Dr. Lew Cantley's 1/18/07 presentation for the Boston LAM/TSC REsearch Seminar in Boston MA.

1. Regulation of Glycolysis downstream of mTOR

2. Germline loss of function mutations in Hamartin (TSC1) or Tuberin (TS2) result in a Hamartoma Syndrome, Tuberous Sclerosis Complex >> Estimated to affect 1 in 6000 individuals. >> Mapped to mutations in one of two tumor suppressor genes TSC1 (hamartin) or TSC2 (tuberin). >> Characterized by widespread benign tumors (hamartomas) most commonly affecting the brain, skin, lungs, and kidneys. >> Clinical manifestations include epilepsy, mental retardation, autism, facial fibromas, lymphangioleiomyomatosis (LAM) and renal complications.

3. PTEN* PI3K Hamartin*/Tuberin* AKT Rheb-GTP Rheb-GDP Cell Growth and Energy Management mTOR Protein Synthesis HIF Glycolysis Cell growth and proliferation Amino Acids Growth Factor AMP LKB1* AMPK Low Nutrient/hypoxia

4. Why does activation of PI3K/AKT/mTOR lead to increased cell growth? (The Warburg Effect: don’t put the cart before the horse)

5. Expression of Activated AKT in the prostate results in neoplasia that can be reversed by an mTOR inhibitor. Genes involved in glucose uptake and glycolysis are elevated in the AKT-induced neoplasia and are reduced within a day in response to the mTOR inhibitor, RAD001 (a rapamycin analog). These same genes are also regulated by HIF (hypoxia inducible factor). Yet hypoxia is unlikely in this mild neoplasia. Days RAD001

6. HIF Induction of Genes for glycolytic pathway Transcription/Translation PI3K AKT mTOR Degradation O 2 Proline Hydroxylase

7. PTEN* PI3K Hamartin*/Tuberin* AKT Rheb-GTP Rheb-GDP Cell Growth and Energy Management mTOR Protein Synthesis HIF Glycolysis Cell growth and proliferation Amino Acids Growth Factor AMP LKB1* AMPK Low Nutrient/hypoxia

8. PI3K AKT mTOR Glucose Pyruvate Pyruvate dehydrogenase Acetyl-CoA Fatty Acid Synthesis ATP ATP ATP Ox. Phos. High rates of ATP production by glycolysis frees the mitochondria from consuming pyruvate for ATP synthesis, allowing pyruvate to be used for macromolecule synthesis (fatty acids, proteins). Glucose Glut1

9. HK PFK1 PK Cancer cells upregulate key enzymes in glycolysis but also switch to a different isoform of pyruvate kinase from that used in most adult tissues: Pyruvate Kinase- Type M2 Why does this happen?

10. Pyruvate Kinase Isoforms L gene M gene L isozyme R isozyme M1 isozyme M2 isozyme tissue-specific promoter alternate splicing Liver -allosteric regulation by FBP -hormonal control Red Blood Cell -allosteric regulation by FBP Multiple tissues - muscle, brain -unregulated activity Fetal tissue/undifferentiated cells -allosteric activation by FBP -reexpressed in tumors 10 th exon 9 th exon 1 st exon 2 nd exon Heather Christofk

11. pY Y pY Y pY Y pY Y M1 M2 KE L eluates flowthroughs Preferential binding to pTyr peptides is specific to the M2 isoform Elute bound protein using 20mM phenylphosphate Run gel, western blot with flag tag antibody pY peptide library Lysates from H1299 cells stably expressing flag-PKs Control: Y peptide library α -flag *interestingly, while M2 binds pTyr peptides, its splice variant, M1, does not

12. Tumor tissues and cell lines express M2 isoform A. α -PKM1 α -PKM2 α -GAPDH B. A549 H1299 293T HeLa MCF10a muscle α -PKM1 α -PKM2 α -actin N T1 T2 T3 T4 Mammary glands from MMTV-neu mice before (N) and after tumor development (T) *Lysates from Momo Bentires-Alj Like tumor tissues, all cell lines tested express M2 isoform of PK Since PKM2 is expressed in growing cells, is M2 isoform expression important for cell growth?

13. M1 M2 PK-M Isoform Expression is Regulated by Alternative Splicing Adult Tissues Embryonic expression Cancer cells ?All Dividing cells

15. Dombrauckas et al, 2005, Biochemistry 44: 9417 FBP FBP FBP FBP Catalytic Pocket

16. Arg436 Lys433 Arg516 Arg455 Arg489 PK-M2

18. Glucose F-1,6-BP PEP Pyruvate F6P Maintaining a balance of glucose products needed for protein/lipid/nucleic acid synthesis while avoiding AMP elevation and oxidative stress is critical for tumor growth. Feed Forward Activation Feedback Inhibition DNA RNA Ribose + NADPH Glycerol Acetyl-CoA Lipid Synthesis Pentose Phosphate Shunt

19. Brendan Manning, Nicole Logsdon Tuberin/RHEB Andrew Tee, John Blenis Reuben Shaw, Monica Kosmatka LKB1/AMPK Nabeel Bardeesy , Ron DePinho, Lee Witters Heather Christofk, Pyruvate Kinase Matthew Vander Heiden Ning Wu Crystallization John Asara Mass Spec Acknowledgements