serves as a precursor for elongation of putrescine to spermidine and from there to spermine 24. Both putrescine and spermine levels are enhanced in cisplatin-treated cells at 4 h. The increase of putrescine, which can be derived from ornithine, could also be explained by enhanced expression of ADC, an arginine decarboxylase that converts arginine to agmatine, a precursor of putrescine. Spermine is the substrate for SMOX, an enzyme that is upregulated at 8 h by cisplatin and catalyzes the breakdown of spermine to spermidine, 3-aminopropanal, and H2O2. Platinum drugs have already been shown to regulate enzymes involved in polyamine catabolism, including spermine N1-acetyltransferase (which is not seen in our study) and SMOX 46; 47.

Moreover, SMOX-produced H2O2 is considered a major source of oxidative stress after induction of polyamine catabolism 47. Therefore it appears that the initially increased levels of polyamines could later be levelled out by oxidation potentially leading to an increased production of H2O2.

Changes in proline/ arginine metabolism and urea cycle We show that cisplatin induces substantial changes in metabolites and enzymes related to urea cycle and proline/ arginine metabolism including an apparent increase in arginine catabolism. Arginine, proline, glutamate and ornithine are all interconvertable provided that glutamine is available as a precursor of carbamoyl phosphate. This metabolism is the basis for synthesis of nitrogen-containing compounds such as ureum, putrescine, agmatine, creatine and even nitrogen oxide and is furthermore crucial for pyrimidine synthesis. Interestingly, changes in pyrimidine metabolites are seen in response to cisplatin treatment, including an increase in the pyrimidine synthesis precursor, UMP 26; 48.

Effects of cisplatin on purine/pyrimidine/nucleotide metabolism Cisplatin affects DNA replication and transcription by causing inter- and intrastrand crosslinks and it can bind free nucleotides. Our integrated networks indicate that pathways potentially involved in DNA metabolism, such as purine/pyrimidine and nucleoside/nucleotide metabolism are significantly increased, including both de novo synthesis and salvage pathways. Notably, interpretation of changes in nucleotide metabolism is complicated by the fact that nucleotides, next to serving as building blocks of DNA and RNA, are involved in a great number of cellular signaling functions, as well as energy metabolism 49. This cluster may be affected by DNA damage in a p53- dependent manner: affected polymerases and enzymes involved in DNA synthesis and repair are regulated by p53.

Possible cisplatin-ROS related metabolism Different studies have reported cisplatin-induced oxidative stress but the relative contribution of this to cisplatin cytotoxicity is unclear. Cisplatin can cause oxidative stress by depleting cellular antioxidant defenses due to its binding to nucleophilic

139 molecules such as glutathione, methionine or cysteine-rich proteins 11. Furthermore, cisplatin-induced (DDR) signaling processes might cause secondary oxidative stress. The cisplatin-regulated metabolic pathways discussed above provide a number of links to generation of oxidative stress. For instance, a generally increased arginine catabolism may lead to nitrogen oxide formation through NO-synthase, which has been associated 47 with ROS, while an increase in SMOX can lead to H2O2 production . Notably, we do not detect elevated ROS at the timepoints and cisplatin concentrations used in our study (whereas H2O2 strongly induces ROS, as expected). This may indicate absence of cisplatin-induced ROS production or effective scavenging of ROS in ES cells. The enhanced levels of glutathione, methionine and proline metabolites may point to the latter explanation and indicate that ES cells cope with cisplatin-induced oxidative stress 41; 50. However, the fact that the ratio reduced versus oxidized glutathione is slightly elevated in cisplatin-treated cells, argues against glutathione-mediated ROS-scavenging.

CONCLUSIONS

By integrating metabolomics and transcriptomics analyses of cisplatin-treated ES cells, we have identified metabolic pathways that are significantly affected by the treatment with this genotoxic compound. Part of this metabolic response is mediated by p53, in agreement with its central role in the DDR. This includes for instance DNA damage repair-related nucleotide metabolism enzymes (e.g. RRM2) and amino acid catabolic enzymes (e.g. PRODH, ALDH4a1, GATM). Changes in individual metabolic enzymes or metabolites have been reported by others to be responsive to genotoxic stress and/ or associated with sensitivity to genotoxic therapy in cancer cells. This holds true for instance for GSTO1, METLL6, PRODH, and SMOX enzymes and metabolites such as carnitine, methionine, and glutathione. However, our current study for the first time provides the signaling networks that integrate these events within the metabolic branch of the DDR.

MATERIAL AND METHODS

Cell culture and materials HM1 mouse ES cells derived from OLA/129 genetic background (provided by Dr. Klaus Willecke, University of Bonn GE) were maintained under feeder free conditions in GMEM medium containing 10% FBS, 5x105 U mouse recombinant leukemia inhibitory factor (LIF; PAA), 25 U/ml penicillin, and 25 µg/ml streptomycin. For metabolomics analysis and micro-arrays ES cells were used at passage 22. Cells were confirmed to be mycoplasma-free using the Mycosensor kit from Stratagene. The DNA cross-

140 Identification of metabolic pathways in the DNA damage response in pluripotent stem cells linkers cisplatin (cisplatin; Cis-PtCl2(NH3)2) was provided by the Pharmacy unit of University Hospital, Leiden NL. Ammonium acetate (NH4Ac), sodium chloride (NaCl) and deuterated chloroform (CDCl3) were obtained from Merck (Darmstadt, Germany); methanol (MeOH) and acetone from Biosolve (Valkenswaard, The Netherlands). All chemicals and solvents were purchased in the highest purity available. Ultra-pure water was obtained using the PureLab equipment from Rossmark (Ede, The Netherlands).

Cell viability, apoptosis and cell cycle analysis To monitor cisplatin induced cell killing, a cell viability assay using ATPlite 1Step kit (Perkin Elmer) was performed according to the manufacturer’s instructions followed by luminescence measurement using a plate reader. For cell cycle and apoptosis analysis cells were exposed to vehicle (PBS) or cisplatin for 8 h or 24 h. Floating and attached cells were pooled and fixed in 80% ethanol overnight. Cells were stained using PBS EDTA containing 7.5 mM propidium iodine and 40 mg/ml RNAseA and measured by flow cytometry (FACSCanto II; Becton Dickinson). The number of cells in the different cell cycle fractions (and in sub G0/G1 for apoptotic cells), as seen in FigS 1 was calculated using the BD FACSDiva software.

Metabolomics – sample preparation HM1 ES cells were treated with 5 μM cisplatin or vehicle control for 4 h and 8 h followed by lysis and fractionation as described 17. Five independent biological replicates were examined in each experiment. Metabolomics analysis was performed on the apolar fraction, containing the membranes and intracellular lipids, and the polar fraction containing the polar and semi-polar intracellular metabolites. In short, the pellet containing the apolar metabolites, was resuspended in 1 M NH4Ac, freeze-dried, extracted with CDCl3, and the organic solvent was evaporated under N2 flow. The dried extract was dissolved in 1 ml of CDCl3, 0.6 ml of which was used for NMR analysis. The supernatant containing the polar intracellular metabolites was freeze-dried, resuspended in 1 ml

MeOH, dried with N2, and re-suspended in 50% MeOH/ 50% H2O ultra-pure water. To remove gelatin derived from the adhesive coating used for the ES cell cultures, this sample was consecutively mixed with cold acetone, incubated for 10 min at 4˚C and centrifuged 15 min at 13,000 x g at 4˚C. The supernatant was collected and dried under nitrogen for further analysis.

Metabolomics – measurements The apolar samples were analyzed by 1H-NMR. The 1H-NMR spectra were recorded at 400.13 MHz at 300.0 (± 0.02) K on a Bruker Avance 400 narrow bore using a 5.0-mm probe. The spectrometer settings were the same as described previously 17. The polar samples were analyzed by ultra-high performance liquid chromatography (U-HPLC)-

Orbitrap-MS. For this the samples were diluted twice with H2O and formic acid was added to a final concentration of 0.1%. The injection sequence was randomized as described 18. U-HPLC was performed on a U-HPLC Accela system (Thermo Fisher Scientific, San

141 Jose, CA, USA), with a 150 mm × 2.1 mm UPLC BEH-C18 column with 1.7 μm particles (Waters). Chromatographic conditions were as described 17. The U-HPLC was directly interfaced to a single stage Orbitrap mass spectrometer (Exactive, Thermo Fisher Scientific). Settings of the Orbitrap mass spectrometer are provided (Suppl. Material 1). Data were recorded using Xcalibur software version 2.1.0.1139 (Thermo Fisher Scientific). When identification of mass peaks was needed, samples were subjected to LC-nanomate-Orbitrap-MS analysis using chromatography conditions as described above. These identification procedures are as previously described17 .

Metabolomics - data analysis NMR data analysis of apolar samples: A method for normalization of data using phospholipid signals in apolar samples was accessed as previously described 17. The NMR data were pre-processed and aligned as described using a for Windows updated version of in-house developed software 19. Based on equal phospholipid content, normalization of data was not required (see Results section). Subsequently, the spreadsheet containing aligned data of the apolar samples was subjected to statistical analysis using Genemaths XT (http://www.applied-maths.com/genemaths/genemaths. htm). Standard initial analysis entailed performing a 2Log transformation and a principal component analysis (PCA) (average of rows and columns subtracted). This was followed by a 2Log transformation, a pre-selection of variables using an ANOVA (p<0.01), followed by a PCA (average of rows and columns subtracted). The grouping in the ANOVA was on the replicates (n=5) per treatment (control and 5 μM cisplatin) for each timepoint (4 and 8 hours) creating 4 groups with 5 replicates or on the treatments, creating 2 groups of 10 replicates.

LC-MS data analysis of polar samples: U-HPLC-Orbitrap-MS data were pre-processed, mass corrected to sub-ppm precision 19 and aligned using MetAlign (http://www. metalign.nl) 20. In short, this software performs a baseline correction, accurate mass calculation, data smoothing, and noise reduction, followed by alignment between chromatograms. Since the NMR data on apolar samples did not indicate a need for normalization (see Results) and the polar samples were derived from the same cell cultures no normalization was applied on U-HPLC-Orbitrap-MS data. The generated spreadsheet of the dataset was subjected to statistical analysis using Genemaths XT. Standard initial analysis entailed performing a 2Log transformation and a PCA (average of rows and columns subtracted). This was followed by a 2Log transformation, a pre- selection of variables using an ANOVA (p<0.01), followed by a PCA (average of rows and columns subtracted). The grouping in the ANOVA was: on the replicates for each time point and treatment (4 groups with 5 replicates), per treatment (2 groups of 10 replicates), on the control samples at both time points (2 groups of 5 replicates), as well as per treatment at each time point (2 groups of 5 replicates at 4 h and 2 groups of 5 replicates at 8 h). The peak loadings responsible for the separation in the different PCAs were selected and exported as described 20. Only those signals with intensity

142 Identification of metabolic pathways in the DNA damage response in pluripotent stem cells higher than 5 times noise and a fold change higher than 1.2 were taken as candidates for further identification.

