Post on 15-Jan-2016
description
Imatinib Mesylatemodulates Regulators of Quiescence in
Gastrointestinal Stromal Tumor (GIST) cells
Joshua A. Parry, Matthew F. Brown, Danushka Seneviratne,Stefan Duensing, Anette Duensing
University of Pittsburgh Cancer Institute,Pittsburgh, PA
Clinical and experimental findings suggesttumor cell quiescence in imatinib-treated GIST
• Major problems of imatinib therapy: therapy resistance incomplete responses
• Detectable tumor mass under treatment
• Relapse when imatinib therapy is terminated
• Tumor cells that do not respond to imatinib by undergoing apoptosis but remain quiescent may be a reservoir for resistant GIST cells
Holdsworth, Am J Roentgenol 2007;189:W324-W330
transgenic KitV558 /+ mouse
(in collaboration with C. Antonescu and P. Besmer)
PE
TC
T
baseline imatinib
Tumor cell quiescence
• Definition:
• lack of growth/proliferation, “non-dividing state”
• exit of cell division cycle in G0/G1
• reversible
• In contrast to senescence (= irreversible)
• Molecular regulators:
• p27Kip1
• DREAM complex
• Problem for cancer therapy:
• quiescent cells do not respond to anticancer agents that target proliferating cells
• quiescent cells are unlikely to undergo apoptosis
Regulation of quiescence
proliferating cellscells in G0/G1
APC inactive
SKP2 high
p27 low
CDK active
SKP2 low
p27 high
CDK inactive
APC active
DREAM active
DREAM inactive
D R E A MD
M
R
E
A
Effects of imatinib on regulators of quiescence
GIST882GIST882
p27Kip1 upregulation in mouse xenografts
GIST882xenografts
p27Kip1 and SKP2 - risk for progression
Risk of progression (NCCN)
< 63.7% p27 pos.
cells
> 63.7% p27 pos.
cellsTotal
None, very low, low, intermediate
6 13 19
High 6 3 9
Total 12 16 28
Risk of progression (NCCN)
≤ 5 SKP2 pos.
cells/HPF
> 5 SKP2 pos.
cells/HPFTotal
None, very low, low, intermediate
17 0 17
High 4 5 9
Total 21 5 26
SKP2 (p<0.0001)p27Kip1 (p=0.0797)
Non-apoptotic cells enter quiescenceafter imatinib
Quiescent cells can re-enter the cell cycle
Imatinib and the DREAM complex
mammalian DREAM complex(DP, RB-like (p130), E2F and MuvB=LIN)
E2F target gene
Conclusions
imatinib leads to cell cycle exit and cellular quiescence• modulation of the APCCDH1 – SKP2 – p27Kip1 axis• modulation of the DREAM complex members
quiescent cells readily re-enter the cell cycle after removal of
imatinib
compounds that modulate these pathways as potential
antitumor agents in GIST?
Acknowledgments
Duensing Lab• Joshua Parry• Danushka Seneviratne• Matthew Brown• Julianne Baron• Ying Liu• Sophie Perdreau• Payel Chatterjee• Stefan Duensing
American Cancer Society(#RSG-08-092-01-CCG)GIST Cancer Research FundLife Raft Group (#UPCC-AD-100108)
University of Pittsburgh• Shih-Fan Kuan
Brigham & Women's Hospital• Jonathan Fletcher
Katolieke Universiteit Leuven• Maria Debiec-Rychter• Patrick Schöffski
Dana Farber Cancer Institute• James DeCaprio• Larisa Litovchick
Memorial Sloan Kettering• Peter Besmer• Cristina Antonescu
Pro-apoptotic Activity of Bortezomib in Gastrointestinal Stromal Tumors (GIST) cells
Sebastian Bauer1, Joshua A. Parry2, Thomas Mühlenberg1,Payel Chatterjee2, Shih-Fan Kuan2, Jonathan A. Fletcher3,
Stefan Duensing2, Anette Duensing2
1University of Duisburg-Essen, Essen, Germany2University of Pittsburgh Cancer Institute, Pittsburgh, PA
3Brigham & Women’s Hospital, Boston, MA
IMATINIB
KIT activity
tumor cell quiescence
risk of relapsewhen taken off imatinib!
GIST
APCCDH1 - SKP2 - p27Kip1
DREAM complex
IMATINIB
apoptosis tumor cell quiescence
risk of relapsewhen taken off imatinib!
GIST
APCCDH1 - SKP2 - p27Kip1
DREAM complex
KIT activity
apoptosis tumor cell quiescence
risk of relapsewhen taken off imatinib!
GIST
OTHEROTHERCOMPOUNDS?COMPOUNDS?
APCCDH1 - SKP2 - p27Kip1
DREAM complex
Histone H2AX is upregulated after imatinib treatment
• variant of core histone H2A• randomly incorporated into nucleosomes • rapidly phosphorylated at serine 139 in
response to genotoxic stress
γ-H2AX• mediates DNA damage response reactions• potential tumor suppressor
Serine 139
1 142
histone H2AXnucleosome
Upregulation of H2AX is causatively involved in GIST cell apoptosis
ima
tinib
72
h
imat
inib
72h
H2AX upregulation is due to increasedprotein stability
IB: ubiquitin
Ubiquitin/26S proteasome pathway
Mani and Gelmann, JCO 2005; 23:4776-4789
Inhibition of the proteasome inducesapoptosis in GIST
dose response time course
Bortezomib:• Velcade™ (Millennium
Pharmaceuticals)
• proteasome inhibitor
FDA-approved for:• multiple myeloma
• mantle cell lymphoma
Bortezomib mechanism of action
Bortezomib mechanism of action
Mechanism of action of Bortezomib
RT-PCR qRT-PCR
Bortezomib inhibits ongoing gene transcription
Bortezomib is effective in imatinib-resistant GIST
24 h
48 h
Bortezomib is effective in imatinib-resistant GIST
GIST004: imatinib-resistant GIST(short-term culture)
Kit+/K641E transgenic mice
Inhibition of NF-kB signaling is not involved in Bortezomib-induced apoptosis in GIST
bortezomib
Conclusions
Bortezomib induces apoptosis in GIST cells
dual mechanism of action• upregulation of H2AX• transcriptional inhibition of KIT
• NOT inhibition of NF-kB signaling
new therapeutic option for imatinib-resistant GIST patients
Acknowledgments
Duensing Lab• Joshua Parry• Matt Brown • Dee Seneviratne• Julianne Baron• Payel Chatterjee• Anna Chin• Stefan Duensing
American Cancer Society(#RSG-08-092-01-CCG)GIST Cancer Research FundLife Raft Group (UPPCC-AD-100108)
Universität Duisburg-Essen• Sebastian Bauer• Thomas Mühlenberg
Brigham & Women's Hospital• Jonathan Fletcher
University of Pittsburgh• Shih-Fan Kuan
Cleveland Clinic• Brian Rubin
Oregon Health & Science University
• Christopher Corless• Michael Heinrich