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Therapeutic Targeting of mTORC1 and mTORC2 Signaling Abrogates TSC2-null Tumor Growth and Prevents Tumor Recurrence

Vera P. Krymskaya, Ph.D. Pulmonary, Allergy & Critical Care Division Department of Medicine Abramson Cancer Center University of Pennsylvania

FROM CHARACTERIZING A TSC2 GENE FUNCTION TO CLINICAL TRIALS

H&E

SM -actin Blood vessel P-S6

Normal LAM V V

45 40 35 30 25 20 15 10 5 0 RAPA (nM) 0 DNA synthesis

LAM nodule Human LAM-derived (LAMD) cells * p<0.04 ** p<0.03 *** **** p<0.02 p<0.02 2 20 200

(N Engl J Med 358, 140-151, 2008) (N Engl J Med 10.1056/on-line, 2011)

0.02

0.2

(JBC, 277: 30958-30967, 2002; J. Cell Biol., 167: 1171, 2004)

Growth factors Insulin

Combinational therapy for TS/LAM?

PIP2

PIP3

PIP3

IRS1

PI3K

Akt

Cytostatic Effects of Rapalogs

TSC1

TSC2

Novel mTORC2 Therapeutic mTOR target? Rictor mLST8

Rheb

mTORC1

mTOR Raptor S6K1

mLST8 4E-BP1

Rho GTPases

Cell growth Cell proliferation

[Goncharova et al., 2004, J Cell Biol] [Goncharova et al., 2006 Am J Resp Cell Mol Biol]

I. mTORC2 and RhoA GTPase in TSC2-null and LAM cell growth and survival

Both mTORC1 and mTORC2 are required for TSC2-null cell proliferation

siRNA Rheb Rheb

-

+

TSC1 TSC2 DNA synthesis, %

Rheb mTORC1 mTOR Raptor 4EBP1 S6K

80 70 60 50 40 30 20 10

Actin siRNA mTOR mTOR

-

+

mTORC2 mTOR Rictor Rho GTPase

?

* * *

Control siRNA Rheb siRNA mTOR siRNA Raptor siRNA

*

Actin siRNA Raptor Raptor Actin siRNA Rictor

-

+

0

-

+

Rictor siRNA

Rictor Actin

Cell proliferation

(Goncharova et al., Mol Cell Biol, 31:2584-2498, 2011)

siRNA Rictor has little effect on S6 phosphorylation

Control siRNA Rheb siRNA mTOR siRNA Raptor siRNA Rictor siRNA

TSC1 TSC2

Rheb mTORC1 mTOR Raptor 4EBP1 S6K mTORC2 mTOR Rictor Rho GTPase

P-S6

GFP

*p < 0.01

100 80

P-S6, %

60 40 20 0 Control siRNA

* *

*

Rheb siRNA

mTOR siRNA

Raptor siRNA

Rictor siRNA

siRNA Rictor inhibits P-Ser473-Akt in TSC2-null ELT3 cells

TSC2-null + siRNA contr + siRNA Rictor Rictor P-Ser473-Akt Akt P-S6 S6 LAMD + + -

TSC1 TSC2

Rheb mTORC1 mTOR Raptor 4EBP1 S6K mTORC2 mTOR Rictor Rho GTPase

P-Ser473Akt

siRNA Rictor

Cell proliferation

Activated RhoA GTPase in TSC2-null ELT3 and LAMD cells

TSC2-null Rho-GTP TSC1 TSC2

Rheb mTORC1 mTOR Raptor 4EBP1 S6K mTORC2 mTOR Rictor Rho GTPase

Total Rho TSC2 TSC2

-

+

(Goncharova et al., 2004, J Cell Biol) (Goncharova et al., 2006 Am J Resp Cell Mol Biol) (Goncharova et al, 2011 Mol Cell Biol)

mTORC2 is required for RhoA GTPase activity in TSC2-null and LAMD cells

TSC2-null Rho-GTP TSC1 TSC2 Total Rho Rictor siRNA Rictor siRNA contr + LAMD

+ TSC2-null

+

+ LAMD

p < 0.05

siRNA Rictor

mTORC2 mTOR Rictor Rho GTPase Rho GTP/total (fold)

siRNA Rictor siRNA contr +

1.2 1 0.8 0.6 0.4 0.2 0

p < 0.01

+ -

+

+ -

mTORC2 modulates RhoA-dependent stress fiber formation in TSC2-null cells

GFP F-actin

Cells w/o stress fibers, %

?