Identification of metabolites: To facilitate further analysis and identification of the selected signals, GM2MS, an application of MetAlign that re-creates “new chromatograms” only containing the peaks exported from the PCA selection, was used 20. Polar metabolites were afterwards identified with commercially available standards, using previous acquired identification information 17, with FT-MS/MS analysis (using the LC-nanomate- Orbitrap-MS method described above), and using databases such as the HMDB [http:// www.hmdb.ca/] (see TableS 1 for the identified metabolites).

Transcriptomics analysis and integration of metabolomics and transcriptomics data Transcriptional microarray data have been published 13 and are available from ArrayExpress. The 2269 genes whose expression differed significantly (parametric p<0.0005) between control ES cells and ES cells treated for 8 hours with 10 μM cisplatin were used as input in Ingenuity pathway analysis (IPA) or the cytoscape plug-in Metscape to identify metabolic enzymes 21; 22. Transcriptional heatmaps were obtained using Multiple Array Viewer (MEV) software. Differentially expressed genes encoding metabolic enzymes and the metabolites that were differentially regulated at 4 h or 8 h (p<0.01) were imported in IPA and Metscape to form integrated metabolic signaling networks.

ROS formation assay For probing intracellular ROS, cells were cultured in µClear 96 well plates to 70% confluence. Cells were washed twice with PBS and incubated for 1 h with 40µM 5-(and-6)-Carboxy-2’,7’-Dichlorofluorescein Diacetate (DCF-DA, Invitrogen) in phenol- red free culture medium. After washing with PBS, cells were exposed to 5 μM cisplatin or 250 μM Hydrogen peroxide (H2O2) in the presence or absence of 10 mM of the ROS scavenger N-acetylcysteine (NAC). Fluorescence was measured at different time points after exposure using a plate reader.

ACKNOWLEDGEMENTS

This work was supported by the Netherlands Genomics Initiative /Netherlands Organization for Scientific Research (NWO): nr 050-060-510.

143 REFERENCES

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146 Identification of metabolic pathways in the DNA damage response in pluripotent stem cells

SUPPLEMENTARY MATERIALS

A CELL VIABILITY CELL VIABILITY APOPTOSIS APOPTOSIS CELL VIABILITY CELL CYCLE CELL CYCLE

4h 8h 24h

5x control 5x control 3x control

5x 5µM 5x 5µM 3x 5µM

LC-MS LC-MS NMR NMR 3x 10µM

Microarray

B C 4h 120000 8h 24h 100 100000 control *** 80000 5μM 50 60000 *** ns 40000 % sub G1/ G0

cell viability *** 0 20000 8h 24h

0 PBS 5μM 10μM

D

G1 S G2/M G1 S/G2

24h PBS 5μM PI

8h

count

Figure S1. Cisplatin does not lead to cell death at 4 h and 8 h of treatment, but causes cell cycle arrest. (A) Schematic representation of the experiments. (B) Cell viability measured by ATPlite in ES cells after treatment with 5 μM and 10 μM cisplatin at 4 h, 8 h and 24 h of treatment. (C) Apoptosis measured by FACS analysis after 8 h and 24 h of treatment with 5 μM cisplatin. (D) Cell cycle profile after 8 h and 24 h treatment with PBS or 5 μM cisplatin.

147 2

Figure S2. Expanded region between 5.5 and 2.5 ppm of a 1HNMR spectrum of the apolar extract of HM1 ESC after 8 h of exposure to cisplatin (blue) and to vehicle (red). Arrows indicate characteristic phospholipid signals.

Figure S3. Regulation of metabolites in cisplatin and control group at different timepoints. PCA plots of aligned U-HPLC-Orbitrap-MS data after pre-selection using 2Log transformation and an ANOVA (p <0.01), Samples grouped on biological replicates.

148 Identification of metabolic pathways in the DNA damage response in pluripotent stem cells

SelenohomocysteineSelenocystathionine Cystathionine

L-Serine

tRNA(Met) L-Methionyl-tRNA L-Cysteine

CBS Betaine L-Cystathionine N,N-Dimethylglycine homocysteine N-Acetylmethionine thiolactone MARS2 MARS

PON2

ACY1 BHMT2 PON1 BHMT

NAGS PON34-Methylthio-2-oxobutanoic acid MTR IL4I1

L-Methionine L-Homocysteine

Methylarsonate Arsenite DNA adenine Methylarsonite DNA 6-methylaminopurine Protein m7G(5')pppR-RNA Dimethylarsinate C-terminal AHCYL2 ProteinS-farnesyl-L-cysteine MAT2B C-terminalmethyl ester S-farnesyl-L-cysteine AHCY G(5')pppR-RNA MAT2A AHCYL1 Protein MAT1A L-isoaspartate Protein tRNA containing N2-methylguanine L-isoaspartate Peptide AS3MT tRNA containing methyl2-[3-carboxy-3-(methylammonio)propyl]-L-histidine ester tRNA N7-methylguaninetRNA containing N6AMT1 5-methylaminomethyl-2-thiouridylate [Methionine 2-(3-Carboxy-3-aminopropyl)-L-histidine synthase]-cob(II)alaminm7G(5')pppm6Am ICMT RNMT (mRNA PCMT1 containing an C1orf25 [MethionineN6,2'-O-dimethyladenosine TRMT1 METTL1 synthase]-methylcob(I)alamincap) DPH5 TRMU m7G(5')pppAm MTRR METTL3

Histone L-arginine Thiopurine S-methylether TPMTP1 CARM1 S-Adenosyl-L-homocysteineS-Adenosyl-L-methionine Histone-L-lysine EHMT1SUV39H2 Procollagen Histone TPMT 5-hydroxy-L-lysine USP8 N-methyl-L-arginine Thiopurine Protein WHSC1L1 ASH1L N6,N6-dimethyl-L-lysineProteinProtein WHSC1 N6,N6,N6-trimethyl-L-lysineN6-methyl-L-lysineHistone USP32 N6-methyl-L-lysine PLOD2 USP10 USP22 PAN2 NSD1 USP36 USP21 STAMBPL1USP6 SETDB2 SUV39H1SETD1A EHMT2 USP47 SETD8SETDB1 DOT1L CERCAM USP11 USP45 USP43 USP48 USP49 N-Acetylneuraminate SETD7 USP37 SMYD3 USP7 Phosphoenolpyruvate PLOD3 USP1 MLL3 PLOD1 USP44 USP4 SETD2 USP9X USP9Y USP30 USP34 USP3 USP18 USP26 Thiol N-Acyl-D-mannosamine USP16USP13 Ubiquitin USP14 USP28 6-phosphate C-terminal USP41 DNA DNMT3B USP5 USP2 DNMT3A AMDP1 thiolester N-Acylneuraminate 5-methylcytosine USP35 9-phosphate NANS USP46 USP24USP40 USP42 TRDMT1DNMT1 AMD1 LOC647341 Protein lysine USP33 USP31 N-Acetylneuraminate N-Acetyl-D-mannosamine Ubiquitin UBE2K USP38 9-phosphate UDP-N-acetyl-D-mannosamine UBE2D4 USP20 USP25 USP51 UBE2G1 USP12 N-Acetyl-D-mannosamine UBE2E4P CYLDUSP15 6-phosphate USP29 UBE2L3 STAMBP UBE2S UBE3A USP19 UBE2A UBE2L6 UBE2WUBE2Z UBE2H UBE2N GNE UBE2I UBE2E1 UBE2B Queuine CDC34 UBE2E3 tRNA queuine UBE2D1 UBE2E2 ProteinUBE2R2 UBE2C UBE2NLUBE2Q2 UBE2Q1N-ubiquityllysine UBE2J1 UBE2T tRNA guanine UBE2D3 UBA1 UBE2D2 UBE2G2 UBE2U

QTRTD1 S-Adenosylmethioninamine NRF1 QTRT1 UBE2M S-Methyl-5-thio-D-ribose 1-phosphate 1-(5'-Phosphoribosyl)-5-amino-4-imidazolecarboxamide UBE2J2

