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