Loss of succinate dehydrogenase activity results in dependency on pyruvate carboxylation for cellular anabolism

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1 Loss of succinate dehydrogenase activity results in dependency on pyruvate carboxylation for cellular anabolism Charlotte Lussey-Lepoutre, Kate ER Hollinshead, Christian Ludwig, Mélanie Menara, Aurélie Morin, Luis-Jaime Castro-Vega, Seth J Parker, Maxime Janin, Cosimo Martinelli, Chris Ottolenghi, Christian Metallo, Anne-Paule Gimenez-Roqueplo, Judith Favier and Daniel A Tennant. List of supplementary Figures and Tables - Supplementary Figure - Supplementary Figure - Supplementary Figure - Supplementary Figure - Supplementary Table (see data sheet) - Supplementary Table

2 Supplementary Figure : Transcriptome of cancer-associated metabolic genes shows altered metabolic profile of SDH-mutated tumours. Gene expression data from 8 PCC/PGL that were profiled on the HG-U Plus. Affymetrix GeneChip were evaluated for a list of 55 genes that encode for metabolic enzymes and transporters, adapted from the list reported by Possemato et al. 58 genes were differentially expressed (adjusted p value <; Bonferroni multiple test comparisons): upregulated and 8 downregulated in SDH-mutated tumours vs SDH mutated tumours. Heatmap shows classification using the differentially expressed genes ( upregulated and downregulated) with a fold change >.7.

3 a Glycine Isoleucine Leucine Proline Serine Valine.5. b. a-kg Alanine 5 Fumarate Glycine non-sdh tumours SDH tumours. Malate Proline. Serine non-sdh tumours SDH tumours non-sdh tumours SDH tumours c d e Supplementary Figure a) Abundance of glycine, isoleucine leucine, proline, serine and valine in imcc Sdhb-null (KO c and c8) cells relative to wild-type () control, as assessed using GC-MS (n=, mean +/- SD). Serine is significantly decreased, while glycine and proline are increased, only in clone 8 b) Steady-state KG, alanine, fumarate, glycine, malate, proline and serine in SDH-mutated patient tumours (n=) compared to SDH tumours (n=). Data are mean +/- SD. As in KO cells, serine is significantly decreased, in SDHmutated tumours. Data are mean+/-sd c) genotyping of the mouse adrenal fibroblast Sdhb knockout cell line (MAF KO) shows homozygous deletion of Sdhb exon ( ex) resulting in d) complete loss of SDHB protein expression. e) Steady-state concentrations of succinate are increased in cells and in the medium from SDH-deficient imcc and MAF (n=, mean +/- SD).

4 a C Incorporation from C-[,]-glucose (mmol/ cells/ hours) Synthesis of C-[,]-acetate from C-[,]-glucose (mmol/ cells/ hours) Succinate MID from Glutamate MID from. Alanine b C-[,]-glucose (%) 9 8 Glutamate imcc imcc KO c imcc KO c8 MAF MAF KO c C-[,]-glucose (%) 9 8 Succinate d C incorporation from C-[,]-glucose (nmol/ cells). Acetate MAF MAF KO e. Acetate.. imcc MAF MAF KO Supplementary Figure a) SDH-deficient imcc and MAF exhibit increased alanine production and excretion. After h of incubation with medium containing C-[,]-glucose, media was extracted and the abundance of different mass isotopomers of alanine excreted were quantified using NMR spectroscopy. C atoms (arising from glucose) are shown as filled circles, while C atoms are empty circles. Incorporation of C into b) glutamate and c) succinate. Reduction in the relative abundance of the C-[,5]-glutamate and increase in C-[,]-glutamate isotopomers suggests increase in PC activity and relative decrease in PDH activity in SDH-deficient cells. C atoms are shown as filled circles, while C atoms are empty circles. MID: mass isotopomer distribution. d-e) SDH-deficient cells produce and excrete significantly more acetate from glucose than wild-type cells. n= replicates, data indicate mean +/- SD.

5 Glutamate MID from m+ succinate from a Succinate MID from Lactate MID from doubling time Doubling time b imcc scr sipc sipc 5 imcc KO c8 KO scr KO sipc KO sipc 8 mrna expression (arbitrary units) mrna expression (arbitrary units) 8 days - 8 days c Slc5a d Slc5a e C -aspartate (%) Succinate f C5 -glutamine (%) Lactate imcc imcc KO c imcc KO c8 g Succinate h Glutamate C5 -glutamine 8 C5 -glutamine 8 m+ m+ m+ m+ m+ m+ m+ m+ m+ m+ m+5 Supplementary Figure a) Reduction in the expression of PC using sipc sequences # and # results b) in a significant reduction doubling time calculated over 7 days in SDH-deficient cells (right panel) while proliferation of wild-type cells (left panel) is unaffected. Data are mean +/- SEM (n=). mrna expression of c) Slc5a and d) Slc5a evaluated by microarray in and SDH-deficient cells e) Amount of m+ label (%) from C- asparate incorporated into succinate was measured using GC-MS (n=, data are mean +/- SD). f) Mass isotopomer distribution (MID) of lactate in media after incubation of cells with C 5 -glutamine suggest incorporation of carbons into lactate from reductive metabolism in SDH-deficient cells. As the C-[,]- lactate and C-[,]-lactate isotopologues are made in equal ratios through the action of oxidative TCA cycle metabolism in wild-type cells, the C-[,]-lactate isotopomer in these cells is an over-estimation of reductive carbon incorporation (n=, data are mean +/- SD). g-h) MID of intracellular succinate and glutamate after cells were incubated for 7 hours with C 5 -glutamine as measured using GC-MS (n=, data are mean +/- SD).

6 Legend to Supplementary Table - Transcriptome data. Data sheet shows a) metabolism-related genes upregulated in SDH-mutated PCC/PGL while b) metabolism-related genes downregulated in SDH-mutated PCC/PGL. Data sheet c) Gene ontology analysis of differentially regulated pathways in SDH-mutated PCC/PGL and the corresponding schematic illustration is shown in d). Data sheet e) shows the expression of the same genes in mouse imcc and MAF, knockout for Sdhb or wild-type (). SPO:sporadic.

7 Supplementary Table - Immunohistochemical evaluation of PC and SLC5A levels in human PGL/PCC. IHC score Tumour ID Mutation Tumour site PC SLC5A CIT_ SDHB LA CIT_8 SDHB Meta CIT_9 SDHB Meta NA.5 PGL SDHB Meta NA PGL SDHB APG CIT_ SDHB APG PGL SDHB RA CIT_7 SDHC TPG CIT_58 SDHD APG PGL SDHD CPG.5 CIT_8 SDHD TPG PGL5 SDHD CPG CIT_8 SDHB LA PGL SDHD APG PGL7 SDHC PGL PGL8 SDHD CPG NA CIT_ RETs RA CIT_ RET LA CIT_8 NF LA PGL9 RET RA CIT_7 RETs LA CIT_ NFs LA CIT_ NF RA CIT_5 NF LA PGL NFs LA CIT_ TMEM7 RA NA CIT_ RET LA LA: Left adrenal; RA: Right adrenal; TPG: Thoracic paraganglioma; CPG: Cervical paraganglioma; APG: Abdominal paraganglioma; PC: pyruvate carboxylase; IHC: immunohistochemistry