UDP-N-acetyl-D-glucosamine Pseudouridine 5'-phosphate 5-Amino-4-imidazolecarboxyamide MTAP PDE6D PDE11A PDE9A PDE6B ADCY1 ADCY9 DNA GDA PDE4D PDE1A NPR1 1D-myo-Inositol1D-myo-Inositol PDE7A ADCY4 3,4,5,6-tetrakisphosphate1,3,4,5,6-pentakisphosphate PDE4BNucleoside PDE1B REV3L GUCY1A3 Polynucleotide APRT PDE6H 5'-phosphate POLA2 GUCY2D AMP NPR2 ADCY6 Lysosomal-enzyme PDE4A PDE10A3',5'-Cyclic 1D-myo-Inositol Guanine PDE3B GUCY1B3 5'-Methylthioadenosine D-mannose RPUSD4 XDH nucleotide 3',5'-Cyclic GMP GUCY2C GUCY1B2 5'-Phosphopolynucleotide 1,3,4,6-tetrakisphosphate POLM PDE8A PDE2A POLE4ADCY7 ADCY5GUCY1A2 Lysosomal-enzyme POLE3 ADCY8 PDE7B POLD4 (5')ppPur-mRNA N-acetyl-D-glucosaminyl-phospho-D-mannose Xanthine Adenine PDE6G 3',5'-Cyclic AMP POLQ DNM3 POLB POLL SMS ADCY2 GALNT8 GALNT11 Hypoxanthine PDE4C POLD1 GUCY2F EEF1A1 DNM1 WBSCR17 HPRT1 AMPD3 GFM2 5,6-Dihydrouracil POLG POLN ADCY3 GALNT12 ITPK1 PDE3A RFC5 POLK RNGTT 1-(5'-Phosphoribosyl)-5-formamido-4-imidazolecarboxamide PDE6A PDE5A POLS POLD2 DNM2 G(5')pppPur-mRNASpermidine GALNTL4 DPYD PDE8B IL18BP POLG2POLA1 POLE DNM1L EEF1A2 GALNT6 GALNTL1 GNPTAB AK2 LOC729197 CDPdiacylglycerol ADK PDE1C POLE2 TUFM GALNTL2 GFM1 EFTUD1 EEF2 GALNT3 GALNT4 Uracil ATIC dATP AMPD1 PDE6C TYMP AMPD2 dCTP POLR3D GALNT9 (GalNAc)1 DAD1 POLH dTTP POLI SRM (Ser/Thr)1 RPN1 GMP dGTP TUT1 Deoxyinosine GTP POLRMT UDP-N-acetyl-D-galactosamine GMPRGMPR2 AK3L1 GALNT13 dIDP GALNT1 POLD3 POLR2E GDP POLR2JPOLR1B Protein serine RPN2 POLR3F alk-1-enyl-2-lyso-glycerophosphate NP ITPA POLR3GL CDS2 CDS1 DGUOK plasmenate DCK ITP AK1 POLR1C GALNT10GALNT5 UPP1 IMPDH2 AK7 POLR2C POLR3K 1-Acyl-sn-glycerol LRGUK NME1 UTP 1D-myo-Inositol UPP2 ADA Nucleoside dADP POLR3C REG3A 3-phosphate TAF9 ENTPD1 POLR2I DDOST AGPAT2 1D-myo-Inositol1,3,4,5-tetrakisphosphate IMPDH1 diphosphate dGDP NME5 ZNRD1POLR3G GALNT2 (Glc)3 (GlcNAc)2 AGPAT7 NT5E POLR2G Pyrophosphate PAPOLB (Gal)4 (GalNAc)1 1,3,4-trisphosphate Sugar IMP GUK2 C6orf199 NME7 GALNT14 (Man)9 (Asn)1 1D-myo-Inositol NME6 dITPNME4dUTP CTP POLR2K (Gal)3(Gal)3 (Glc)1 (Glc)1 (Gal)3 (GalNAc)1 1-phosphate XanthosineDeoxyguanosine ENTPD4 ENTPD6 NME2 (Glc)1 (GlcNAc)1 1,4-bisphosphate NME2P1 POLR3APOLR1APOLR3E RNAPOLR3H (GlcNAc)2(GlcNAc)3 (Cer)1 (Glc)1 (GlcNAc)1 AGPAT5 NT5C1B IDP AK5 (Cer)1 AGPAT6 5'-phosphate CANT1 Nucleoside PAPD1 (LFuc)1 (Cer)1 (Gal)3 (Glc)1 (LFuc)1 (Cer)1 DeoxyuridineDeoxyadenosine ENTPD8 POLR2Htriphosphate PAPOLG AGPAT1 AGPAT3 Uridine NT5C1A ENTPD3 NME3 ATP5B Spermine (Gal)3 (Glc)1(GlcNAc)2 dUDP POLR2L ATP5O Type A glycolipid IsoGlobotriasyl(GlcNAc)2 NUDT14 Inosine dAMP ENTPD5 POLR3B POLR2D (GlcNAc)2(LFuc)1 (Cer)1 AGPAT4 NT3 GUK1 RRM1 POLR2F isoglobopentaosyl(Glc)3 (GlcNAc)2(Gal)3(Glc)2(Glc)1 (Glc)1 (GlcNAc)2 (GlcNAc)2 Ethanolamine(Man)4ceramide (PP-Dol)1 Adenosine dTMP POLR2A KATNAL1 PAPOLA (LFuc)3 (Cer)1 phosphate UDP-sugar NT5C2 dGMP C9orf98 RRM2 ATP6V1A (Man)9ceramide(GlcNAc)2 EtNP-Man-Man-(EtNP)Man-GlcN-acyl-PI(PP-Dol)1(Man)9(Man)9 (Cer)1 (PP-Dol)1(Gal)4 (PP-Dol)1 (Glc)1 SKIP INPP5A Xanthosine NT5C3 CDPUDP (Gal)2 (GlcNAc)4 (alpha-D-Mannosyl)4-beta-D-mannosyl-(Gal)3 (GalNAc)1 (GlcN)1(GlcN)1 PIB5PA CDA Thymidine POLR2B POLR1D mannosealpha1-6(ethanolaminephosphate-2)mannosealpha1-4(acyl)glucosaminylphosphatidylinositol(Gal)3 (Glc)1 GALNT7(GlcNAc)3 (Cer)1 POLR1E ATP4BFAM65A ATP8 (LFuc)1 (Man)3 (Glc)1 (Cer)1(Gal)4 (Glc)1(Ino(acyl)-P)1 (Ino(acyl)-P)1 DCTD dCMP Nucleotide ATP6V0C (GlcNAc)1 (Cer)1 Nicotinate dCDP RRM2B ATP2C1 4-Aminobutanal (Neu5Ac)2 (Asn)1 (Gal)3 (Glc)1 (GlcNAc)3B4GALT1(Gal)4 (Cer)1(Man)2 (Glc)1 (EtN)1 INPPL1 INPP5B Cytidine ATP10D ST6GAL1 (Man)1 (EtN)1 APOBEC4 D-ribonucleosideN-Ribosylnicotinamide ATP5IATP5D ATP4A (GlcNAc)1 (GlcNAc)3(P)1 dTDP ATP5L2 B4GALT7 (GlcNAc)2(P)1 DGKG DGKB KATNA1 (Gal)2 (GlcNAc)4 (LFuc)1 (Cer)1 B3GNT6ALG3STT3B(LFuc)1ALG2 (Cer)1 Deoxycytidine (Man)3 (PP-Dol)1 AICDA UMP CMPK1 TCIRG1 ATP6 ATP2A3ATP9A AOC2 MAOA (LFuc)1 (Man)3 B4GALT4 Phosphatidate ATP6V0A1 ATP6V1H spermine N-Acetyl-D-glucosaminyl-1,3-beta-D-galactosyl-1,4-N-acetyl-beta-D-ALG12 DGKQ ATP5A1 EtNP-Man-(EtNP)Man-(EtNP)Man-GlcN-acyl-PI(Asn)1 (Gal)3 (Glc)1 B4GALT3 FUT9 NicotinamideTK2 UCK2 ATP monoaldehyde alpha-mannose-glucosaminyl-acyl-phosphatidylinositol(Gal)4 (GalNAc)1 STT3AB3GNT2GBGT1 DGKI Nicotinate Guanosine ATP1B1 ATP5F1 ATP6V1G3 ABP1 UCK1 SAT2 N4-Acetylaminobutanal (GlcNAc)3(Glc)1 (GlcNAc)2B4GALT5 (Cer)1 B4GALT2 (Gal)4 (Glc)1 DGKA D-ribonucleotideD-ribonucleotide Thioredoxin ATP10AATP6AP1 N-Acetylputrescine (GlcNAc)4 Glucosylceramide CMP ADP ATP11A DYNC2H1 (Gal)3 (GalNAc)1(LFuc)1 (Cer)1 Digalactosylceramide(GlcNAc)3 KIF3B (LFuc)1 (Man)3 DGKD ADSSL1 TK1 Reduced ATP11C AOC3 FUT8 ethanolaminephosphate-2-mannosealpha1-4glucosaminyl-acyl-phosphatidylinositol(Glc)1 (GlcNAc)2 (Gal)3 (Glc)1 GMPS DBH ATP8B2ATP8B4 MAOB (Asn)1 beta-D-Galactosyl-1,4-beta-D-glucosylceramide(LFuc)2 (Cer)1 NT5M DTYMK ATP5J SAT1 N1-Acetylspermine (LFuc)1 (Cer)1 Galactosylceramide(GlcNAc)2isoglobotetraosyl DGKK glutaredoxin ATP2A1 alpha-D-Mannosyl-beta-D-mannosyl-(Gal)3 (GalNAc)1 DGKE NT5C ATP6V1E2 (GlcNAc)4 mannosealpha1-2mannosealpha1-6(ethanolaminephosphate-2)mannosealpha1-4glucosaminyl-acyl-phosphatidylinositolN-Acetyl-D-glucosaminyl-1,3-beta-D-galactosyl-1,4-D-(Gal)3 (Glc)1(LFuc)2 (Cer)1ceramide ADSS ATP6V1D (Glc)1(alpha-D-Mannosyl)8-beta-D-mannosyl- (GlcNAc)1 MOXD1 ATP6V0A2 (Man)3 (Asn)1 (GlcNAc)2 Sulfate ATP6V0E2 ATP1A2 beta-D-Galactosyl-1,4-N-acetyl-beta-D-glucosaminyl-1,3-beta-D-(Cer)1 ATP6V0D1ATP2B1 (LFuc)2 (Cer)1 DGKH ATP5BL2 ATP1A4 beta-D-FructoseSucrose 1,2-Diacyl-sn-glycerol D-myo-Inositol ATP6V1C2ATP6V1E1ATP8A2 (Gal)4 (Glc)1 DGKZ 1-lyso-2-arachidonoyl-phosphatidate Adenosine ATP6V0E1NSF 1,4,5-trisphosphate ATP6V1B2ATP1B2 alpha-Maltose Phosphatidylethanolamine 3',5'-bisphosphate N6-(1,2-Dicarboxyethyl)-AMP ATP8A1 ATP5BL1 ATP6V1G1 SI (GlcNAc)3 ST3GAL4ST3GAL1 ATP1A3 (LFuc)3 (Cer)1 ATP2C2ATP1A1 AGK ST3GAL2 ATP5C2 ATP8B1 ATP6V0A4 ATP6V0BATP5G3 Putrescine Dephospho-CoA dUMP ATP5C1 ATP6V1B1 ATP6V1G2 D-Glucose ENPP3 ATP2B2 ATP5L PLCD4 2-Arachidonoylglycerol ATP5G1 ATP5G2 MGAM PLCD3 ENPP1 ENPP2 ATP1B4 ATP6V0D2 PLCH1 PPAP2C ST6GALNAC2 ATP6V1F ATP8B3 ST6GALNAC4 ENTPD2 N1-Acetylspermidine cis-beta-D-Glucosyl-2-hydroxycinnamate PPAP2A NUDT12 GLA GANC GANAB alpha-D-Glucose ATP9B PAOX Melibiose D-Glucoside GBA PLCB2 PLCB4 ATP2B4 PLCG1 PPAP2B FAD ATP6V1C1 GAA PLCG2 ATP5E GBA3 Ceramide ATP2A2 ATP12A D-Fructose CMP-N-acetylneuraminate ATP5H Cyanoglycoside PLCL2 PLCZ1 PLCH2 UDPglucoseSphingosine1-phosphate ATP5J2 D-Galactose PLCE1 Deamino-NAD+ ATP10B ATP2B3ATP11B cis-2-Hydroxy L-Aspartate Pantetheine ATP1B3 acetamidopropanal cinnamate 4'-phosphate Sphinganine PLCD1 PLCB3 3'-Phosphoadenylyl Sphinganine L-Iditol PLCB1 sulfate Sphingosine1-phosphate alpha-N-Acetylneuraminyl-2,3-beta-D-galactose-1,3-(alpha-N- Cyanohydrin alpha-N-Acetylneuraminyl-2,3-beta-D-galactosyl-1,3-N-acetyl-D- SORD 1-phosphate N-Acylsphingosine FMN SMPD2 TYMS D-Sorbitol SMPD4

1-Phosphatidyl-D-myo-inositol SMPD1 4,5-bisphosphate L-Sorbose ENPP7 PIK3CB Sphingomyelin PIK3C2G INPP4B SMPD3 PIK3CG 1-Phosphatidyl-1D-myo-inositol1-Phosphatidyl-D-myo-inositol Peptide INPP4A PIK3CA TPTE2 L-Glutamine 3-phosphate PIK3C2A AGMAT OXSM PIP5K1A 1-phosphatidyl-myo-inositolPIP5K1B LAP3 NAD+ 5-phosphate DUSP11 PYCR1 PIP5K3 Dihydropteroate PARP14 PIP4K2A P4HA2 1-phosphatidyl-myo-inositol CHMP4A (S)-1-Pyrroline-5-carboxylate PIK3CD UMPS L-erythro-4-Hydroxyglutamate TNKS2 PARP12 PIP4K2C3,5-bisphosphate PAPSS2 PYCRL PIP4K2B L-Proline PARP11 PIK3C2B ODC1 ASNS PIP5K1C PARS2 L-Asparagine Phosphatidylinositol-3,4,5-trisphosphate ITPKA PYCR2 L-4-Hydroxyglutamate PARP1 PARP2 tRNA(Pro) P4HB L-1-Pyrroline-3-hydroxy-5-carboxylate PARP8 semialdehyde D-myo-Inositol Nicotinamide 1-Phosphatidyl-1D-myo-inositol PIK3C3 ITPKB PARP9 3,4-bisphosphate SPHK1 trans-4-Hydroxy-L-proline 4-phosphate Orotidine EPRS PARP16 PARP15 Dihydrofolate 5'-phosphate PRODH ALDH4A1 P4HA1 L-Glutamate GLS L-Prolyl-tRNA(Pro) P4HA3 TNKS 5-semialdehyde FPGS PARP10 PARP3 SPHK2 Orotate D-Glutamate GLS2 PARP4 TIPARP PARP6

