METABOLIC VULNERABILITIES OF CANCER. Eyal Gottlieb

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1 METABOLIC VULNERABILITIES OF CANCER Eyal Gottlieb

2 METABOLIC VULNERABILITIES OF CANCER Eyal Gottlieb

3 Cancer and metabolism: the anabolic angle glucose glucose-6-phosphate Ribose-5-phosphate ADP + Pi Serine Cysteine Glycine Alanine NAD + ATP NADH LIPIDS FATTY ACIDS acetyl-coa NADPH NUCLEOTIDES Aspartate pyruvate pyruvate citrate Glutamate Proline Arginine acetyl-coa Glutamine Aspartate Asparagine oxaloacetate citrate Glutamate malate α-ketoglutarate FH fumarate FADH 2 O 2 H 2 O FAD succinate NAD + NADH ATP synthase H + SDH (complex II) H + H + H +

4 Cancer and metabolism: historic perspective IDH mutations are shown to be oncogenic 2008

5 Cancer and metabolism: the bioenergetic angle glucose glucose-6-phosphate ADP + Pi NAD + ATP NADH pyruvate lactate pyruvate acetyl-coa Glioma/AML citrate oxaloacetate isocitrate HLRCC FH malate fumarate IDH α-ketoglutarate O 2 H 2 O H + HPGL FADH 2 SDH (complex II) FAD succinate NADH NAD + H + H + H +

6 VHL Succinate Phaeochromocytoma SDH Paraganglioma HIFα Fumarate RCC

7 1) HIF regulation by oxygen VHL OH O 2 PHD Hifα SDH

8 2) HIF regulation by VHL VHL OH O 2 PHD Hifα SDH

9 3) HIF regulation by succinate VHL OH O 2 PHD Hifα α-ketoglutarate SDH succinate Germline LoF mutations in SDH, VHL or PHD or GoF mutations in HIF2α can lead to paraganglioma! Hypoxia is a positive phenotypic modifier of paraganglioma

10 The birth of a new concept: Oncometabolites SDH FH O O O O 2 O O O O succinate O fumarate IDH1* O OH O Dioxygenase HIF O O 2-hydroxyglutarate Epigenome α-ketoglutarate

11 Fumarate hydratase deficient (Fh1 -/- ) cells Fh1fl/fl Fh1fl/fl Fh1-/- ahcre metabolite intensity (log10, normalized to Fh1 fl/fl ) citrate -/- CL1 Fh1 -/- CL19 Fh1 malate fumarate succinate

12 Metabolic adaptation and vulnerabilities of FHdeficient cancers Frezza et al, Nature, 477: 225, 2011 Zheng et al, Cancer Metab, 1: 12, 2013 Zheng et al, Nature Commun, 6: 6001, 2015

13 The biosynthetic pathway of succinic-gsh Fumarate Electrophilic attack Glutathione (γglu-cys-gly)

14 Fumarate attack on GSH induces oxidative stress

15 Metabolic adaptation and vulnerabilities of FHdeficient cancers Frezza et al, Nature, 477: 225, 2011 Zheng et al, Cancer Metab, 1: 12, 2013 Zheng et al, Nature Commun, 6: 6001, 2015

16 Cysts-bearing, FH-deficient mice excrete metabolites (biomarkers) in the urine Urine metabolic profile 2-succinic cysteine

17 A new in vitro model for SDH-deficient cancers SV40 T-Ag

18 SDH deficient cells depend on Pyruvate Carboxylase for sustaining aspartate levels

19 Aspartate limitation and PC expression in SDHdeficient tumors

20 H-Ras G12V transform SDH-deficient cells Succinate A spartate TPA /total cellular volum e on w ell SDHB fl/fl SDHB / SDHB fl/fl, H RAS G12V SDHB /, H RAS G12V colony 1 colony 2 colony 9 TPA /total cellular volum e on w ell SDHB fl/fl SDHB / SDHB fl/fl, H RAS G12V SDHB /, H RAS G12V colony 1 colony 2 colony H-Ras G12V H-Ras G12V

21 Metabolic vulnerabilities of SDH-deficient tumours

22 Conclusions Metabolomics is a robust and efficient tool for mechanistic studies of metabolic adaptabilities and vulnerabilities, but also as an important sensor of biomarkers for early prognosis and detection of recurrence disease FH or SDH loss of function leads to the accumulation of fumarate or succinate, respectively, and to the inhibition of dioxygenases, including DNA demethylases SDH loss of function renders cells more dependent on PC to sustain aspartate and nucleotide biosynthesis SDH loss in the kidney leads to hyperproliferative benign cyst formation in-vivo (in GEMMs) HRas activation in the kidney does not demonstrate any phenotype, but it dramatically accelerate cyst formation and size in SDHB ablated genotype

