Dr. Brendan Mannings Powerpoint Presentation from the October 26, 2006 LAM/TSC Research Seminar in Boston MA

1. Signaling events triggered by inactivation of the TSC1-2 Complex Implications for TSC/LAM pathology and treatment

2. X TSC1-TSC2 Rheb mTORC1 Other effectors?? (B-Raf) 2. Aberrantly high mTORC1 signaling OFF ON UNKNOWN Amino Acids cell growth cell proliferation cyclin D c-Myc etc… TIF-IA UBF ribosome biogenesis cap-dependent translation 4E-BP1 S6K1/2 eIF4E eIF4B SKAR S6 1. Constitutive Rheb -mTORC1 signaling Pol I cell survival Growth Factors PI3K Akt others GSK3 FOXO NF  B PTEN eIF2B cell proliferation

3. X TSC1-TSC2 Rheb mTORC1 Other effectors?? (B-Raf) 2. Aberrantly high mTORC1 signaling Growth Factors PI3K Akt others GSK3 FOXO NF  B PTEN cell survival eIF2B OFF ON UNKNOWN Amino Acids cell growth cell proliferation cyclin D c-Myc etc… TIF-IA UBF ribosome biogenesis cap-dependent translation 4E-BP1 S6K1/2 eIF4E eIF4B SKAR S6 1. Constitutive Rheb -mTORC1 signaling Pol I

4. X TSC1-TSC2 Rheb mTORC1 cell growth cell proliferation ribosome biogenesis cap-dependent translation 4E-BP1 S6K1/2 eIF4E Pol I Growth Factors PI3K Akt others GSK3 FOXO NF  B PTEN 2. Aberrantly high mTORC1 signaling 1. Constitutive Rheb -mTORC1 signaling cell proliferation OFF ON UNKNOWN cell survival eIF2B

5. cap-dependent translation X TSC1-TSC2 Rheb mTORC1 cell growth cell proliferation Growth Factors PI3K Akt others GSK3 cell survival FOXO NF  B ribosome biogenesis PTEN 4E-BP1 S6K1/2 eIF4E Pol I 2. Aberrantly high mTORC1 signaling 1. Constitutive Rheb -mTORC1 signaling OFF ON UNKNOWN eIF2B

6. GSK3  is constitutively phosphorylated on S9 in TSC cells and tumors Tsc2+/+ v. Tsc2-/- MEFs pGSK3  (S9) IHC Tsc2+/- Liver Hemangioma

7. 15 min pretreatment : w = 100 nM wortmannin; r = 20 nM rapamycin Reconstituted Tsc2-/- MEFs PI3K-independent / mTOR-dependent Phosphorylation of GSK3 in TSC-deficient cells

8. S6K1 is the GSK3 kinase in TSC null cells -5 -4 -3 -2 -1 P +1 GSK3  R A R T S S 21 F GSK3  R P R T T S 9 F eIF4B R S R T G S 422 E rS6 R R R L S S 236 L

9. S6K1 IP-kinase assays from serum-starved cells S6K1 is the GSK3 kinase in TSC null cells In Vitro S6K1 siRNA knockdown in serum-starved TSC null cells In Vivo

10. S6K1 is the GSK3 kinase and S6K2 is the S6 kinase in TSC null MEFs S6K1 v. S6K2 siRNA knockdown in serum-starved TSC null cells

11. cap-dependent translation TSC1-TSC2 Rheb mTORC1 cell growth cell proliferation Growth Factors PI3K Akt GSK3 ribosome biogenesis 4E-BP1 S6K1 eIF4E Pol I X eIF2B

12. cap-dependent translation X TSC1-TSC2 Rheb mTORC1 cell growth cell proliferation Growth Factors PI3K Akt ribosome biogenesis 4E-BP1 S6K1 eIF4E Pol I X GSK3 eIF2B 1) GSK3 activity is lower in serum-starved TSC cells. 2) Rapamycin activates GSK3 in TSC cells. 3) Aberrant regulation of GSK3 contributes to the proliferation properties of TSC1/2 null cells.

13. GSK3 kinase activity is reduced in Tsc2-/- cells and is activated by rapamycin Serum starved littermate-derived MEFs IP Kinase Assay

14. Constitutive inhibition of GSK3 contributes to the serum-free proliferation of Tsc2-/- cells 48h-no serum 48h-no serum

15. Constitutive inhibition of GSK3 contributes to the serum-free proliferation of Tsc2-/- cells Serum-starved Tsc2 null cells

16. cap-dependent translation X TSC1-TSC2 Rheb mTORC1 cell growth cell proliferation GSK3 ribosome biogenesis eIF2B 4E-BP1 S6K1 eIF4E Pol I neuronal polarity/survival metabolic processes

17. GSK3 is phosphorylated in TSC “giant cells” H&E pS6 Tuber SEGA Vimentin pGSK3  (S9) Merge Mustafa Sahin

18. H&E Periodic acid Schiff (PAS) Cardiac rhabdomyomas are common in newborns with TSC Comprised of vacuolated myocytes filled with glycogen S6K1 GSK3 glycogen synthase