ADP-D-ribosyl-acceptor Agmatine Tetrahydrofolyl-[Glu](2) NH3 Dibenzo[a,l]pyrene-11,12-epoxide5,6-DHET D-Glutamine 10-Formyltetrahydrofolylpolyglutamate 5,6-EET 10-Formyltetrahydrofolyl 7,12-Dimethylbenz[a]anthracene-3,4-diol L-glutamate Lipoxin B4 Tetrahydrofolyl-[Glu](n) Benzo[a]pyrene-4,5-diol8,9-EET DHFR 11,12-EETBromobenzene-2,3-dihydrodiol FASN 5(S),6(S)-epoxy-15(S)-hydroxy-7E,9E,11Z,13E-eicosatetraenoic JAK3 5(S),6(S)-epoxy-15(R)-hydroxyeicosatetraenoateDibenzo[a,l]pyrene-11,12-diol PTP4A1 acid anion12(13)-EpOME 8,9-DHET15-epi-lipoxin A5 L-Glutamate 1,2-Dihydronaphthalene-1,2-diol 11,12-DHET 14,15-DHET 10-Formyltetrahydrofolate 9,10-hydroxyoctadec-12(Z)-enoate Tetrahydrofolate EPHA6 7,12-Dimethylbenz[a]anthracene-3,4-oxide CDC25A 15-epi-lipoxin15-epi-lipoxin B5Benzo[a]pyrene-9,10-diol B4 PTK7 15-epi-lipoxin A4 L-Amino acid INSR 1-Nitro-5,6-dihydroxy-dihydronaphthalene Benzo[a]pyrene-7,8-oxide TYK2 FGFR1 Benzo[a]pyrene-2,3-oxide L-Ornithine EPHA1 Benzo[a]pyrene-2,3-diol (5-L-Glutamyl)-L-amino GAD2 5-Phospho-alpha-D-ribose PTPN18 5(S),6(S)-epoxy-15(R)-HEPE Bromobenzene-3,4-dihydrodiol GGT1 1-diphosphate PTPN23 acid ADC Folate ERBB4 PTEN PTPRM EPHX1 GGTL4 (S)-2-Aminobutanoate GAD1 EPHA2 Benzo[a]pyrene-9,10-oxide PTPRE TEC FLT4 12,13-hydroxyoctadec-9(Z)-enoateEPHX2 LipoxinBenzo[a]pyrene-4,5-oxide A4 EPHB6 INSRR GGT3 MTHFD1L INSL3 Cys-Gly MTHFD1 PTPRAPTPRC GPX4 GPX5 GGTL3 PTPRR (5Z,9E,14Z)-(8xi,11xi,12S)-8,11,12-Trihydroxyicosa-5,9,14-trienoate GGT2 MTHFD2 9(10)-EpOME GPT FLJ32658NTRK3 MET GGTLA1 PRAGMIN PDGFRB 5-oxo-6-trans-leukotriene LCK MATK Prostaglandin GPT2 Malonyl-[acyl-carrier ACP1 B4 ERBB3 PTPRUTYRO3 GSR Oxidized protein] RYK F2alpha 12-oxo-20-carboxy-leukotriene 4-Aminobutanoate ALK HCK ITK 12-oxo-10,11-dihydro-20-COOH-LTB45-oxo-(6E,8Z,11Z,14Z)-eicosatetraenoic GPX3 glutathione PEPB PTPN11 B4 5,10-Methylenetetrahydrofolate PTPRD EPHA4 PTPN13FYN FGFR3 acid S-(PGA2)-glutathione 2-Oxoglutarate EYA3 CDC25B TXK BLK R-CHOH-R'10,11-dihydro-12R-hydroxy-leukotriene Formate SGK269 NTRK2 5-HEPE PTPN7 PTPN3 EPHB2EPHA3 RET SPRYD3 C4 AKR1CL1 (5Z,9E,14Z)-(8xi,11R,12S)-11,12-Epoxy-8-hydroxyicosa-5,9,14-trienoic SYK FGFR2 2-hydroxy-17beta-estradiol-1-S-glutathione 5,10-MethenyltetrahydrofolateARG1 PDGFRA PTP4A2PTPN5 PTPDC1 Leukotriene B4 5-oxo-EPE GPX7 PRPS1 PRPS1L1 5(S)-HETE 6-trans-leukotriene PTPN1 S-(PGA1)-glutathione Pyruvate PTPRZ1 STYK1 PTPN2 PTPN14EPHB1 B4 Bromobenzene-2,3-oxide GPX2 EYA4 IGF1R FLT1 PTK6 FRK 5-oxo-6E-12-epi-LTB46,7-dihydro-leukotriene 4-hydroxyestrone-2-S-glutathione17beta-estradiol-2,3-quinone L-Alanine2-Oxobutanoate ARG2 EGFR EPHA8 EPHA5 ABL1 17beta-estradiol-3,4-quinoneS-(1,2-Dichlorovinyl)glutathione Glutathione PRPS2 KDR SRMS MERTK 6,7-dihydro-12-epi-LTB4 B4 (1S,2R)-NaphthaleneBromobenzene-3,4-oxideHydrobromic acid PTPN21 ABL2 1-Nitronaphthalene-5,6-oxide Benzo[a]pyrene-7,8-diol FRMPD2 FES MUSK TIE1 EPHB3 EYA2 CBR3 (1R,2S)-NaphthaleneProstaglandin1,2-oxideadrenochrome(1S)-Hydroxy-(2S)-glutathionyl-1,2-dihydronaphthalene J2 5-S-glutathionyl-noradrenochrome GPX1 PTPN12 BTK 5-S-glutathionyl-adrenochrome PTPRN2 PTPRHEPHB4 20-COOH-10,11-dihydro-LTB45-oxo-12(S)-hydroxy-eicosa-6E,8Z,10E,14Z-tetraenoate hydroquinone GPX6 L-Arginine CSK 1,2-oxide 1-Nitro-7-hydroxy-8-glutathionyl-7,8-dihydronaphthaleneProstaglandin A2 EYA1 Protein tyrosine SRC 5,12-DiHETE hydroquinone 2-hydroxyestrone-4-S-glutathione Protein tyrosine DHRS4 DNAJC6 CBR1 3,4-Dihydro-3-hydroxy-4-S-glutathionylhepoxilin1,2-Dibromoethane A3-C DDR1 ERBB2 phosphate FLT3 10,11-dihydro-12-epi-leukotriene GSTO2 TPTE TEK 5-S-glutathionyl-aminochromeChloroacetylestrone-3,4-quinone GSTZ1 dopaminebromobenzene B4 12-oxo-leukotriene GSTM1 2-(S-Glutathionyl)acetyl MCAT Phosphoribosyl-ATP TNK2 PTPN9PTPRO R-CO-R' estrone-2,3-quinonechloride MGST3 GSTT1o-quinone FGR PTPRG PTPRN B4 reduced GSTM2 GSTA4 Prostaglandin1-Nitronaphthalene-7,8-oxide A1 chloride KIT GSTA3GSTA2GSTM5 6E-12-epi-LTB4 12-oxo-c-LTB3 GSTT2GSTM4 GSTK15-S-glutathionyl-L-DOPAR-S-GlutathioneMGST2 JAK2 FER JAK1 PTPRJ PTPRFLTK PTPN4 S-(PGJ2)-glutathione GSTA1 ROR1 ROR2 2,2-DichloroacetaldehydeGSTM3GSTO1 RX D-Ribose CDC25C S-(2,2-Dichloro-1-hydroxy)ethyl MGST1GSTA5 ROS1 HSD17B3 7,8-Dihydro-7-hydroxy-8-S-glutathionyl-benzo[a]pyreneGSTP1 9-deoxy-delta12-PGD2 5-phosphate AXL MTM1 TNK1 HSD17B1RDH8 6,7-dihydro-5-oxo-leukotriene 4-hydroxy-17beta-estradiol-2-S-glutathioneglutathione Glutathione GATM Urea EPHA7 DDR2 S-(Formylmethyl)glutathione2-hydroxyestrone-1-S-glutathioneTrichloroethene ZAP70 MST1R PTPN6BMX B4 S-(9-deoxy-delta12-PGD2)-glutathioneglutathionyl-3-hydroxykynurenine1-Nitro-7-glutathionyl-8-hydroxy-7,8-dihydronaphthalene3-hydroxykynurenine-O-beta-D-glucosideepisulfonium ion PTPRB Prostaglandin E2 2-(S-Glutathionyl)acetyl PTK2BPTPRS FGFR4 EPHA10 HSD17B2 glucoside noradrenochrome1-Nitro-5-hydroxy-6-glutathionyl-5,6-dihydronaphthalene5-S-glutathionyl-dopamine PTPRT MTMR3 PTP4A3 HSD17B8 (1R)-Glutathionyl-(2R)-hydroxy-1,2-dihydronaphthaleneglutathione CSF1R PTK2 2-hydroxy-17beta-estradiol-4-S-glutathione1-Nitro-5-glutathionyl-6-hydroxy-5,6-dihydronaphthalene2,3-Dihydro-2-S-glutathionyl-3-hydroxy GSS AKR1C3 6,7-dihydro-5-oxo-12-epi-LTB420-carboxy-leukotriene-B4 L-Dopachrome LYN 10,11-dihydro-12-oxo-LTB4 bromobenzene NUDT9 DUSP18 delta12-Prostaglandin2-BromoacetaldehydeS-(2-Chloroacetyl)glutathione Estrone 10,11-dihydro-leukotriene HSD17B7 tetracosa-6,9,12,15,18-all-cis-pentaenoyl-CoAtimnodonate J2 Guanidinoacetate ADPRH 5-S-glutathionyl-dopachromedopaminochrome1,1-Dichloroethylene