23 CML is a stem cells disease Chronic Normal myeloid haemopoiesis leukaemia Self-renewal Differentiation HSC LSC BCR ABL Imatinib CD34 CD38 CLP MEP MPP CMP GMP Survival and Proliferation Granulocytes Macrophages Platelets Erythrocytes T cells B cells

24 TKIs do not target CML stem cells Death CD34

25 TKIs do not target CML stem cells Progenitors cells Short-term colony formation assay 150 CML Stem cells Long- term colony formation assay 150 (LTC-IC) number colonies (% of untreated) number colonies (% of untreated) Untreated Imatinib 0 Untreated Imatinib CML CD34+ cells Single drug treatment Remaining cells plated into colony assay Feeder cells 5 weeks in culture Colonies count after 2weeks Measures drug effect on stem cells

26 Metabolomics in CML CD34+ cells versus CML CD34- cells MNC CD34+ Stem+ Progenitors cells CML Patient at diagnosis CD34 - Differentiated cells

27 Fatty acid oxidation is increased in CML CD34+ cells 12 C (m) 13 C (m+1) 13 C 16 Palmitate Acetyl-CoA Aspartate Oxaloacetate Malate Fumarate Succinate Citric Acid Cycle Citrate Isocitrate CO 2 Succynil-CoA α-ketoglutarate Glutamate Glutamine CO 2

28 Fatty acid oxidation is increased in CML CD34+ cells

29 CML CD34+ cells have an increase in oxidative metabolism 13 C 6 Glucose Aspartate PC Oxaloacetate Pyruvate CO 2 Acetyl-CoA PDH Malate Fumarate Succinate Citric Acid Cycle Citrate Isocitrate CO 2 Succynil-CoA α-ketoglutarate Glutamate CO 2

30 increased oxidative metabolism in CML CD34+ Vs. normal CD34+ cells CD34+ CML Patient at diagnosis CD34+ Healthy donor

31 increased oxidative metabolism in CML CD34+ Vs. normal CD34+ cells

32 increased oxidative metabolism in CML stem cells

33 PHARMACOLOGICAL INHIBITION OF MITOCHONDRIAL METABOLISM WITH TIGECYCLINE FDA approved antibiotic Inhibitor of bacterial protein synthesis Inhibitor of mitochondrial protein synthesis

34 Inhibitor of mitochondrial oxidative metabolism decreases proliferation of CD34+ CML cells

35 Inhibition of mitochondrial oxidative metabolism targets CML stem cells

36 In vivo model to study drug effect on CML Stem Cells Tail vein injection CML CD34+ cells Sub-lethal irradiation 6 weeks engraftment NSG MICE NOD scid gamma N=24 CD45 Human Tigecycline Imatinib Confirmation of engraftment Vehicle (n=6) TIG (n=6) IM (n=5) IM+TIG (n=6) In vivo drug treatment for 4 weeks

37 Inhibition of mitochondrial oxidative metabolism in-vivo is tolerated in treated mice

38 Inhibition of mitochondrial oxidative metabolism eradicates CML stem cells in-vivo

39 Inhibition of mitochondrial oxidative metabolism delays disease relapse after Imatinib withdrawal 60 After 3 weeks of treatment Number of human CD34 + cells (x10 3 ) Vehicle TIG IM TIG+IM 2 or 3 weeks of treatment discontinuation After 2-3 weeks of treatment discontinuation Number of human CD34 + cells (x10 3 ) Experiment 2 Experiment 1 0 Vehicle TIG IM TIG+IM

40 Acknowledgements Gottlieb Lab Current members Elaine MacKenzie Simone Cardaci Henry Däbritz Jiska van der Reest Elodie Kuntz Johan Vande Voorde Metabolomics Gillian McKay Niels van den Broek Past members Mary Selak Christian Frezza Leon Zheng

41 Acknowledgements Gottlieb Lab Current members Elaine MacKenzie Simone Cardaci Henry Däbritz Jiska van der Reest Elodie Kuntz Johan Vande Voorde WOLFSON WOHL CANCER RESEARCH CENTRE Vignir Helgason PAUL O GORMAN LEUKAEMIA RESEARCH CENTRE Tessa Holyoake Metabolomics Gillian McKay David Sumpton