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Imatinib Mesylate modulates 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. - PowerPoint PPT Presentation
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Imatinib Mesylatemodulates Regulators of Quiescence inGastrointestinal 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 GISTMajor problems of imatinib therapy: therapy resistance incomplete responses
Detectable tumor mass under treatmentRelapse 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 cellsHoldsworth, Am J Roentgenol 2007;189:W324-W330transgenic KitV558 /+ mouse(in collaboration with C. Antonescu and P. Besmer)PETCTbaselineimatinib
Tumor cell quiescenceDefinition:lack of growth/proliferation, non-dividing stateexit of cell division cycle in G0/G1 reversibleIn contrast to senescence (= irreversible)Molecular regulators:p27Kip1DREAM complex
Problem for cancer therapy:quiescent cells do not respond to anticancer agents that target proliferating cellsquiescent cells are unlikely to undergo apoptosis
Regulation of quiescenceproliferating cellscells in G0/G1APC inactiveSKP2 highp27 lowCDK activeSKP2 lowp27 highCDK inactiveAPC activeDREAM activeDREAM inactiveD R E A MDMREA
Effects of imatinib on regulators of quiescenceGIST882GIST882
p27Kip1 upregulation in mouse xenograftsGIST882xenografts
p27Kip1 and SKP2 - risk for progressionSKP2 (p 63.7% p27 pos. cellsTotalNone, very low, low, intermediate61319 High639Total121628
Risk of progression (NCCN) 5 SKP2 pos. cells/HPF> 5 SKP2 pos. cells/HPFTotalNone, very low, low, intermediate17017 High459Total21526
Non-apoptotic cells enter quiescenceafter imatinib
Quiescent cells can re-enter the cell cycle
Imatinib and the DREAM complexmammalian 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?
AcknowledgmentsDuensing LabJoshua ParryDanushka SeneviratneMatthew BrownJulianne BaronYing LiuSophie PerdreauPayel ChatterjeeStefan Duensing
American Cancer Society(#RSG-08-092-01-CCG)GIST Cancer Research FundLife Raft Group (#UPCC-AD-100108)University of PittsburghShih-Fan Kuan
Brigham & Women's HospitalJonathan Fletcher
Katolieke Universiteit LeuvenMaria Debiec-RychterPatrick Schffski
Dana Farber Cancer InstituteJames DeCaprioLarisa Litovchick
Memorial Sloan KetteringPeter BesmerCristina Antonescu
Pro-apoptotic Activity of Bortezomib in Gastrointestinal Stromal Tumors (GIST) cells
Sebastian Bauer1, Joshua A. Parry2, Thomas Mhlenberg1,Payel Chatterjee2, Shih-Fan Kuan2, Jonathan A. Fletcher3,Stefan Duensing2, Anette Duensing2
1University of Duisburg-Essen, Essen, Germany2University of Pittsburgh Cancer Institute, Pittsburgh, PA3Brigham & Womens Hospital, Boston, MA
IMATINIBKIT activity tumor cell quiescencerisk of relapsewhen taken off imatinib!GISTAPCCDH1 - SKP2 - p27Kip1
DREAM complex
IMATINIBapoptosistumor cell quiescencerisk of relapsewhen taken off imatinib!GISTAPCCDH1 - SKP2 - p27Kip1
DREAM complexKIT activity
apoptosistumor cell quiescencerisk of relapsewhen taken off imatinib!GISTOTHERCOMPOUNDS?APCCDH1 - SKP2 - p27Kip1
DREAM complex
Histone H2AX is upregulated after imatinib treatmentvariant of core histone H2Arandomly incorporated into nucleosomes rapidly phosphorylated at serine 139 in response to genotoxic stress -H2AXmediates DNA damage response reactionspotential tumor suppressorSerine 1391142histone H2AXnucleosome
Upregulation of H2AX is causatively involved in GIST cell apoptosis
H2AX upregulation is due to increasedprotein stabilityIB: ubiquitinUbiquitin/26S proteasome pathwayMani and Gelmann, JCO 2005; 23:4776-4789
Inhibition of the proteasome inducesapoptosis in GISTdose responsetime courseBortezomib:Velcade (Millennium Pharmaceuticals)proteasome inhibitor
FDA-approved for:multiple myelomamantle cell lymphoma
Bortezomib mechanism of action
Bortezomib mechanism of action
Mechanism of action of BortezomibRT-PCRqRT-PCR
Bortezomib inhibits ongoing gene transcription
Bortezomib is effective in imatinib-resistant GIST24 h48 h
Bortezomib is effective in imatinib-resistant GISTGIST004: imatinib-resistant GIST(short-term culture)Kit+/K641E transgenic mice
Inhibition of NF-kB signaling is not involved in Bortezomib-induced apoptosis in GISTbortezomib
Conclusions Bortezomib induces apoptosis in GIST cells
dual mechanism of actionupregulation of H2AXtranscriptional inhibition of KIT
NOT inhibition of NF-kB signaling
new therapeutic option for imatinib-resistant GIST patients
AcknowledgmentsDuensing LabJoshua ParryMatt Brown Dee SeneviratneJulianne BaronPayel ChatterjeeAnna ChinStefan Duensing
American Cancer Society(#RSG-08-092-01-CCG)GIST Cancer Research FundLife Raft Group (UPPCC-AD-100108)Universitt Duisburg-EssenSebastian BauerThomas Mhlenberg
Brigham & Women's HospitalJonathan Fletcher
University of PittsburghShih-Fan Kuan
Cleveland ClinicBrian Rubin
Oregon Health & Science UniversityChristopher CorlessMichael Heinrich
***********but what happens to these cells? can we really mimic what happens in a patient that goes off imatinib? can if they are really quiescent, they should be able to re-enter the cell cycle this is indeed what happens following experiment: treat cells with imatinib for 3 days (most, but not all cells undergo apoptosis) remove imatinib-containing media, replace with regular media follow cells over a period of up to 11 days each day (later every other day) take cells for BrdU and protein extracts what we see is that proliferative activity of cells rapidly declines under imatinib as expected from about 60% to less than 5% on treatment day 3 after removal of imatinib, cells re-enter the cell cycle aproximately after 5 days being in regular media even a rebound effect with a higher percentage of proliferating cells after 9 days, before proliferative index returns to same level as control illustrated here: DMSO control, 3 days imatinib, 9 days after removal of imatinib in parallel, we can see that phosphoKIT immediately rebounds after removal of imatinib levels of SKP2, p27 and cyclin A respond in parallel to what is seen in the BrdU incorporation somewhat delayed
this experiment shows that we are really able to mimic in the tissue culture dish what can possibly happen in a patient who goes off imatinib despite having residual tumor ****************************