inhibition of the mtor and mapk pathways in the treatment of osteosarcoma

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