gamma-L-Glutamyl-L-cysteine Malonyl-CoA YES1 PTPN22 Estradiol-17betaEstriol B4 CoA-20-COOH-LTE4 S-(9-deoxy-delta9,12-PGD2)-glutathione NTRK1 (9Z,12Z,15Z)-Octadecatrienoic hydroquinoneepoxide TRPM2 Androst-4-ene-3,17-dione 10,11-dihydro-LTB4-CoA ACSL3 (1R)-Hydroxy-(2R)-glutathionyl-1,2-dihydronaphthalene PTPRK Testosterone acid NEK1 CDC14BDUSP16 Linoleate DUSP2 DUSP10 Cholesterol ester Desmosterol 16alpha-Hydroxyestrone ACSL1 alpha-linolenoyl-CoA CDKN3 DUSP19 nisinate 4-Guanidinobutanoate NUDT5 SOAT1 trans-eicos-2-enoyl-CoA ADPribose DUSP3DUSP15 ACSL5 DUSP5 DUSP7 CDC14A (2S)-pristanoyl-CoA3-Hydroxypropanoate trans-2-cis,cis,cis-8,11,14-eicosatetraenoyl-CoA DUSP12 DUSP9 all-cis-10,13,16,19-docosatetraenoyl-CoA 6,9,12,15,18-all-cis-tetracosapentaenoate5-oxo-12(S)-hydroxy-eicosa-8E,10E,14Z-trienoyl-CoAACSL4 (2S,6R,10R)-pristanate ACVR1 EPM2A DUSP21 cis-13-docosenoyl-CoAcis,cis,cis-10,13,16-docosatrienoyl-CoA ACSL6 Glycine DUSP14 ATM DUSP4 5-oxo-12(R)-hydroxy-eicosa-8E,10E,14Z-trienoyl-CoA CDC14C dihomo-gama-linolenoyl-CoAtrans,cis-2,11-eicosadienoyl-CoA N(omega)-(ADP-D-ribosyl)-L-arginine SSH2 SOAT2 Linoleoyl-CoA Dopaquinone SSH3 Oleoyl-CoA 20-COOH-leukotriene Taurolithocholate PPM1E SSH1PTPMT1 DUSP22 (8Z,11Z,14Z,17Z)-eicosatetraenoyl-CoAtetracosa-9,12,15,18,21-all-cis-pentaenoyl-CoA 20-CoA-20-oxo-leukotriene DUSP23 IRAK4 trans,cis-2,9-octadecadienoyl-CoA2-trans-9,12,15,18,21-all-cis-tetracosahexaenoyl-CoA palmitoleoyl-CoA ALLC Cholesterol B4 eicosa-5,8,11,14,17-all-cis-pentaenoyl-CoApalmitoleateE4 PPM1M DUSP13 dehydrodolichol glycolithocholate3alpha-Hydroxy-5beta-cholanate LMTK2 DUSP1EIF2AK1 3(S)-hydroxy-docosa-10,13,16,19-all-cis-tetraenoyl-CoA Arachidonate PPEF2 WEE1DUSP6STK32B tetracosa-9,12,15,18-all-cis-tetraenoyl-CoA tetracosa-6,9,12,15,18,21-all-cis-hexaenoyl-CoASLC27A2 MARK1 HIPK1MAP3K11 DUSP8 ULK2 PPP3CA trans,cis,cis,cis-2,10,13,16-docosatetraenoyl-CoAcis-15-tetracosenoyl-CoA Dolichol3(S)-hydroxy-all-cis-8,11,14,17-eicosatetraenoyl-CoA Arachidonyl-CoA PPP2R3A CSNK1A1LMAPK13 CDK10 STK3 3-oxo-docosa-10,13,16,19-all-cis-tetraenoyl-CoA TYR PPP6C IKBKB SNF1LKPPEF1 LMTK3 ROCK1 3alpha,7alpha,12alpha,25-tetrahydroxy-5beta-cholestane-24-one Taurine PPM1A NLK PLK1 CDKL5PRKAA2 trans-tetracos-2-enoyl-CoA 13'-carboxy-alpha-tocotrienol PPP2CB PPP1R3A PPP2R3B ACVRL1CLK4 trans,cis-2,13-docosadienoyl-CoA3-oxo-tetracosa-9,12,15,18-all-cis-tetraenoyl-CoA13,14-dihydro-15-oxo-lipoxin 3-hydroxyeicosanoyl-CoA HSD17B10 CIT trans,cis-2,15-tetracosadienoyl-CoA AKT2 EIF2AK2PPP2R1A ACVR1C CTDP1 Eicosanoyl-CoA 5beta-cholestane-3alpha,7alpha,12alpha,24S,25-pentolA4 BUB1B PAK3 FASTKMARK4 3-oxo-docosa-7,10,13,16-all-cis-tetraenoyl-CoA 3-Hydroxypropionyl-CoApristanate MAPK4 RAGEMYO3A SRPK2 13,14-dihydro-15-oxo-PGE13-oxo-docosa-7,10,13,16,19-all-cis-pentaenoyl-CoA TTBK1RPS6KL1PKN3 AMHR2PIM2 MAP4K3 RIPK2 (3S)-hydroxy-eicosa-cis,cis-11,14-dienoyl-CoA MAPK9 PPM1L SBK1NUAK1 CDK3RIPK3 TESK2 trans,cis,cis,cis,cis-2,12,15,18,21-tetracosapentaenoyl-CoAcis,cis-11,14-eicosadienoyl-CoAtetracosanoyl-CoA 3-oxo-tetracosa-12,15,18,21-all-cis-tetraenoyl-CoAHSD17B12 ACOT714,15-EETHADHA (-)-Ureidoglycolate TBK1 PRKCG 3-oxo-docosa-cis,cis,cis-10,13,16-trienoyl-CoA Allantoate HUNK ILK TGFBR2 PPP2CAMAP3K8 15-oxo-lipoxin A4 (2R)-pristanoyl-CoA AMACR BMP2K PRKCB1 MAP3K13 PHLPP Stearoyl-CoA 3-keto-eicosa-8,11,14,17-all-cis-tetraenoyl-CoA ACOT6 L-Tyrosine BAAT MKNK2 STK38L 3(S)-hydroxy-cis-15-tetracosaenoyl-CoA 6(S)-hydroxy-tetradeca-2E,4E,8Z-trienoate RPS6KA1 NEK3 3(S)-hydroxy-docosa-7,10,13,16,19-all-cis-pentaenoyl-CoA 11'-carboxy-alpha-tocotrienol ACVR2A CTDSP1 docosa-7,10,13,16,19-all-cis-pentaenoyl-CoA 3-oxooctadecanoyl-CoA 3(S)-hydroxy-dihomo-gama-linolenoyl-CoA 5,6-epoxy-(8Z)-tetradecenoic ACOT2ECHS1 2-trans-cis,cis,cis,cis-4,8,11,14-eicosapentaenoyl-CoA MAK TRPM7 TSSK2PAK1RPS6KC1 ceramide 3alpha,7alpha,12alpha,24-Tetrahydroxy-5beta-cholestanoyl-CoA EHHADH ACOT8 8(R)-hydroxy-hexadeca-2E,4E,6E,10Z-tetraenoate PRKG2 VRK2 PRKCSH MYO3B docosanoyl-CoA 3-hydroxydocosanoyl-CoA acid PPP3CB SNRK PRKCQ d18:1(4c) ACACB STK33 LIMK2 PCTK2IRAK1 SPEGPRKAR1B TAOK3 CHEK1AURKC MAPK3 Alprostadil 9-cis-4-oxo-13,14-dihydro-retinoate 3-oxo-cis-15-tetracosaenoyl-CoA HSD17B47,8-epoxy-(4Z,10Z)-hexadecadienoic PPP2R1B SGK1 CDC7KALRN 10,11-epoxy-(4Z,7Z)-hexadecadienoic Holo-[carboxylase] MAST3MAPKAPK3 DMPK PPP1CC trans-octadec-2-enoyl-CoA 15-oxo-PGE1alpha-hydroxyisocaproic HSD11B2AKR1C4 acid trans,trans-2,3,4,5-Tetradehydroacyl-CoAtrans-2,3-Didehydroacyl-CoA ACVR1B STK4 RPS6KA4GSG2 MAP3K2 MOS PLK3 ACOT4 acid GAK PCTK1 3(S)-hydroxy-11-cis-eicosenoyl-CoAacid DCI CoA-18-COOH-15E-dinor-LTE4 RIPK1 AKT1 EIF2AK4RIPK4 LRRK1CSNK1A1 STK16 AKR1C1 8,9-epoxy-(5Z)-tetradecenoic7'-carboxy-gama-tocotrienol ACACA MAP3K9 STK17B BCR CASK trans-docos-2-enoyl-CoAall-cis-12,15,18,21-tetracosatetraenoyl-CoA 5'-carboxy-alpha-chromanolACOT1 MINK1 CSNK1G2 STK10 STK32A 3(S)-hydroxy-docosa-7,10,13,16-all-cis-tetraenoyl-CoA vitamin A2 acid7'-carboxy-alpha-tocotrienol SNF1LK2 MAPK1TLK1 CDK5 MAP3K7PFTK1 MKNK1 CSNK1G3 trans,cis,cis-2,11,14-eicosatrienoyl-CoA 3(S)-hydroxy-13cis-docosenoyl-CoA3-oxo-13cis-docosenoyl-CoA 3-oxo-dihomo-gama-linolenoyl-CoAcis-laur-5-enoyl-CoA CDC2L5 LIMK1 TRPM6STK32C STK36 TTBK2 MAP3K14 3(S)-hydroxy-10,13,16-all-cis-docosatrienoyl-CoA HADH trans-3-cis-8,11,14-eicosatetraenoyl-CoA3Z,7Z,10Z-hexadecatrienoyl-CoA PPP3CC TTN DYRK2CHEK2NRKMAP3K12 12,13-epoxy-(6Z,9Z)-octadecadienoic4-methyl-pentanoyl-CoA5'-carboxy-gama-chromanol PAK2BMP2KL PRKY ERN1 PLK4 RIPK5 