1. A crossroad to tumor metabolome: pyruvate kinase type M2 Sybille Mazurek 1 Institute of Biochemistry & Endocrinology, Veterinary Faculty, University of Giessen, 35392 Giessen, Germany 2 ScheBo Biotech AG, Giessen, Germany

2. This lecture is dedicated to Prof. Dr. med. vet. Erich Eigenbrodt (1949 - 2004) Head of the Department Comparable Biochemistry of Animals Institute of Biochemistry & Endocrinology, Veterinary Faculty, University of Giessen, Giessen, Germany

3. glucose PEP lactate pyruvate Pyruvate kinase ADP ATP Glycolysis Nucleogenesis M2-PK lung, proliferating cells: embryonic cells, adult stem cells, tumor cells Gluconeogenesis L-PK liver, kidney Energy production M1-PK muscle, brain

8. Tumor M2-PKLung M2-PK

11. kinase 3´ gagpolenvsrc pp60 v-src p19p27p12p15 gp35gp85 core proteinsreverseenvelope tyrosine proteinstranscriptase Genome and gene products of the Rous Sarcoma Virus NH 2 COOH tyr P ATPADP U3U3 R 5´ b (a) pyruvate kinase type M2 enolase lactate dehydrogenase R U 5

12. Gelpermation of purified M2-PK in NIH 3T3 cells control pp60 v-src 30405060 0 20 40 60 80 100 120 140 fractions [mU/ml]

13. Genome of the HPV 16

14. HPV-16 E7 Tumor M2-PKM2-PK E7 oncoprotein of HPV-16

15. 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 10 fractions Gel permeation of M2-PK in NRK cells NRK 30204050 [U/ml] NRK + ras NRK + ras + E7

16. Glycolytic enzyme-complex glycolytic enzymes: HK, GAPDH PGK, PGM, PK, LDH other enzymes: NDPK, AK, G6PDH components of the protein kinase cascades: RAF, MEK, ERK AU rich mRNA components: Isoelectric focusing of NRK cells pyruvate kinase activity [U/ml] glycolytic enzyme complex pI 6.0 pI 6.7 0 2 4 6 8 10 12 3545 fractions [U/ml] 304550 NRK NRK + ras

17. PEP affinity of the tetrameric and dimeric M2-PK 0.0 0.2 0.4 0.6 0.8 1.0 012345 phosphoenolpyruvate [mM] v vmax 543210 phosphoenolpyruvate [mM] 0.2 0.4 0.6 0.8 1.0 0.0 Km + FBP FBP v vmax

18. glucose fructose-1,6 P 2 pyruvate lactate ADP ATP normal proliferating cells: DNA Energy production M2-PK

19. glucose ADP ATP pyruvate lactate fructose-1,6 P 2 oncoproteins + nucleic acids phospholipids amino acids tumor cells: PEP

20. temperature sensitive RSV transformed chicken embryo cells 0 1 2 3 4 5 35°C42°C nmoles/mg *** fructose 1,6-P 2 42°C35°C P-ribose-PP ***

21. fructose 1,6-bisphosphate 0 10 20 30 40 [nmoles/mg protein] *** NRKNRK + ras * NRK + ras + E7 x + SD

22. glucose ADP ATP pyruvate lactate fructose-1,6 P 2 oncoproteins + nucleic acids phospholipids amino acids tumor cells: PEP GDP GTP (UTP, CTP)

23. NRK cells 0 1 2 3 4 5 6 7 parental+ rasras and E7 transformed Correlation between the tetramer : dimer ratio of M2-PK and the (ATP+GTP) : (UTP+CTP) ratio (ATP+GTP) : (UTP+CTP)

24. truncated citrate cycle citrate  -keto- glutarate oxalo- acetate  -keto- glutarate pyruvate fatty acids PEP glucose DNA, amino acids, phospholipids fatty acids M2-PK acetyl-CoA oxalo- acetate  -keto- glutarate pyruvate malate glutaminolysis lactate aspartate glutamine glutamate glutaminase malic enzyme glutamate proline glutamine phospholipids

25. Correlation between glucose consumption and fatty acid release in xenotransplanted human mammary carcinoma glucose consumption [nmole/(g min)] fatty acid release [nmole/(g min)] slope = 58 r = 0.706

26. glucose ADP ATP pyruvateglutaminelactate fructose-1,6 P 2 tumor cells: oxygen starvation oncogenes + DNA glutaminolysis + oxygen starvation

27. lactate production glucose consumption 010203040102030 uptakerelease glutamine [nmoles/g min] 0.5 1.0 1.5 2.0 Correlation between the lactate production : glucose consumption ratio and glutamine release in xenotransplanted human cervix carcinomas slope = 0.02 r = 0.86

28. Metabolic characteristics of tumor cells Expression of the pyruvate kinase isoenzyme type M2 which is mainly in the dimeric form High phosphometabolite pools High channelling of glucose carbons to synthetic processes A low (ATP+GTP) : (UTP+CTP) ratio A high glutaminolytic capacity The pyruvate kinase isoenzyme type M2 which can switch between a highly active tetrameric form and an inactive dimeric form, is an important metabolic sensor to adapt tumor metabolism to varying nutrient and oxygen supply.