19. GSK3 inhibition can enhance the survival of smooth muscle cells under hypoxic conditions

20. GSK3 is constitutively phosphorylated in Tsc2-/- smooth muscle cells Serum-starved ELT3 Cells Alex Lipovsky

21. cap-dependent translation X TSC1-TSC2 Rheb mTORC1 cell growth cell proliferation Growth Factors PI3K Akt others GSK3 cell survival FOXO NF  B ribosome biogenesis PTEN 4E-BP1 S6K1/2 eIF4E Pol I 2. Aberrantly high mTORC1 signaling 1. Constitutive Rheb -mTORC1 signaling eIF2B

22. X TSC1-TSC2 Rheb mTORC1 cell growth cell proliferation Growth Factors PI3K Akt GSK3 cell survival FOXO NF  B 4E-BP1 S6K1/2 eIF4E TORC2 2. Aberrantly high mTORC1 signaling 1. Constitutive Rheb -mTORC1 signaling HIF1  VEGF VEGFR PI3K Akt TORC1 Angiogenesis Endothelial Cell TORC2 Tumor Cell

23. X TSC1-TSC2 Rheb mTORC1 cell growth cell proliferation Growth Factors PI3K Akt GSK3 cell survival FOXO NF  B 4E-BP1 S6K1/2 eIF4E TORC2 HIF1  VEGF Effects of mTORC1 inhibitors on tumors with loss of TSC1-2? RAP VEGFR PI3K Akt TORC1 Angiogenesis Endothelial Cell TORC2 Tumor Cell

24. X TSC1-TSC2 Rheb mTORC1 cell growth cell proliferation 4E-BP1 S6K1/2 eIF4E VEGFR PI3K Akt TORC1 Angiogenesis Endothelial Cell TORC2 Tumor Cell PI3K Akt GSK3 cell survival FOXO NF  B TORC2 Effects of mTORC1 inhibitors on tumors with loss of TSC1-2? RAP 12h rap allows reactivation of Akt

25. X TSC1-TSC2 Rheb mTORC1 cell growth cell proliferation Growth Factors PI3K Akt GSK3 cell survival FOXO NF  B 4E-BP1 S6K1/2 eIF4E TORC2 VEGFR PI3K Akt TORC1 Angiogenesis Endothelial Cell TORC2 Tumor Cell Effects of mTORC1 inhibitors on tumors with loss of TSC1-2? RAP

26. mTORC1 inhibitors block the “feedback loop” and allow reactivation of PI3K-Akt signaling. Are they effective LAM/TSC therapeutics?

27. mTORC1 inhibitors can enhance survival of TSC null cells RAD001 rescues lethality of dTsc1 -/- larvae Radimerski et al. 2002 Genes Dev Rap restores IGF1-induced survival of Tsc2 -/- MEFs Harrington et al. 2004 J. Cell Biol. Rap protects Tsc2 -/- MEFs from DNA damage- induced cell death Ghosh et al. 2006 Cancer Cell

28. Before Rapamycin for 2.5 Mos Rapamycin’s effects on TSC SEGAs Franz et al. 2006 Ann Neurol 4 Mos off Rapamycin Rapamycin an additional 8 Mos

29. X TSC1-TSC2 Rheb mTORC1 cell growth cell proliferation Growth Factors PI3K Akt GSK3 cell survival FOXO NF  B 4E-BP1 S6K1/2 eIF4E TORC2 HIF1  VEGF VEGFR PI3K Akt TORC1 Angiogenesis Endothelial Cell TORC2 Tumor Cell Effects of mTORC1 inhibitors on tumors with loss of TSC1-2? RAP

30. X TSC1-TSC2 Rheb mTORC1 cell growth cell proliferation Growth Factors PI3K Akt GSK3 cell survival FOXO NF  B 4E-BP1 S6K1/2 eIF4E TORC2 VEGFR PI3K Akt TORC1 Angiogenesis Endothelial Cell TORC2 Tumor Cell Effects of mTORC1 inhibitors on tumors with loss of TSC1-2? RAP RAP

32. X TSC1-TSC2 Rheb mTORC1 cell growth cell proliferation Growth Factors PI3K Akt GSK3 cell survival FOXO NF  B 4E-BP1 S6K1/2 eIF4E TORC2 VEGFR PI3K Akt TORC1 Angiogenesis Endothelial Cell TORC2 Tumor Cell Effects of mTORC1 inhibitors on tumors with loss of TSC1-2? RAP RAP

33. X TSC1-TSC2 Rheb mTORC1 cell growth cell proliferation Growth Factors PI3K Akt GSK3 cell survival FOXO NF  B 4E-BP1 S6K1/2 eIF4E TORC2 VEGFR PI3K Akt TORC1 Angiogenesis Endothelial Cell TORC2 Tumor Cell Effects of mTORC1 inhibitors on tumors with loss of TSC1-2? RAP RAP RAP ? ?

34. Rapamycin increases basal and growth factor-stimulated activation of Akt in Tsc2-/- smooth muscle cells Serum-starved ELT3 Cells Alex Lipovsky

35. X TSC1-TSC2 Rheb mTORC1 cell growth cell proliferation Growth Factors PI3K Akt GSK3 cell survival FOXO NF  B 4E-BP1 S6K1/2 eIF4E TORC2 VEGFR PI3K Akt TORC1 Angiogenesis Endothelial Cell TORC2 Tumor Cell RAP RAP RTK inhibitor PI3K inhibitor Akt inhibitor mTOR KINASE inhibitor TORC2