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CYCLING MULTI-KINASE INHIBITORS IN
IMATINIB-RESISTANT GASTROINTESTINAL STROMAL
TUMORS TO MAXIMIZE DISEASE CONTROL:
PRECLINICAL AND CLINICAL RATIONALE
César Serrano, Grant Eilers, Meijun Zhu, Anu Gupta, George D. Demetri,
Suzanne George, Sebastian Bauer, Brian P. Rubin, Jonathan A. Fletcher
Brigham and Women’s Hospital; Dana-Farber Cancer Institute; Harvard Medical School, Boston, MA, USA; Lerner
Research Institute and Cleveland Clinic, Cleveland, OH;
West German Cancer Center, Essen, Germany
CTOS 18th Annual Meeting
Oct 30 - Nov 2, 2013
New York
Paper 037
Background
•
KIT and PDGFRA are primary drivers of oncogenic
signaling in GISTs.
•
KIT inhibition with tyrosine-kinase inhibitors
(TKIs)improves outcomes in most GIST patients.
•
Resistance to TKIs eventually emerges in virtually
all GIST patients.
•
KIT secondary resistance mutations are the main
mechanism of TKI failure.
Secondary resistance in GIST
SECONDARY
MUTATIONS
Exon 13
V654
FREQUENCY
40%
ATPbinding
pocket
30%
Activation
Loop
Exon 14
Exon 9
Exon 11
D816
Exon 17
Debiec-Rychter M, 2005
Antonescu CR, 2005
Wardelmann E, 2006
Heinrich MC, 2008
Liegl B, 2008
D820
N822
Y823
Secondary resistance in GIST
SECONDARY
MUTATIONS
Exon 13
V654
FREQUENCY
40%
ATPbinding
pocket
30%
Activation
Loop
Exon 14
Exon 9
Exon 11
D816
Exon 17
Debiec-Rychter M, 2005
Antonescu CR, 2005
Wardelmann E, 2006
Heinrich MC, 2008
Liegl B, 2008
D820
N822
Y823
Secondary resistance in GIST
SECONDARY
MUTATIONS
FREQUENCY
DRUG
SENSITIVITY
Imatinib Sunitinib
Exon 13
V654
40%
Exon 14
Exon 9
Exon 11
D816
Exon 17
Debiec-Rychter M, 2005
Antonescu CR, 2005
Wardelmann E, 2006
Heinrich MC, 2008
Liegl B, 2008
D820
N822
Y823
30%
Sensitive
Resistant
Secondary resistance in GIST
SECONDARY
MUTATIONS
FREQUENCY
DRUG
SENSITIVITY
Imatinib Sunitinib
Exon 13
V654
40%
Exon 14
Exon 9
Exon 11
D816
Exon 17
Debiec-Rychter M, 2005
Antonescu CR, 2005
Wardelmann E, 2006
Heinrich MC, 2008
Liegl B, 2008
D820
N822
Y823
30%
Sensitive
Resistant
Second- and third-line treatment in GIST
• Regorafenib (REGO) has recently obtained FDAapproval in GIST patients after failure of imatinib
(IM) and sunitinib (SU).
• There is substantial heterogeneity of secondary KIT
resistant mutations between and within metastases
from individual patients after progression on TKIs.
• Progression-free survival after imatinib failure is 4 to
6 months irrespective of the second- or third-line TKI
used.
Aims
We investigated novel strategies to overcome
heterogeneity of resistant clones in TKI-resistant
GIST patients.