29. ® Metabolome ProteomeGenomeTumor- http://www.metabolic-database.com

30. melanoma Ugurel et al.: Int. J. Cancer (2005), 117: 825-830 pancreatic cancer Ventrucci et al.: Dig. Dis. Sci. (2004), 49: 1149-1155 cervical carcinoma Kaura et al.: J. Obstet. Gynaecol. Res. (2004), 30: 193-196 thyroid carcinoma Bena-Boupda et al.: Anticancer Res. (2003), 23: 5237-5240 gastric cancer Kim et al.: Korean J. Gastroenterol. (2003), 42: 387-393 breast cancer Hoopmann et al.: Cancer Lett. (2002), 187: 223-228 Lüftner et al.: Anticancer Res. (2000), 20: 5077-5082 lung cancer Schneider et al.: Cancer Detec. Prev. (2000), 24: 531-535 bile duct cancer Kim et al.: Korean J. Gastroenterol. (2003), 42: 387-393 oesophageal carcinoma Schulze: Anticancer Res. (2000), 20: 4961-4964 colorectal cancer Zhang et al.: World J. Gastroenterol. (2004), 10: 1643-1646 Increase of Tumor M2-PK in EDTA-Plasma renal cell carcinoma Wechsel et al.: Anticancer Res. (1999), 19: 2583-2590 Oremek et al.: Anticancer Res. (1999), 19: 2599-2601

31. Robson score Tumor M2-PK (U/ml) 0 100 200 300 400 Nephritis I II III IV 42 Cut off Tumor M2-PK concentrations in EDTA plasma samples from patients with renal cell carcinomas Correlation between Tumor M2-PK values and staging Oremek et al., Anticancer Res. (2000), 20: 5095-5098. 11 36 55 14 n =

32. Tumor M2-PK concentrations in EDTA plasma samples from patients with lung tumors Correlation between Tumor M2-PK values and staging J. Schneider in: Tumor Markers, AACC Press (2002), 471-475 0 20 40 60 80 100 120 Stage IStage IIStage IIIStage IV n =2365155 Tumor M2-PK [U/ml] Cut off [15 U/ml] 25 % 50 % 75 % 241189 sensitivity 28 %33 %64 %73 %

33. Tumor M2-PK concentrations in EDTA-Plasma samples of patients with Colorectal, Gastric,Oesophageal and Pancreatic Cancer Schulze, Anticancer Res. (2000), 20: 4961-4964 0 20 40 60 80 100 120 140 controls 141 Cut off [U/ml] 50 % 75 % colorectal cancer gastric cancer oesophageal cancer pancreatic cancer n =1631378726 25 %

34. N Tumor M2-PK CEA CA 19-9CA 72-4 Best Combination Non- metastasized 117 48% 34% 18% n.d. Metastasized 46 54% 72% 50% n.d. Colorectal cancer Total 163 50% 42% 27% n.d. Tumor M2-PK + CEA 67 % Non- metastasized 65 63% 12% 31% 25% Metastasized 72 71% 39% 54% 57% Gastric cancer Total 130 67% 26% 45% 41% Tumor M2-PK + CA 72- 4 82 % Non- metastasized 77 57% 9% 16% 10% Metastasized 10 60% 20% 70% 14% Oesoph. cancer Total 87 59% 15% 43% 12% Tumor M2-PK + CA 19-9 Pancreatic cancer Total 26 73% 42% 85% 43% Tumor M2-PK + CA 19-9 96 % 65 % Sensitivity of Tumor M2-PK in comparison to CEA, CA 19-9, CA 72-4 Schulze, Anticancer Res. (2000), 20: 4961-4964

35. Tumor M2-PK concentrations in EDTA-plasma samples Follow-up study of a patient with a RCC with metastases Complete Remission 0 20 40 60 80 100 120 140 0 months3 months6 months9 months 1 st cycle 2 nd cycle 3 rd cycle 4 th cycle Cut off Immunochemo [U/ml] W. Wechsel : in: Tumor Markers, AACC Press (2002), 471-475

36. x x x x 364612 0 20 40 60 80 100 120 0weeks [ng/ml] Tumor M2-PK [U/ml] CEA CYFRA-21 SCC NSE tumor resection diagnosisrelapse Schneider et al., Anticancer Res. (2002), 22: 311-318 Follow-up study of a patient with a squamous cell carcinoma of the lung

37. 973530n = NORMALCANCERADENOMA 60 50 40 30 20 10 0 Tumor M2-PK concentrations in stool samples of patients with colorectal tumors McLoughlin et al. Dublin, Ireland Poster abstract P395, NCRI Cancer Conference, October 2005, Birmingham, UK [U/ml] sensitivity:97 %76 % Cut off 25 % 50 % 75 %

38. 0 10 20 30 40 50 60 70 80 90 100 110 120 Controls n =191 1.7 Dukes A n = 23 52.2% Dukes B n = 24 76.0% Dukes C n = 26 80.8% 12.2 46.3 Dukes D n = 17 82.4% 16.6 Sensitivity: Cut off [U/ml] 5.5 Tumor M2-PK concentrations in stool samples of patients with colorectal tumors 25 % 50 % 75 % Hardt, et al., Brit. J. Cancer (2004), 91: 980-984

39. Collaborators Robert R. Friis RSV transformed cells University of Bern, Switzerland Werner Zwerschke, Pidder Jansen-Dürr E7 transformed cells Tyrolean Cancer Institute, Innsbruck Austria Erich Eigenbrodt University of Giessen, Germany Metabolome analysis Peter Vaupel Xenotransplanted human mammary carcinomas University of Mainz, Germany ScheBo Biotech AG Giessen, Germany ELISA Tumor M2-PK