Inhibition of the mTOR and MAPK pathways in the treatment of osteosarcoma
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Transcript of 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