Imatinib, sunitinib and regorafenib are active
against primary KIT exon 11 mutation
IM
nM
50 100
REGO
SU
500
50
100 500
50
100 500
p-KIT (Y703)
p-AKT S473
p-S6 (S235/236)
Actin
Imatinib, sunitinib and regorafenib are active
against primary KIT exon 11 mutation
IM
nM
50 100
REGO
SU
500
50
100 500
50
100 500
p-KIT (Y703)
p-AKT S473
p-S6 (S235/236)
Actin
Imatinib, sunitinib and regorafenib are active
against primary KIT exon 11 mutation
IM
nM
50 100
REGO
SU
500
50
100 500
50
100 500
p-KIT (Y703)
p-AKT S473
p-S6 (S235/236)
Actin
Sunitinib and regorafenib have complementary
activity against imatinib-resistant GIST cell lines
KIT Mutation
IC50 (nM)
Cell line
Primary
Secondary
SU
REGO
GIST430/654
Ex 11
Ex 13 (V654A)
194
3,341
GIST-T1/816
Ex 11
Ex 17 (D816E)
3,111
395
GIST-T1/820
Ex 11
Ex 17 (D820A)
2,599
368
*IC50s: Green = predictive of clinical efficacy
Red = predictive of clinical resistance
Sunitinib and regorafenib have complementary
activity against imatinib-resistant GIST cell lines
KIT Mutation
IC50 (nM)
Cell line
Primary
Secondary
SU
REGO
GIST430/654
Ex 11
Ex 13 (V654A)
194
3,341
GIST-T1/816
Ex 11
Ex 17 (D816E)
3,111
395
GIST-T1/820
Ex 11
Ex 17 (D820A)
2,599
368
*IC50s: Green = predictive of clinical efficacy
Red = predictive of clinical resistance
Sunitinib and regorafenib have complementary
activity against imatinib-resistant GIST cell lines
KIT Mutation
IC50 (nM)
Cell line
Primary
Secondary
SU
REGO
GIST430/654
Ex 11
Ex 13 (V654A)
194
3,341
GIST-T1/816
Ex 11
Ex 17 (D816E)
3,111
395
GIST-T1/820
Ex 11
Ex 17 (D820A)
2,599
368
*IC50s: Green = predictive of clinical efficacy
Red = predictive of clinical resistance
Progression of KIT Exon 13
imatinib-resistant subclone on regorafenib
KIT exon 13 (V654A). Radiographic and metabolic progression on regorafenib
Baseline
C12D21
Resection biopsy
exon 11 + exon 13
(V654A)
Response of KIT Exon 17
imatinib-resistant subclone on regorafenib
KIT exon 17 (D820Y). Radiographic and metabolic response on regorafenib
Baseline
Pre-regorafenib
exon 11 + exon 17
(D820Y)
C4D21
Sunitinib and regorafenib have complementary
activity against IM-resistant KIT mutations
KIT Mutation
Primary
Secondary
Activity
IM
SU
REGO
Ex 11
Sensitive
Sensitive
Sensitive
Ex 11
Sensitive
Sensitive
Sensitive
Ex 11
Ex 13 (V654A)
Resistant
Sensitive
Resistant
Ex 11
Ex 17 (D816)
Resistant
Resistant
Sensitive
Ex 11
Ex 17 (D820)
Resistant
Resistant
Sensitive
Targeting TKI-resistance heterogeneity in GIST
SUNITINIB
REGORAFENIB
ATP-binding
pocket
Activation
Loop
Cycling sunitinib and regorafenib might suppress a
broader spectrum of imatinib-resistant GIST clones
and achieve prolonged long-term disease control
Time-frame for restoration of kinase signaling
and proliferation after TKI withdrawal
TKI withdrawal
 Phosphorylation of KIT
 Phosphorylation of downstream
signal intermediates (AKT and ERK)
 Increase of Cyclin A expression
 Increase of Ki-67 expression
 Mitotic activity
Sunitinib washout: reactivation of
KIT, downstream pathways, and cell cycle
GIST430/654
exon 11 + exon 13
Sunitinib treatment
SU 500nM
Days of drug
withdrawal
pKIT Y703
pAKT S473
pRB S795
Cyclin A
Actin
Sunitinib washout: reactivation of
KIT, downstream pathways, and cell cycle
GIST430/654
exon 11 + exon 13
Sunitinib treatment
SU 500nM
Days of drug
withdrawal
pKIT Y703
pAKT S473
pRB S795
Cyclin A
Actin
Sunitinib washout: reactivation of
KIT, downstream pathways, and cell cycle
GIST430/654
exon 11 + exon 13
Sunitinib treatment
SU 500nM
Days of drug
withdrawal
pKIT Y703
pAKT S473
pRB S795
Cyclin A
Actin
Sunitinib washout:
reactivation of proliferation
GIST430/654
exon 11 + exon 13
Untreated
Sunitinib treatment
Day 0
KI-67 expression
Day 1
Mitotic Count (per 5 mm2)
UT
100nM
500nM
Day 0
