Clinical and experimental findings suggest tumor cell quiescence in imatinib-treated GIST

1. Imatinib Mesylatemodulates Regulators of Quiescence inGastrointestinal Stromal Tumor (GIST) cellsJoshua A. Parry, Matthew F. Brown, Danushka Seneviratne,Stefan Duensing, Anette DuensingUniversity of Pittsburgh Cancer Institute,Pittsburgh, PA

2. Clinical and experimental findings suggesttumor cell quiescence in imatinib-treated GIST baseline imatinib • 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 PET CT Holdsworth, Am J Roentgenol 2007;189:W324-W330 transgenic KitV558 /+ mouse (in collaboration with C. Antonescu and P. Besmer)

3. 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

4. Regulation of quiescence APC active APC inactive SKP2 low SKP2 high p27 high p27 low CDK inactive CDK active D D R E A M E DREAM active DREAM inactive M A R proliferating cells cells in G0/G1

5. Effects of imatinib on regulators of quiescence GIST882 GIST882

6. p27Kip1 upregulation in mouse xenografts GIST882 xenografts

7. p27Kip1 and SKP2 - risk for progression SKP2 (p<0.0001) p27Kip1 (p=0.0797)

8. Non-apoptotic cells enter quiescenceafter imatinib

9. Quiescent cells can re-enter the cell cycle

10. Imatinib and the DREAM complex mammalian DREAM complex (DP, RB-like (p130), E2F and MuvB=LIN) E2F target gene

11. 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?

12. Acknowledgments • 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 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 Fund Life Raft Group (#UPCC-AD-100108)

14. 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 Duensing21University of Duisburg-Essen, Essen, Germany2University of Pittsburgh Cancer Institute, Pittsburgh, PA3Brigham & Women’s Hospital, Boston, MA

15. IMATINIB KIT activity GIST APCCDH1 - SKP2 - p27Kip1 DREAM complex tumor cell quiescence risk of relapse when taken off imatinib!

16. IMATINIB KIT activity GIST ? APCCDH1 - SKP2 - p27Kip1 DREAM complex apoptosis tumor cell quiescence risk of relapse when taken off imatinib!

17. OTHER COMPOUNDS? GIST ? APCCDH1 - SKP2 - p27Kip1 DREAM complex apoptosis tumor cell quiescence risk of relapse when taken off imatinib!

18. 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 1 142 Serine139 nucleosome histone H2AX

19. Upregulation of H2AX is causatively involved in GIST cell apoptosis imatinib 72h imatinib 72h

20. H2AX upregulation is due to increasedprotein stability Mani and Gelmann, JCO 2005; 23:4776-4789 Ubiquitin/26S proteasome pathway IB: ubiquitin

21. Inhibition of the proteasome inducesapoptosis in GIST • Bortezomib: • Velcade™ (Millennium Pharmaceuticals) • proteasome inhibitor • FDA-approved for: • multiple myeloma • mantle cell lymphoma dose response time course

22. Bortezomib mechanism of action

23. Bortezomib mechanism of action

24. Mechanism of action of Bortezomib RT-PCR qRT-PCR

25. Bortezomib inhibits ongoing gene transcription

26. Bortezomib is effective in imatinib-resistant GIST 24 h 48 h

27. Bortezomib is effective in imatinib-resistant GIST GIST004: imatinib-resistant GIST (short-term culture) Kit+/K641E transgenic mice

28. Inhibition of NF-kB signaling is not involved in Bortezomib-induced apoptosis in GIST bortezomib

29. 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

30. 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 Fund Life 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