Pyruvate Kinase M2 : Linking Glycolysis to Amino Acid Biosynthesis Disclosure Information 2012 AACR Annual Meeting

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Pyruvate Kinase M2 : linking glycolysis to amino acid biosynthesis Disclosure Information 2012 AACR annual meeting Eyal Gottlieb The presented work was done in collaboration with Astex Pharmaceuticals I am a consultant of Astex Pharmaceuticals I will not discuss off label use and/or investigational use in my presentation Glucose G6P GA3P 3PG PEP PK Pyruvate Lactate Respiration Pyruvate Kinase L/R isoforms CAAT TATA PKLR Gene E1 E2 E3 E4 E5 E12 1q21 PKR mRNA E1 E2 E3E4E5 E6-E12 PKL E2 E3E4E5 E6-E12 mRNA Pyruvate Kinase M1/M2 isoforms PKM gene E8 E9 E10 E11 E12 15q22 E8E9 E11 E12 E8E10 E11 E12 E10 PKM1 mRNA PKM2 mRNA B Domain Active Site A Domain Inter-subunit interface Tyr105 Lys305 (acetylation) 180º (phosphorylation) Cys358 (oxidation) FBP Pro403/ 408 (hydroxylation) C Domain Nucleotides Glucose PPP G6P GA3P Phospholipids ROS 3PG Serine PEP Glycine PKM2 Pyruvate Lactate Respiration Cancer cell lines express high levels of PKM2 Silencing PKM2 expression does not change ATP levels and proliferation capacity of HCT116 cells Metabolic adaptation to PKM2 silencing in HCT116 cells PKM2 silencing diverts more glucose-derived carbons to serine/glycine biosynthesis and oxidative phosphorylation Metabolic adaptation to PKM2 silencing in HCT116 cells Serine and glycine starvation decreases PK activity Serine binds to and activates PKM2 in vitro Mutation of the amino acid binding pocket abolishes serine activation PKM2 H464A PKM2 S437Y 4000 800 Ser Ser FBP FBP 3000 600 2000 400 % activation % activation 1000 200 0 0 10 -10 10 -8 10 -6 10 -4 10 -2 10 0 10 -10 10 -8 10 -6 10 -4 10 -2 10 0 [compound] (M) [compound] (M) ITC data PKM2 WT PKM2 H464A PKM2 S437Y Kd for Ser 200µM N/B 210µM Serine specifically activates PKM2 but not PKM1 or PKLR PKLR PKM1 Ser 15 FBP 20 15 10 10 Fold-activation 5 Fold-activation 5 Ser FBP (relative to no enzyme control) (relative to noenzyme control) 0 0 10 -10 10 -8 10 -6 10 -4 10 -2 10 0 10 -10 10 -8 10 -6 10 -4 10 -2 10 0 [compound] (M) [compound] (M) Of all proteinogenic amino acids only serine 600 400 activates PKM2 RLU 200 0 Serine 10mM Lysine 10mM Tryptophan 10mM Tyrosine 10mM Threonine 10mM Asparagine 10mM Glutamate 10mM Basal activity Alanine 10mM Arginine 10mM Histidine 10mM Isoleucine 10mM Leucine 10mM Aspartate 10mM Glutamine 10mM noPEP Glycine 10mM PKM2 Proline 10mM Valine 10mM Phenylalanine 10mM Methionine 10mM Cystine 10mM FBP 50 uM FBP 50 uM Two known modifier amino acids of PKM2 (Tyr105 and Cys358) are in the vicinity of the serine binding domain Acknowledgements Barbara Chaneton Marc O’Reilly Liang (Leon) Zheng Petra Hillmann Christian Frezza Agnès Martin Elaine MacKenzie Joe Coyle Finn Holding Adam Hold.

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Phenotypic and Molecular Genetic Analysis of Pyruvate Kinase Deficiency in a Tunisian Family
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