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