Pyruvate Kinase M2 : linking 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 L/R isoforms

CAAT TATA

PKLR 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 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) S acidsonlyserine amino Of allproteinogenic e Tryptophanrine 10mM RLU

Lysi 200 400 600

n 10m 0 Tyr e 10mM M T o hr sin Aspare oni e 1 Gl n 0mM ag e 10mM utamain e B 10m asa te Ala 10 M l Ar n ac mM ine 10mti g vi Histidinein 10mMty activates PKM2 Isoleucinein 10mM e 10mM Leu M As c Glutaminep in 10mM artatee 10mM

Glyci n PhenylP n oPEP ro e 10mM lin Val e M al in 10m e aninee 10mM th 10 M io n mM Cystin e in 10m e FBP 1050 uMM F mM B P 50 u

M PKM2 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