62
4
1
Day 1
60
3
0
Day 3
63
45
1
Day 7
68
65
11
Day 3
Day 7
SU 100nM
SU 500nM
Sunitinib washout:
reactivation of proliferation
GIST430/654
exon 11 + exon 13
Untreated
Sunitinib treatment
Day 0
KI-67 expression
Day 1
Mitotic Count (per 5 mm2)
UT
100nM
500nM
Day 0
62
4
1
Day 1
60
3
0
Day 3
63
45
1
Day 7
68
65
11
Day 3
Day 7
SU 100nM
SU 500nM
Regorafenib washout: reactivation of
KIT, downstream pathways, and cell cycle
GIST48/820
exon 11 + exon 17
Regorafenib treatment
REGO 500nM
Days of drug
withdrawal
pKIT Y703
pAKT S473
pRB S795
Cyclin A
Actin
Regorafenib washout: reactivation of
KIT, downstream pathways, and cell cycle
GIST48/820
exon 11 + exon 17
Regorafenib treatment
REGO 500nM
Days of drug
withdrawal
pKIT Y703
pAKT S473
pRB S795
Cyclin A
Actin
Regorafenib washout: reactivation of
KIT, downstream pathways, and cell cycle
GIST48/820
exon 11 + exon 17
Regorafenib treatment
REGO 500nM
Days of drug
withdrawal
pKIT Y703
pAKT S473
pRB S795
Cyclin A
Actin
Regorafenib washout:
reactivation of proliferation
GIST48/820
exon 11 + exon 17
Untreated
Regorafenib treatment
Day 0
KI-67 expression
Day 1
Mitotic Count (per 5 mm2)
UT
100nM
500nM
Day 0
42
15
1
Day 1
44
13
0
Day 3
54
63
2
Day 7
35
42
15
Day 3
Day 7
REGO 100nM
REGO 500nM
Regorafenib washout:
reactivation of proliferation
GIST48/820
exon 11 + exon 17
Untreated
Regorafenib treatment
Day 0
KI-67 expression
Day 1
Mitotic Count (per 5 mm2)
UT
100nM
500nM
Day 0
42
15
1
Day 1
44
13
0
Day 3
54
63
2
Day 7
35
42
15
Day 3
Day 7
REGO 100nM
REGO 500nM
Once a KIT inhibitor is withdrawn:
 Phosphorylation of KIT
2 days
 Phosphorylation of downstream
signal intermediates (AKT and ERK)
4 days
7 days
 Increase of Cyclin A expression
 Increase of Ki-67 expression
 Mitotic activity
Recovery of mitotic activity in GIST patients
responding to TKI therapy after withdrawal of the KIT
inhibitor
PATIENT
DRUG
#DAYS after
last TKI
KIT
MUTATION
2ND MUTATION
Mitosis
1a
SU
3
Exon 11
no
0
1b
SU
3
Exon 11
Exon 13 (V654A)
0
2a
REGO
9
Exon 11
Exon 17 (Y823D)
13
2b
REGO
9
Exon 11
Exon 17 (D820Y)
7
Rapid alternation regimen
3 days SU
4 days REGO
3 days SU
4 days REGO
Rapid alternation regimen might
minimize toxic effects.
Alternation of complementary drugs
increases the spectrum of effective
inhibition of IM-resistant clones.
Conclusions
1. Sunitinib and regorafenib have complementary
activity against secondary KIT mutations.
2. After withdrawal of an effective KIT inhibitor,
target re-activation occurs in 1 to 3 days.
3. Proliferation markers are re-activated between
3 to 7 days, and mitotic activation is observed
in vitro and in clinical correlates between 4 to
7 days.
4. These observations define a rational schedule
for alternation of sunitinib and regorafenib in a
heterogeneous GIST population that will be
shortly tested in a Phase Ib clinical trial.
Co-authors / Acknowledgments
Brigham and Women’s Hospital
Jonathan Fletcher Lab
Jonathan A. Fletcher
Grant Eilers
Albert Ha
Adrián Mariño-Enríquez
Anna Quattrone
Gloria Ravegnini
Inga-Marie Schaefer
Derrick Tao
Yue-Xiang Wang
Mei-Jun Zhu
Pathology Department
Christopher D.M. Fletcher
Leona A. Doyle
Jason Hornick
Division of Surgical Oncology
Chandrajit P. Raut
Ludwig Center at Dana-Farber Cancer Institute
George D. Demetri
James E. Butrynski
David R. D’Adamo
Suzanne George
Jeffrey A. Morgan
Andrew J. Wagner
Lerner Research Institute and Cleveland Clinic
Anu Gupta
Brian P. Rubin
West German Cancer Center
Sebastian Bauer
Vall d’Hebron University Hospital
Joan Carles Galcerán
ASCO Young Investigator Award
Spanish Society of Medical Oncology Translational Award
Faculty from the 2013 Flims Workshop
GIST Cancer Research Fund, The LifeRaft Group
Virginia and Daniel K. Ludwig Trust for Cancer Research