+

40 30 20 10 0

GFP

siRNA raptor

4EBP1 S6K

Rho GTPase

siRNA rictor

mTORC1 mTOR Raptor

mTORC2 mTOR Rictor

siRNA mTOR Stress fiber formation

TSC2

TSC1 TSC2

GFP

* * *

TSC2 siRNA siRNA siRNA mTOR rictor raptor

mTORC2 modulates RhoA-dependent stress fiber formation in TSC2-null cells

GST GST F-actin

TSC1 TSC2

siRNA rictor

siRNA Rictor V14RhoA

mTORC2 mTOR Rictor Rho GTPase

Cells w/o stress fibers, %

Stress fiber formation

siRNA rictor + GST-V14RhoA

GSTV14RhoA

40 30 20 10 0

GST siRNA GST siRNA rictor+ rictor V14RhoA V14RhoA *p < 0.001 *p < 0.001

RhoA GTPase is necessary for TSC2-null and LAMD cell proliferation

60 50 40 30 20 10 0

TSC2-null

LAMD

p < 0.001

p < 0.001

DNA synthesis, %

DNA synthesis, %

16 12 8 4 0 0.2 1 2 0 C3 transferase, µg/ml

0

0.2 1 2 C3 transferase, µg/ml 32.9 31.6

RhoA , % of total 0 siRNA contr, h 72 siRNA RhoA, h RhoA Actin p-S6 S6

-

-

48

72 100 80 60 40 20 0 siRNA DNA synthesis, % of control

TSC2-null

p < 0.01

LAMD

p < 0.001

Contr siRhoA

Contr siRhoA

V14RhoA reverses anti-proliferative effects of TSC2 and siRNA Rictor in TSC2-null cells

80 DNA synthesis, % 60

p < 0.005 p < 0.001

TSC1 TSC2

Rheb mTORC1 mTOR Raptor 4EBP1 S6K mTORC2 mTOR Rictor Rho GTPase

TSC2

40 20 0 GFP GST GST- GFP- TSC2 + V14RhoA TSC2 V14RhoA

p < 0.001

DNA synthesis, %

siRNA Rictor V14RhoA

80 60 40 20 0 GST

Cell proliferation

GSTsiRNA V14RhoA rictor

siRNA rictor + V14RhoA

RhoA GTPase is required for TSC2-null cell survival

Control siRNA, 72 h Phase siRNA RhoA 48h 72h

TSC1 TSC2

mTORC2 mTOR Rictor

Apoptosis, %

siRNA RhoA

Rho GTPase

Annexin V

40 30 20 10 0

* *

Control 48h 72h siRNA siRNA RhoA

Cell survival

Can Rho GTPase activity be therapeutically targeted?

Acetyl-CoA + Acetoacetyl-CoA

Rho Rho GTP

HMG-CoA

GDP

Statins

HMG-CoA reductase

- Reduces melanoma, colorectal and breast cancer - Inhibit breast cancer cell proliferation via prevention of RhoA GTPase geranylgeranylation and decrease tumor formation in mouse mammary tumor models; - Inhibit geranylgeranylation of RhoA and induce cell apoptosis in mouse melanoma model.

Mevalonate

Ras -GDP

Farnesyl-PP (FPP) Squalene

Geranyl-Geranyl-PP (

GGPP)

Proliferation Survival Migration

Ras -GTP Proliferation

Cholesterol

GDP Rac

GTP Rac

Simvastatin inhibits RhoA GTPase activity is TSC2-null cells

Simvastatin, µM

TSC1 TSC2

0

0.1

1

10

Rho-GTP Total Rho Rho activity, %

mTORC2 mTOR Rictor

100

0

80 60 40 20 * ** 0 0.1 1 10

Simvastatin

Rho GTPase

0

0

Simvastatin, µM

Constitutively active RhoA GTPase rescues simvastatin-induced TSC2-null cell apoptosis