trans,cis,cis,cis,cis-2,10,13,16,19-docosapentaenoyl-CoAall-trans-3,4-didehydroretinoate Carboxybiotin-carboxyl-carrier STK31 MAPK12 DCLK1CDK4EIF2AK3NEK11STK38 3(S)-hydroxy-tetracosa-12,15,18,21-all-cis-tetraenoyl-CoA3-oxoeicosa-cis,cis-11,14-dienoyl-CoA acid DECR2trans-2-cis,cis-4,8-tetradecatrienoyl-CoA PPM1G RIOK3 3(S)-hydroxy-cis-9-octadecenoyl-CoA3-oxo-all-cis-6,9,12,15,18-tetracosapentaenoyl-CoA ACOX3 PECI protein MAP3K15 PhosphoproteinProteinPRKD3 COL4A3BP 3(S)-hydroxy-tetracosa-9,12,15,18,21-all-cis-pentaenoyl-CoA 3alpha,7alpha-Dihydroxy-5beta-cholestanoyl-CoA DAPK2 ACVR2B RIOK2 SLK MLKL 2-trans-9,12,15,18-all-cis-tetracosapentaenoyl-CoANAD(P)H 3-oxotetracosanoyl-CoA4-oxo-9-cis-retinoate cis,cis-3,6-Dodecadienoyl-CoA ACOX2 DECR1 CDC2L2 PRKAR1A ATRRIOK1CRKRSCDC42BPGPRKCA 3,4-Dihydroxy-L-phenylalanine PIK3R4 PASK 4-Methyl-2-oxopentanoate 3-oxo-8(R)-hydroxy-hexadeca-6E,10Z-dienoate 6(R)-hydroxy-tetradeca-2E,8Z-dienoateBDH2 PPP3R1 LATS1 ULK1 PRPF4 PKMYT1 BMPR1B 3-hydroxyoctadecanoyl-CoA3-hydroxytetracosanoyl-CoA3alpha,7alpha,12alpha-Trihydroxy-5beta-24-oxocholestanoyl-CoA 3-oxolaur-cis-5-enoyl-CoA 8(R)-hydroxy-hexadeca-2E,6E,10Z-trienoate PPP1R1ACTDSP2 CSNK2A1PPRPF4B MARK2FLJ25006MAPKAPK2O-PhosphoprotamineMAP3K3 NEK4 PIM3 eicosa-2E,8Z,11Z,14Z,17Z-pentaenoyl-CoA3-oxoeicosanoyl-CoA 3-oxo-10(R)-hydroxy-octadeca-6E,8E,12Z-trienoyl-CoACoA-omega-COOH-tetranor-LTE3 8(S)-hydroxy-hexadeca-2E,6E,10Z-trienoate PHLPPL CDKL4 TAOK2 12-hydroxyeicosatetraenoate 3-oxo-11-cis-eicosenoyl-CoA adrenoyl-CoA cis-6-dodecenoyl-CoA3-oxo-10(S)-hydroxy-octadeca-6E,8E,12Z-trienoate NEK8LRRK2 NEK7 HIPK3 ARAF 3-oxo-cis-9-octadecenoyl-CoA CDKL1 TAOK1Protamine PDPK1 STK11 CDC2L6 12-HETrE (2R,6R,10R)-trimethyl-hendecanoyl-CoA BUB1 TSSK4 CLK3DYRK4SMG1 11-dehydro-15-keto-TXB23-oxodocosanoyl-CoA ACOX1 ULK4 11-dehydro-13,14-dihydro-15-oxo-TXB2 cis,cis-myristo-5,8-dienoyl-CoA8(S)-hydroxy-hexadeca-4E,6E-10Z-trienoateCoA-omega-COOH-dinor-LTE4 trans-3-cis-8-tetradecadienoyl-CoA 2-Carboxy-2,3-dihydro-5,6-dihydroxyindole PRKAR2B UHMK1 CSNK2A2PSKH2 11-cis-eicosenoyl-CoA 3-oxolaur-6-cis-enoyl-CoA 3-oxo-6(S)-hydroxy-tetradec-8Z-enoate CoA-18-COOH-16E-dinor-LTE5 CDC2L1LATS2CDK8 ERN2 NEK2 AURKB PRKD1 PAK4 PKN2 3-oxo-cis,cis-5,8-tetradecadienoyl-CoA 2E,4Z,7Z,10Z-hexadecatetraenoyl-CoA AATKTTKTGFBR1ROCK2 cis,cis-palmito-7,10-dienoyl-CoA 2(R),6-dimethyl-heptanoyl-CoA CLK2 MAPK14 BMPR1ACLK1 3-oxo-5(S),12(R)-dihydroxy-eicosa-8-trans-6,14-cis-trienoyl-CoAChenodeoxyglycocholoyl-CoA 3-oxo-cis,cis-7,10-hexadecadienoyl-CoA PIM1 MARK3 PPP2R2AYSK4 2-trans-7,10,13,16,19-all-cis-docosahexaenoyl-CoA 3-cis-Dodecenoyl-CoA PPP2CBP MASTL ICK PRKCD PAK7 AKT3 CDC2 docosa-4,7,10,13,16,19-all-cis-hexaenoyl-CoA3-Oxodecanoyl-CoA trans,cis-deca-2,4-dienoyl-CoA PRKCE GTF2F1 BRSK1 RPS6KA3 4(R)-hydroxy-dodec-6Z-enoateACAA1 6(R)-hydroxy-tetradeca-2E,4E,8Z-trienoate PINK1 WNK4 BRAFPSKH1MAPK7 PRKAR2A PAK6 16,18-oxo-18-CoA-dinor-LTE4 8(S)-hydroxy-hexadeca-2E,4E,6E,10Z-tetraenoate STK40 TP53RK MAPK15 PRKCH MAST1 SRPK1 PRKD2 HADHBACAA2 6(S)-hydroxy-tetradeca-2E,8Z-dienoate DYRK3 RPS6KA6 MAP3K4 3-oxo-10(R)-hydroxy-octadeca-6E,8E,12Z-trienoate18,20-dioxo-20-CoA-leukotriene CSNK1G1CDKL2 MAP4K1PKN1DYRK1B CDC42BPA DAPK3PPP1R3C 3-oxo-(2S)-methylisocapryloyl-CoA PRKAA1MAP3K10WNK2 CCRK DAPK1WNK1KIAA1804 B4 3-oxo-4(R),8-dimethyl-nonanoyl-CoA ANKK1 RPS6KB1MAP4K2 MAP4K4 Chenodeoxycholoyl-CoA4(S)-hydroxy-dodec-6Z-enoate STK24MGC42105PCTK3 8(R)-hydroxy-hexadeca-4E,6E,10Z-trienoate (R)-3-Hydroxybutanoate RPS6KB2 OXSR1PRKCZ PDIK1L SGK3 3-oxomyrist-7-enoyl-CoA 3-Oxooctanoyl-CoA3-oxo-6Z,9Z,12Z-octadecatrienoyl-CoA RAF1 AURKA gamma-Linolenoyl-CoA SRPK3 MAPK8 CDK7 CDKL3 DCLK3 TAF1TSSK3KIAA0999PRKACB MAP4K5 RPS6KA2 3-Oxododecanoyl-CoA TNIK TESK1 CoA-20-COOH-18-oxo-LTE4 PPP3R2 NEK10WNK3 BRSK2PPP1R2IKBKE TRIO Hexanoyl-CoA PPP1R1B CDK9 SGK2 TLK2PRKG1 VRK1 3-oxo-8(S)-hydroxy-hexadeca-6E,10Z-dienoate3-Oxohexanoyl-CoA LOC646643CHUK CSNK2A1 NEK5 cis-myrist-7-enoyl-CoA 3-oxo-6(R)-hydroxy-tetradec-8-cis-enoateAcetoacetyl-CoA PRKACA 6(S)-hydroxy-tetradeca-4E,8Z-dienoate 18-CoA-18-oxo-dinorleukotrienecis-deca-4-enoyl-CoA STK35 NEK6 3(S),10(R)-OH-octadeca-6-trans-4,12-cis-trienoyl-CoA 2-Methylacetoacetyl-CoA TNNI3KCDK2HIPK4 ULK3 2-Methylpropanoyl-CoAB4 NUAK2 AAK1 CSNK1E HIPK2 CSNK2B PPP5C STK39 MAPKAPK5 NEK923227 3-Oxotetradecanoyl-CoA3-Oxoadipyl-CoA Acetyl-CoA STK25 TSSK6MAP3K5 6(R)-hydroxy-tetradeca-4E,8Z-dienoate 3-oxolinoleoyl-CoA3-oxo-4-methyl-pentanoyl-CoA UBLCP1 PRKDC MELK 3,5-dioxo-12(R)-hydroxy-eicosa-8E,10E,14Z-trienoyl-CoA HTATIP RPS6KA5 PFTK2 MAPK6 3-oxo-(4R,8R,12R)-trimethyl-tridecanoyl-CoAButanoyl-CoA ACAT2 PPM1B STK17A Tetradecanoyl-CoA Lauroyl-CoA3-oxo-all-cis-6,9,12,15,18,21-tetracosahexaenoyl-CoA3-oxopalmitoleoyl-CoA4Z,7Z,10Z-hexadecatrienoyl-CoA CDY1 MAPK10 PLK2 PPM1FPRKACG trans-Hex-2-enoyl-CoA 3-oxo-cis-8-tetradecenoyl-CoA BMPR2 MAP3K1STK19 IRAK2 Decanoyl-CoA CDK6 GTF3C4 BDH1 PPM1K PPP1CBPRKCI ILKAP NCOA3 HMGCLL1 DCLK2 3alpha,7alpha-Dihydroxy-5beta-24-oxocholestanoyl-CoA CoA NCOA1 MYST2 CSNK1D MAPK11CDC42BPB 3,5-dioxo-12(S)-hydroxy-eicosa-8E,10E,14Z-trienoyl-CoACrotonoyl-CoA ACAT1 PPM1D PPP2R2B PPP1CA 3-oxo-10(S)-hydroxy-octadeca-6E,8E,12Z-trienoyl-CoACholoyl-CoA CDY2A PPP4CTSSK1B 3-Oxopalmitoyl-CoA CDY1B MYST1 MGEA5 VRK3 MAST2 PCAF Succinyl-CoA AACS HMGCL EP300 Acetoacetate IRAK3 ACADM CDY2B ACADLACADSB PRKX Octanoyl-CoA Propanoyl-CoA MYST3 Histone DYRK1A (S)-3-Hydroxy-3-methylglutaryl-CoA GCN5L2 MAP3K6 CREBBP Acetylhistone PECR ACADS MECR HAT1 PPP2R2C trans-Tetradec-2-enoyl-CoA MYST4

