Inhibition of the mTOR and MAPK pathways in the treatment

of osteosarcomaKathleen M. Diehl, M.D. FACS

Assistant Professor

University of Michigan

Background

• Osteosarcoma cell lines– SAOS-2, COL, OS-187

• Rapamycin– Sirolimus– Natural macrolide antibiotic (anti-fungal)– Binds to FKBP12 inhibiting mTORC1– Analogues

• CCI-779 (Wyeth)• RAD001 (Novartis)• AP23573 (Ariad)

Clinical Trials • CCI-779

– I/II lung, breast, neuroendocrine, uterine, cervical, soft tissue sarcomas– III (RCCA)– PR 7-9%– SD 26-36%

• RAD001– I/II, RCCA, solid tumors– PR 5-33%– SD 7.3-23.5%– Very high PR or SD rate soft tissue sarcoma

• AP23573– I/II hematologic, solid tumors, sarcoma– PR 3-11%– SD 25%

– 100% of sarcoma patients had PR or SD– 56% clinical improvement

PI3k

IFG-1Growth Factor

ReceptorsNutrientsHypoxiaStress

PTENIRS Ras Ras

bRaf

ERK1/2(p-MAPK)

ProliferationProliferation

Akt

p70s6K

Survival and Survival and Cell Cycle ProgressionCell Cycle Progression

MEK1/2TSC 1/2

Rheb

AKT4EBP

mTORTORC1

mTORTORC2

elF4E

uo-126Rapamycin

IC50 Comparing Sensitivity Between Cell Lines

IC50 of Rapamycin

0.0%10.0%20.0%30.0%40.0%50.0%60.0%70.0%80.0%90.0%

100.0%

1E-07 1E-05 0.001 0.1 10 1000

Rapamycin Concentration (uM)

Survival %

COL

OS187

SAOS-2

Flow Cytometry of Cells Treated for 48 hrs with Rapamycin

Cell Line G1 % S-phase % G2/G1 OS-187

Control 42.64 40.42 1.86 Rapa 67.31 26.64 1.86

% decrease S-phase 34% COL

Control 54.09 36.78 1.86 Rapa 64.79 27.96 1.86

% decrease S-phase 24% SAOS-2

Control 70.46 22.94 1.86 Rapa 68.48 16.77 1.86

% decrease S-phase 27% Flow cytometry results. Cells were were trypsinized, fixed in 70% alcohol, stained with Propidium Iodide, and analyzed with flow cytometry.

Decrease in cell cycle proteins cyclin D1 and cdk4 in OS-187 cells

Control Rapa

A

B C

A

B C

Control Treated

A = Cyclin D1B = cdk4C = Cyclin D3

Western blot 4EBP

Cont 50 100 200 50 100 2001hr 24 hr

p-4EBP

4EBP

Cont 50 100 200 50 100 200

1 hr 24 hrs

p-4EBP1

4EBP1

Cont 50 100 200 50 100 200 1 hr 24 hrs

p-4EBP1

4EBP1

COL

OS-187

SAOS-2

Western blot 70S6k

Note: lack of activity in COL and OS187 cells confirmed with 2-D gels for T389 and T421-424 at 0-24-48-72 hrs.

Cnt 1hr 8hr 24hr 1hr 8hr 24hr 1hr 8hr 24hr50nM 100nM 200nM

p-70 S6k

70 S6k

Cont 50 100 200 50 100 2001 hr 24 hrs

p-70 S6k

70 S6k

200 100 50 200 100 50 200 100 50 Cont

24hrs 8hrs 1hr

p-70 S6k

70 S6k

COL

OS-187

SAOS-2

Summary Treatment Osteosarcoma Cells with Rapamycin

• Concentration dependent decrease in cell growth and proliferation

• Associated with G1 arrest but not apoptosis

• Cell line dependent decrease in the phosphorylation of proteins of the mTOR pathway

• Decrease in cell cycle proteins

Proliferation Assays showing effectiveness of uo-126 in decreasing proliferation in these cells

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0 1 2 3 4 5

Days of Treatment

Fluorescence

Control

Rapa 50nM

Rapa 100nM

uo-126 10uM

uo-126 20uM

AKTI 3uM

AKTI 5uM

COL OS-187 SAOS-2

uo-126

• Synthesized, in-vitro use

• Inhibits active and inactive MEK1/2 of the Mitogen Activated Protein Kinase Pathway

• Cellular proliferation

COL

IC50 of Rapamycin with 10 uM U0126 (COL, 5 days)

0.00%

20.00%

40.00%

60.00%

80.00%

100.00%

120.00%

0.0000001uM 0.0000100uM 0.0010000uM 0.1000000uM10.0000000uM1000.0000000uM

Rapamycin Concentration (uM)

Survival % R+U R alone

OS-187 cells

IC50 of Rapamycin with 10 uM U0126 (OS187, 5 days)

0.00%

20.00%

40.00%

60.00%

80.00%

100.00%

120.00%

0.0000001uM 0.0000100uM 0.0010000uM 0.1000000uM10.0000000uM1000.0000000uM

Rapamycin Concentration (uM)

Survival % R+U R alone

SAOS-2 cells

IC50 of Rapamycin with 20 uM U0126 (SAOS-2, 5 days)

0.00%

20.00%

40.00%

60.00%

80.00%

100.00%

120.00%

0.0000001uM 0.0000100uM 0.0010000uM 0.1000000uM10.0000000uM1000.0000000uM

Rapamycin Concentration (uM)

Survival % R+U R alone

2-phase Flow Cytometry showing apopotosis with the addition of uo-126 to Rapa in COL and OS-187 cells

OS-187 Control OS-187 RapaOS-187 Rapa uo-126

COL Control COL Rapa COL Rapa uo-126

Summary

• The addition of the MAPK pathway inhibitor uo-126 to Rapamycin resulted in– Synergistic decrease in proliferation in COL

and OS-187 cells– Additive decrease in proliferation in SAOS-2

cells– Apoptosis

Conclusions

• The combination of inhibition of the mTOR and MAPK pathways shows promise for the treatment of osteosarcoma

Next Steps

• Confirmation with in-vivo model

• Comparison with inhibitors of other cell survival and proliferation pathways

• Comparison with other mTOR inhibitors

Acknowledgements

• Laurence BakerLaurence Baker• Qi Wu• Zhiyu Wang• Dafydd Thomas

• Rashmi Chugh• Kenine Comstock• Carolyn Hoban • Scott Schuetze• David Lucas

Thank You