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GIST

The DREAM complex mediates GIST cell quiescence and is a novel therapeutic target to enhance imatinib -induced apoptosis

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GIST

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  1. The DREAM complex mediates GIST cell quiescence and is a novel therapeutic target to enhance imatinib-induced apoptosis Sergei Boichuk, Joshua A. Parry, Kathleen R. Makielski, Larisa Litovchick, Julianne L. Baron, James P. Zewe, Agnieszka Wozniak, Keith R. Mehalek, Nina Korzeniewski, Danushka S. Seneviratne, Patrick Schoffski, Maria Debiec-Rychter, James A. DeCaprio and AnetteDuensing Cancer Virology Program University of Pittsburgh Cancer Institute Dana-Farber Cancer Institute at Harvard Katholisch University Leuven, Belgium University of Heidelberg, Germany Cancer Research in press 2013

  2. GIST Gastrointestinal stromal tumors ~5,000 cases in US per year Most common stromal tumor (sarcoma) ~80% of tumors are associated with a c-Kit mutation (an RTK)

  3. GIST c-Kit can be inhibited by imatinibmesylete (Gleevec) However, 14% of GIST patients don’t respond to imatinib about 50% of patients develop resistance in 2 years tumor size often stabilizes (but doesn’t disappear) on imatinib

  4. Imatinib induces Quiescence Does imatinib cause GIST cells to become quiescent? p27Kip1 (inhibits Cdk2/CyclinE and Cdk4/CyclinD) as a marker of quiescence 3 cell sources GIST882 cell line Kit K642E [exon 13] GIST from patient biopsy Kit V650D [exon 11] GIST from patient biopsy Kit A502_Y503dup [exon 9] Implanted into athymic “nude” mouse (xenograft)

  5. Imatinib induces Quiescence • When tumors 1 cm3, treat with imatinib or placebo for 12 days • Mice sacrificed; tumors collected • p27Kip1 identified by immunofluorescence (IF) • Fix cells. Permeabilize membrane. • Add anti-p27Kip1 antibody • Add secondary antibody connected to fluorescein • Fluorescein absorbs light at lmax = 494 nm • and fluoresces at lmax = 518 nm (green) • Also stain with DAPI • binds DNA and fluoresces blue

  6. Imatinib induces Quiescence Scale bar is 50 mm Fig. 1A

  7. Imatinib induces Quiescence Fig. 1A

  8. Imatinib induces Quiescence Fig. 1B

  9. Imatinib induces Quiescence Is this quiescence or senescence? Is it reversible? Treat with imatinib 3 days then stop treatment Look at proliferation by BrdU staining Placebo Control Fig. 1C

  10. Imatinib induces Quiescence Placebo Control Imatinib 3d 9 d after removal Fig. 1C

  11. Imatinib induces Quiescence Fig. 1C

  12. Imatinib induces Quiescence Senescent cells secrete b-galactosidase Stain with ONPG (O-nitrophenylgalactoside; cleaved to NPG, blue) No increase in b-galactosidase-positive cells Fig. S3A

  13. Imatinib induces Quiescence Senescent cells express high levels of p16INK4A IF Fig. S3C

  14. How does Imatinib induce Quiescence? What causes p27Kip1 levels to rise? p27Kip1 is usually destroyed by polyubiquitination The E3 ubiquitin ligase is Skp2

  15. How does Imatinib induce Quiescence? Why do the cells become quiescent? Fig. 2A

  16. How does Imatinib induce Quiescence? Imatinib c-Kit ??? Skp2 p27Kip1 What else is in this pathway? Hypothesis: The DREAM complex needed to induce quiescence DREAM Complex (DP, RB-like, E2F4, and MuvB) Represses transcription to hold cells in G0

  17. How does Imatinib induce Quiescence? GIST882 cells treated with imatinib If the hypothesis is correct, what do we expect to see? Fig. 3A

  18. How does Imatinib induce Quiescence? What should we find in a washout experiment? Fig. 3B

  19. How does Imatinib induce Quiescence? What should we find with DYRK1A and LIN52? Fig. 4AB

  20. How does Imatinib induce Quiescence? What should we find in an IF experiment? p<0.0015 Fig. 3C

  21. How does Imatinib induce Quiescence? What should we find in a coIP experiment? Fig. 3D

  22. How does Imatinib induce Quiescence? Hypothesis: The DREAM complex needed to induce quiescence Fig. 3, 4AB

  23. Imatinib and Apoptosis • Imatinib induces quiescence but does it affect the frequency of apoptosis? • 3 different assays of apoptosis: • Cell Morphology • Sub-G1 DNA content • Cleavage of PARP is a marker of apoptosis • Poly (ADP) Ribose Polymerase • Activated to assist with DNA repair (single-strand breaks) • Cleaved by Caspase-3 and Caspase-7

  24. Imatinib and Apoptosis GIST882 cells Fig. S2A

  25. Imatinib and Apoptosis GIST882 cells hours with imatinib Fig. 2B

  26. Imatinib and Apoptosis Gene Expression Knockdowns in GIST882 cells Fig. 4C

  27. Imatinib and Apoptosis Gene Expression Knockdowns in GIST882 cells. FACS profiling Fig. 4DE

  28. Imatinib and Apoptosis Gene Expression Knockdowns in GIST882 cells. FACS profiling Fig. 5C

  29. Imatinib and Apoptosis Gene Expression Knockdowns in GIST882 cells. FACS profiling Fig. 5D

  30. Imatinib and Apoptosis GIST882 cells Inhibition of DYRK1A activity with 10mM Harmine Conclusion? Fig. 5A

  31. Imatinib and Apoptosis GIST882 cells. Visualize by DAPI staining Fig. 5B

  32. Model: Quiescence DREAM Imatinib Apoptosis

  33. Model: Quiescence Quiescence DREAM DREAM Imatinib Imatinib Apoptosis Apoptosis

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