1 µM simvastatin + GST-V14Rho Cleaved Caspase 3 GST Merge

TSC1 TSC2

mTORC2 mTOR Rictor

V14RhoA

Rho GTPase

Apoptosis, %

Simvastatin

25 20 15 10 5 0

p < 0.001

Cell survival

Simvastatin, 1 µM V14RhoA

+ -

+ +

RhoA GTPase modulates Bcl-2 family protein levels in TSC2-null and LAMD cells

TSC2-null

TSC1 TSC2

Rheb mTORC1 mTOR Raptor 4EBP1 S6K mTORC2 mTOR Rictor Rho GTPase

LAMD Bcl2 Bim P-Bad Puma P-S6 S6 P-Akt Akt

Bcl2 Bim P-Bad Bok P-S6 S6 P-Akt Akt C3 +

Bcl-2 proteins Cell survival

C3

-

+

Combined targeting of mTORC1 and mTORC2 signaling pathways in vivo

Schematic representation of experimental design in vivo

Treatment groups: Control; Simvastatin; Rapamycin; Simvastatin + Rapamycin

Days of treatment 0 10 20 30 40 50

Monitoring for tumor recurrence

Tumor tissue collection 5x106 TSC2-null ELT3 cells IHC and IB analysis

End of treatment

Combination of simvastatin and rapamycin abrogates TSC2-null tumor growth and promote survival

400 350 300 250 200 150 100 50 0 0

100 Survival, % 80 60 40 20 10 20 30 Treatment, days 40 50 0 0 10 20 30 40 Treatment, days 50

Tumor volume, mm3

Control (20 mice) Simvastatin (20 mice) Rapamycin (21 mice) Simvastatin + rapamycin (18 mice)

(Goncharova et al., Mol Cell Biol, 31:2584-2498, 2011)

Rapamycin, but not simvastatin, inhibits S6 phosphorylation in TSC2-null tumors

Control RAPA Simvastatin RAPA + Simvastatin

120 100 80 60 40 20 0 Treatment, days RAPA Simvastatin P-S6, %

DAPI

P-S6

*

*

**

20 20

**

20

-

0

10

-

20

-

10

+ -

10

+

10

+ +

+ -

+

+ +

Simvastatin and rapamycin cooperate in inhibition of DNA synthesis in vivo

Control RAPA Simvastatin RAPA + Simvastatin

20 18 16 14 12 10 8 6 4 2 0 Treatment, days RAPA Simvastatin

Ki67, %

DAPI

Ki67

* ***

*

**

***

0

-

10

-

20

-

10

+ -

10

+

10

+ +

20

+ -

20

+

20

+ +

Simvastatin promotes apoptosis in TSC2-null tumors

Control RAPA Simvastatin RAPA + Simvastatin

DAPI

8 7 6 5 4 3 2 1 0

TUNEL

Apoptosis, %

*

* * *

Treatment, days RAPA Simvastatin

0

-

10

-

20

-

10

+ -

10

+

10

+ +

20

+ -

20

+

20

+ +

Schematic representation of experimental design in vivo

Treatment groups: Control; Simvastatin; Rapamycin; Simvastatin + Rapamycin

Days of treatment 0 10 20 30 40 50

Monitoring for tumor recurrence

Tumor tissue collection 5x106 TSC2-null ELT3 cells IHC and IB analysis

End of treatment

Tumor recurrence in rapamycin- but not simvastatin+rapamycin-treated mice

Day 21 post-treatment 350 300 250 200 150 100 50 0 0 10

Tumor volume, mm3

* * * *

20 30 40 50

Rapamycin

* * *

4 months post-treatment Rapamycin + Simvastatin

Post-treatment, days Rapamycin Rapamycin + simvastatin

(Goncharova et al., Mol Cell Biol, 31:2584-2498, 2011)

Rapamycin

TSC1 TSC2

Rheb mTORC1 mTOR Raptor 4EBP1 S6K mTORC2 mTOR Rictor Rho GTPase

Conclusions:

RhoA GTPase is downstream target of TSC2 and mTORC2 Activation of mTORC2/RhoA GTPase is necessary for LAMD and TSC2-null cell survival Combined targeting of growth with cytostatic rapamycin and survival with pro-apoptotic simvastatin abrogates TSC2-null tumor growth, improve survival, and prevents tumor recurrence

Cell growth

Cell survival

Simvastatin

University of Pennsylvania Elena A. Goncharova* Irene Khavin* Hua Li* Melane Fehrenbach Dmitry A. Goncharov* Wittaya Pimtong Andrew Eszterhas Gregory Cesarone Melane Fehrenbach Seung-Beom Hong LAM patients

Brigham & Women's Hospital Harvard University David J. Kwiatkowski University of Texas MD Anderson Cancer Center Cheryl L. Walker NIH/NHLBI The LAM Foundation University of Pennsylvania Research Fund Abramson Cancer Center

LAM registry NDRI

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