trans-Hexadec-2-enoyl-CoA SUCLG1 3-Methylcrotonyl-CoA OXCT1 3-Methylbutanoyl-CoA ECHDC2 CROT 2-trans-Dodecenoyl-CoAtrans-Dec-2-enoyl-CoA SUCLG2 trans-Oct-2-enoyl-CoA OXCT2 FAH Succinate 2-Methylbutanoyl-CoA PCCA PCCB 2-Methylbut-2-enoyl-CoA MLYCD 2-Methylprop-2-enoyl-CoA 4-Fumarylacetoacetate

Palmitoyl-CoA (R)-3-Hydroxybutanoyl-CoA IVDL-Octanoylcarnitine Fumarate

L-Carnitine

(S)-2-Methyl-3-oxopropanoyl-CoA

Methylmalonyl-CoA CPT2 CPT1A

CPT1B CPT1C

Acyl-CoA

MCEE acylcarnitine L-Palmitoylcarnitine

(R)-2-Methyl-3-oxopropanoyl-CoA

Sedoheptulose L-Fucose NDUFA12 GlcAbeta1-3GalNAcbeta1-4IdoAbeta1-3GalNAcbeta1-4GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-Ser-peptide(Gal)2 (GalNAc)1 D-Fructose PAFAH1B2 PAFAH2 PHKG1 Methylglyoxal 2-Amino-4-hydroxy-6-(erythro-1,2,3-trihydroxypropyl)dihydropteridine EXT2 N-Trimethyl-2-aminoethylphosphonate Peptidyl(2-hydroxyglycine) L-Phenylalanyl-tRNA(Phe) 3alpha,7alpha,26-Trihydroxy-5beta-cholestane PMM2 NDUFV3 1,7-bisphosphate PHKG2 (Gal)2 (GlcA)1 1-phosphate ND4 (GlcA)2 (Xyl)1 1,6-bisphosphate (GlcNAc)1 (Xyl)1 (Ser)1 Hydroxypyruvate NDUFS7 (Ser)1 tRNA(Phe) D-Mannose NDUFV2 FARS2 FUCA2 NDUFS5 1-Alkyl-2-acetyl-sn-glycero-3-phosphocholine (Gal)2 (GlcA)2 EXTL3 NDUFS2 NDUFS4 PCYT1A MYCBP2 1-phosphate NDUFS6 D-Fructose (GlcNAc)2 (Xyl)1 Choline FUK PTS Dehydroascorbate NDUFC1 6-phosphate 1-Organyl-2-lyso-sn-glycero-3-phosphocholine PHKA1 (Ser)1 phosphate 3alpha,7alpha,12alpha-Trihydroxy-5beta-cholestane NDUFS8 NDUFA3 6-Pyruvoyltetrahydropterin CDPcholine Ascorbate FBP1 PLA2G7 Phosphorylase a FARSB D-Mannose NDUFB5 NDUFB4IdoAbeta1-3GalNAcbeta1-4GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-Ser-peptide beta-D-Fructose Acetate Tetrahydrobiopterin NDUFB10 Phosphorylase b Glycolate GRHPR (Gal)2 (GlcA)2 PCYT1B PAM 6-Deoxy-L-galactose CYP27A1 6-phosphate NDUFA7 1,6-bisphosphate alpha-D-Fucoside PMM1 NDUFS1 ND1 PHKA2 Glyoxylate (GlcNAc)1 (Xyl)1 EXT1 DSE FBP2 SPR D-Tagatose PAFAH1B1 EXTL1 FARSA 3alpha,7alpha,12alpha,26-Tetrahydroxy-5beta-cholestane NDUFA11 CHSY1 GLYD (Ser)1 CHST11 1,6-bisphosphate NDUFA10 NDUFB11 Sedoheptulose L-Phenylalanine NDUFB8NDUFA4 CMP-N-trimethyl-2-aminoethylphosphonate Peptidylglycine PGM2 NDUFB1 7-phosphate PFKM Triphosphate FUCA1 UbiquinoneUbiquinol NDST2 PAFAH1B3 NDST4 PHKB CALM1 (R)-Lactaldehyde NDUFS3 ND4L NDUFB7 beta-D-Fructose PFKL 3alpha,7alpha-Dihydroxy-5beta-cholestane NDUFA9 NDUFB3 NDUFB2 6-phosphate NDST1 D-Glycerate 6-Lactoyl-5,6,7,8-tetrahydropterin CHST12 Dermatan sulfate PFKP ND5 IdoAbeta1-3GalNAcbeta1-4IdoAbeta1-3GalNAcbeta1-4GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-Ser-peptide NDUFA4L2NDUFB6 NDUFB9 D-Tagatose NDUFA8 NDUFV1 6-phosphate NDUFA2 NDUFC2 NDUFA13 CHST7 ND3 NDUFAB1 NDST3 ND2 NDUFA6 ND6 CSGlcA-T NDUFA1 CHST13 NDUFA5 CHPF CHSY3

Thiamin sn-Glycerol SEPHS2 Selenide GPD1 PGLS 6-Phospho-D-gluconateVitamin K epoxide Xylitol Protoporphyrin (R)-5-Diphosphomevalonate triphosphate 3-phosphate

VKORC1 DCXR FECH PMVK Glycerone D-Glucono-1,5-lactone Selenophosphate SEPHS1 TPK1 GPD2 H6PD phosphate 6-phosphate Thiamin

Thiamin Vitamin K L-Xylulose Heme (R)-5-Phosphomevalonate diphosphate

Figure S4. Metscape “gene-compound metabolic network”. Highlighted in yellow are compounds and genes showing a significant regulation after 4 h cisplatin treatment. Metabolic enzymes were retrieved from this network (Fig 2A, TableS 2).

149 Purine metabolism

Figure S5. Purine metabolism. Upregulated enzymes in green, downregulated enzymes in red. Upregulated metabolites in dark red, downregulated metabolites in blue.

150 Identification of metabolic pathways in the DNA damage response in pluripotent stem cells

Pyrimidine metabolism

Figure S6. Pyrimidine metabolism. Upregulated enzymes in green, downregulated enzymes in red. Upregulated metabolites in dark red, downregulated metabolites in blue.

151 A

-6 0 6

M ettl6 RNA M ettl11a methyltransferase Setd1b Sm yd2

Ezh2 histone methyltransferase Sm yd3 Jm jd6 histone Jm jd7 demethylases Jarid2

B 40

* 30 * *

20 normalized fluorescence fluorescence normalized 10

0 PBS CP H2O2 H2O2 PBS CP H2O2 H2O2 PBS CP H2O2 H2O2 +NAC +NAC +NAC

1h 4h 8h

Figure S7. (A) Regulation of (de)methylases. Heatmap showing regulation of methyltransferases and demethylases after cisplatin treatment. (B) ROS formation is caused by hydrogen peroxide but not cisplatin treatment. Bar graph shows normalized fluorescence indicating intracellular ROS levels measured using 40 μM DCF-DA probe. Cells were preincubated with DCF-DA for 1 h and exposed to 5 μM cisplatin or 250 μM H2O2 in the presence or absence of 10 mM of the ROS scavenger NAC for the indicated times. Bars represent average and SEM of at least 3 independent experiments.

152 Identification of metabolic pathways in the DNA damage response in pluripotent stem cells

.8

158458 803968 479502 187734 32724.4 60254.4 36480.8 17957.6 2739346 4393332 1920786 t 8h 155566.4 125298.2 126605.2 142592.4 119938.2 139221.6 141058.6 215285.4 146548.6 509529 891853.6 920139.4 22618056 P 2103885.8 1979258.2 1992020.2 9986466.2 9615623.6 1439971.4 2057904.6 33234441.6 11983405.8 16813324.4 21220009.2 is - C

189373 151089 69237.4 27447.8 43317.2 42819.8 16772.4 2184768 1029575 108002.2 169188.4 108300.6 131602.8 190853.4 185212.2 537241.4 133855.8 454726.8 227666.4 449850.2 808426.2 12442760 24880710

2383394.2 2567759.8 2605548.6 3986165.6 2625698.4 1292005.6 1696029.4 10484320.6 24374196.8 42463695.2 10570783.8 14295301.6 t 4h P is - C

nsity 40046 e 155167 69975.6 85536.6 43998.2 1972786 2045477 162741.6 137013.2 440818.6 186960.2 107660.8 414232.6 123233.2 198542.2 756566.8 116946.6 815119.2 572516.4 314212.4 528474.6 19642998 10122938 2372360.2 2325012.6 4541230.8 1805556.6 1470363.8 2226354.2 1082367.4 l int 11633406.8 28493909.6 14786181.2 10818675.2 19608510.4 a ontrol 8h C

nd sign nt a 43659 66868 30950 161337 228957 121369 328136 673773 952727 74118.2 98387.2 66191.4 82934.4 27491.2 1955741 2820315 5045863 9654137 114393.4 147380.8 131137.6 450473.2 785843.6 504363.2 936447.6 tm e 17201366 2017249.6 2096198.4 1919764.2 7706583.8 1266479.2 a 13526183.4 15901506.4 29952953.2 13456075.2 ontrol 4h C Tr e nts

gm e - > - >

fr a /M S 112.0501 M S (M+1) 126.0652 126.0649 112.0501 152.0563 135.0296, 110.0343 152.0562 135.0297, 110.0349

97.0279 113.0341

2

ormula

2O8 3O5 3O5 5O7P 2O8P 6O12 S 3O6S 5O8P 5O5 4O8P 5O3 S 4O6 6O5S 6O5S 4 l F

O3 3O5 3O8P 3O7P O2S 2O

2O2 2O9P 2O6 5 3O 5O O2S O3 4O O5 O2 2O2 2O 16 N 15 N 15 N 14 N 15 N 32 N 17 N 14 N 13 N 13 N 15 N 12 N 22 N 20 N 26 N 15 N 5 N 13 N 14 N 5 N 14 N 5 N 9 N 9 N 4 N 11 N 9 N 14 N 10 N 13 N 9 N 4 N 12 N mic a 4 N 11 H h e 7 H 5 H 9 H 10 H 10 H 10 H 9 H 4 H 9 H 10 H 20 H 10 H 10 H 5 H 10 H 4 H 4 H 5 H 10 H 5 H 6 H 6 H 11 H 10 H 9H17NO5 4 H 15H H 9 H 5 H 14 H 10 H 4 H 9 H C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C

m p P 0.1558 0.1988 - 0.2582 - 0.7005 0.33432 0.35778 0.20579 0.03277 0.30607 0.30607 1.16912 0.30838 0.10834 0.11723 0.45407 0.80789 0.33233 1.62449 0.04937 0.30252 0.47779 0.57311 0.02684 0.25957 0.57104 2.98996 0.48246 0.15254 1.07932 0.06121 0.13175 ------0.51109 - 0.02769 - 0.18955 0.086189461

lta D e 0.0001 0.0001 0.0002 - 0.0001 - 0.00006 0.00003 0.00001 0.00008 0.00008 0.00003 0.00013 0.00009 0.00003 0.00004 0.00005 0.00013 0.00012 0.00003 0.00021 0.00002 0.00009 0.00005 0.00007 0.00009 0.00001 0.00007 0.00007 0.00006 0.00003 0.00012 0.00001 0.00036 0.00003 ------0.00001 - 0.00007

) dduct .03455 l a MW (D a 244.0928 348.0704 119.0815 162.11247 136.06177 305.09794 258.10845 258.10845 324.05913 112.05054 308.06421 323.06388 308.09108 364.06528 152.05669 284.09895 136.04268 120.06552 137.04579 349.05438 150.05833 176.05535 131.11789 298.09684 285.08296 399.14451 325.04314 220.11795 116.07061 385.12886 203.22302 113 245.07681 tic a or e 307.083257 (2M+1) 121.072002 (2M+1) Th e

) dduct l a MW (D a 162.11241 244.09279 258.10837 258.10837 348.07037 324.05914 112.05041 308.06412 323.06384 364.06537 152.05673 284.09882 136.04274 120.06542 137.04588 349.05426 121.07164 220.11792 136.061798 305.097839 307.083221 308.091034 150.058411 176.055313 131.117676 298.096832 285.082886 399.144714 325.043152 11 9.081436 116.070549 385.128937 203.222992 113.034431 245.076797 nt a rim e e Ex p

- - x d a S N ine ine ine ine ine ine ine ine ine ine r e MP MP MP MP MP line _ o rine IMP cine s s s cine s racil nine nine nine _r e TMP t e A t e U C C e U G s s e anine r o ri d rmine U d n e s d yti d n o u d tamate n e partate P yt o U arnitine nic acid thi o e tr e an o thi o tr e t ab olite C p e A cy s cy s G xanthine l u C C u u m o S anth o a d o P th e G M e thylcyti d thylcyti d M e X m o m o tathi o tyla s H yp o tathi o o tylp u ylm e l u H s c e l u H c e G ant o partyl g ylh o G 3 - M e 4 - M e - A n o A P s - thylthi o e N d N n o tyla s e M e - A d c e S A A

Table S1. Identified metabolites. Identification of masses found to be significantly different (p<0.01) between control and cisplatin-treated samples.

153 gene symbol gene name fold change

MLL3 myeloid/lymphoid or mixed-lineage leukemia 3 -7.69

EXT1 exostoses (multiple) 1 -6.67

FUT8 fucosyltransferase 8 (alpha (1,6) fucosyltransferase) -5.26

Ppp3ca protein phosphatase 3, catalytic subunit, alpha isoform -4.76

DSE dermatan sulfate epimerase -4.76

PIK3CB phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit beta -3.70

ATP2B1 ATPase, Ca++ transporting, plasma membrane 1 -3.70

PPAP2B phosphatidic acid phosphatase type 2B -3.57

PPAP2B phosphatidic acid phosphatase type 2B -3.57

ENPP3 ectonucleotide pyrophosphatase/phosphodiesterase 3 -3.45

FARS2 phenylalanyl-tRNA synthetase 2, mitochondrial -3.23

PLCH1 phospholipase C, eta 1 -3.13

UCK2 uridine-cytidine kinase 2 -3.12

PIP4K2A phosphatidylinositol-5-phosphate 4-kinase, type II, alpha -3.03

OXCT1 3-oxoacid CoA transferase 1 -3.03

POLA1 polymerase (DNA directed), alpha 1, catalytic subunit -2.94

ST6GAL1 ST6 beta-galactosamide alpha-2,6-sialyltranferase 1 -2.86

SMYD3 SET and MYND domain containing 3 -2.78

ATP11C ATPase, class VI, type 11C -2.78

PTEN phosphatase and tensin homolog -2.78

GALNT1 UDP-N-acetyl-alpha-D-galactosamine:polypeptide -2.70 N-acetylgalactosaminyltransferase 1 (GalNAc-T1)

POLR3A polymerase (RNA) III (DNA directed) polypeptide A, 155kDa -2.70

ITPK1 inositol-tetrakisphosphate 1-kinase -2.63

GPD2 glycerol-3-phosphate dehydrogenase 2 (mitochondrial) -2.63

GALNT7 UDP-N-acetyl-alpha-D-galactosamine:polypeptide -2.63 N-acetylgalactosaminyltransferase 7 (GalNAc-T7)

PCYT1B phosphate cytidylyltransferase 1, choline, beta -2.44

POLR3B polymerase (RNA) III (DNA directed) polypeptide B -2.44

POLR3B polymerase (RNA) III (DNA directed) polypeptide B -2.44

ELOVL6 ELOVL fatty acid elongase 6 -2.38

MBOAT2 membrane bound O-acyltransferase domain containing 2 -2.38

PFKP phosphofructokinase, platelet -2.33

WHSC1L1 Wolf-Hirschhorn syndrome candidate 1-like 1 -2.33

IP6K1 inositol hexakisphosphate kinase 1 -2.33

Lclat1 lysocardiolipin acyltransferase 1 -2.17

TPK1 thiamin pyrophosphokinase 1 -2.17

PCMT1 protein-L-isoaspartate (D-aspartate) O-methyltransferase -2.08

B4GALT5 UDP-Gal:betaGlcNAc beta 1,4- galactosyltransferase, polypeptide 5 -2.08

PCCA propionyl Coenzyme A carboxylase, alpha polypeptide -2.08

ADK adenosine kinase -2.08

PDE3B phosphodiesterase 3B, cGMP-inhibited -2.08

PLCB4 phospholipase C, beta 4 -2.04

SEPHS1 selenophosphate synthetase 1 -2.04

CDS2 CDP-diacylglycerol synthase (phosphatidate cytidylyltransferase) 2 -2.00

ATP10A ATPase, class V, type 10A -1.85

GRK5 G protein-coupled receptor kinase 5 -1.79

FARSB phenylalanyl-tRNA synthetase, beta subunit -1.75

154 Identification of metabolic pathways in the DNA damage response in pluripotent stem cells

gene symbol gene name fold change

BDH2 3-hydroxybutyrate dehydrogenase, type 2 -1.72

PLCG2 phospholipase C, gamma 2 (phosphatidylinositol-specific) -1.69

SLC27A2 solute carrier family 27 (fatty acid transporter), member 2 -1.69

PAFAH1B1 platelet-activating factor acetylhydrolase, isoform Ib, subunit 1 (45kDa) -1.67

PLCB1 phospholipase C, beta 1 (phosphoinositide-specific) -1.67

STT3B STT3, subunit of the oligosaccharyltransferase complex, homolog B (S. -1.64 cerevisiae)

MTHFD1L methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1-like -1.64

HSD17B12 hydroxysteroid (17-beta) dehydrogenase 12 -1.59

DGKH diacylglycerol kinase, eta -1.52

PTPN13 protein tyrosine phosphatase, non-receptor type 13 (APO-1/CD95 (Fas)- -1.52 associated phosphatase)

POLR1A polymerase (RNA) I polypeptide A, 194kDa -1.49

SMS spermine synthase -1.41

ASNS asparagine synthetase -1.39

GBA glucosidase, beta; acid (includes glucosylceramidase) -1.33

POLB polymerase (DNA directed), beta 1.31

FECH ferrochelatase (protoporphyria) 1.39

FUK fucokinase 1.40

ACAA2 acetyl-Coenzyme A acyltransferase 2 1.41

PRODH proline dehydrogenase (oxidase) 1 1.42

NPR2 natriuretic peptide receptor B/guanylate cyclase B (atrionatriuretic peptide 1.42 receptor B)

ENTPD5 ectonucleoside triphosphate diphosphohydrolase 5 1.43

POLR3K polymerase (RNA) III (DNA directed) polypeptide K, 12.3 kDa 1.44

PYCR2 pyrroline-5-carboxylate reductase family, member 2 1.45

POLR2G polymerase (RNA) II (DNA directed) polypeptide G 1.45

PPM1F protein phosphatase, Mg2+/Mn2+ dependent, 1F 1.46

SGPP1 sphingosine-1-phosphate phosphatase 1 1.47

GRHPR glyoxylate reductase/hydroxypyruvate reductase 1.47

CYP27A1 cytochrome P450, family 27, subfamily A, polypeptide 1 1.49

TK1 thymidine kinase 1, soluble 1.49

AGPAT5 1-acylglycerol-3-phosphate O-acyltransferase 5 (lysophosphatidic acid 1.50 acyltransferase, epsilon)

AGPAT5 1-acylglycerol-3-phosphate O-acyltransferase 5 (lysophosphatidic acid 1.50 acyltransferase, epsilon)

NANS N-acetylneuraminic acid synthase 1.50

GAA glucosidase, alpha; acid 1.51

SMPD4 sphingomyelin phosphodiesterase 4, neutral membrane (neutral 1.52 sphingomyelinase-3)

APRT adenine phosphoribosyltransferase 1.53

RRM2 ribonucleotide reductase M2 1.53

MCAT malonyl CoA:ACP acyltransferase (mitochondrial) 1.54

ECHDC2 enoyl Coenzyme A hydratase domain containing 2 1.55

FPGS folylpolyglutamate synthase 1.55

MLYCD malonyl-CoA decarboxylase 1.58

PGLS 6-phosphogluconolactonase 1.58

MECR mitochondrial trans-2-enoyl-CoA reductase 1.58

DPM2 dolichyl-phosphate mannosyltransferase polypeptide 2, regulatory subunit 1.60

ALDH4A1 aldehyde dehydrogenase 4 family, member A1 1.61

HMGCL 3-hydroxymethyl-3-methylglutaryl-Coenzyme A lyase 1.63

155 gene symbol gene name fold change

CANT1 calcium activated nucleotidase 1 1.65

Prkcd protein kinase C, delta 1.65

ACAT2 acetyl-Coenzyme A acetyltransferase 2 1.66

ACADS acyl-Coenzyme A dehydrogenase, C-2 to C-3 short chain 1.66

PMM1 phosphomannomutase 1 1.69

GPT glutamic-pyruvate transaminase (alanine aminotransferase) 1.69

DGKA diacylglycerol kinase, alpha 80kDa 1.70

CBR3 carbonyl reductase 3 1.70

GLS glutaminase 1.70

PARS2 prolyl-tRNA synthetase 2, mitochondrial (putative) 1.71

MDP1 magnesium-dependent phosphatase 1 1.73

POLE3 polymerase (DNA directed), epsilon 3 (p17 subunit) 1.73

PLOD3 procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3 1.75

POLL polymerase (DNA directed), lambda 1.76

NDUFB2 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 2, 8kDa 1.77

DGKZ diacylglycerol kinase, zeta 104kDa 1.78

DHFR dihydrofolate reductase 1.78

UMPS uridine monophosphate synthetase 1.78

PAFAH2 platelet-activating factor acetylhydrolase 2, 40kDa 1.80

FAH fumarylacetoacetate hydrolase (fumarylacetoacetase) 1.80

ENTPD6 ectonucleoside triphosphate diphosphohydrolase 6 (putative function) 1.80

POLR2D polymerase (RNA) II (DNA directed) polypeptide D 1.85

TPMT thiopurine S-methyltransferase 1.87

MCEE methylmalonyl CoA epimerase 1.89

PRPS2 phosphoribosyl pyrophosphate synthetase 2 1.89

AMPD2 adenosine monophosphate deaminase 2 (isoform L) 1.89

NME4 non-metastatic cells 4, protein expressed in 1.90

NT5C 5', 3'-nucleotidase, cytosolic 1.93

GATM glycine amidinotransferase (L-arginine:glycine amidinotransferase) 1.98

GSTO1 glutathione S-transferase omega 1 2.04

PMVK phosphomevalonate kinase 2.05

IVD isovaleryl Coenzyme A dehydrogenase 2.08

CERCAM cerebral endothelial cell adhesion molecule 2.11

FBP2 fructose-1,6-bisphosphatase 2 2.14

LPIN1 lipin 1 2.22

CBS cystathionine-beta-synthase 2.22

SPR sepiapterin reductase (7,8-dihydrobiopterin:NADP+ oxidoreductase) 2.39

DECR2 2,4-dienoyl CoA reductase 2, peroxisomal 2.39

ABHD5 abhydrolase domain containing 5 2.40

AACS acetoacetyl-CoA synthetase 2.41

PTS 6-pyruvoyltetrahydropterin synthase 2.49

VKORC1 vitamin K epoxide reductase complex, subunit 1 2.66

NME3 non-metastatic cells 3, protein expressed in 2.72

SORD sorbitol dehydrogenase 2.76

GNE glucosamine (UDP-N-acetyl)-2-epimerase/N-acetylmannosamine kinase 2.82

ADA adenosine deaminase 2.99

CPT1C carnitine palmitoyltransferase 1C 3.01

AK1 adenylate kinase 1 3.08

156 Identification of metabolic pathways in the DNA damage response in pluripotent stem cells

gene symbol gene name fold change

SMOX spermine oxidase 3.27

ELOVL2 ELOVL fatty acid elongase 2 3.38

ADC arginine decarboxylase 3.75

Apobec1 apolipoprotein B mRNA editing enzyme, catalytic polypeptide 1 3.76

EPHX1 epoxide hydrolase 1, microsomal (xenobiotic) 4.74

Table S2. Significantly regulated metabolic enzymes. List of metabolic enzymes identified by Metscape and Ingenuity pathway analysis from 2269 genes that are differentially regulated by cisplatin.

157 Suppl. Material 1

Orbitrap Mass Spectrometer settings

Heated electrospray interface (HESI): operating in positive mode (ESI+). Data acquisition: between m/z 100 and m/z 1000 Resolving power: 50.000 (FWHM) Scan time: 0.5 s Spray voltage: 2800 V Capillary voltage: 47.5 V Capillary temperature: 250 ˚C Sheath gas flow: 19 arbitrary units Auxiliary gas flow: 7 arbitrary units Instrument calibration: externally, prior to sequence by infusion of calibration solution (m/z 138 to m/z 1822) containing caffeine, MFRA (Met-Arg-Phe-Ala), ultramark 1621, acetic acid in acetonitrile/methanol/water (2:1:1, v/v) (Sigma-Aldrich).

Material S1. MS Instrument Settings.

158