cycling multi-kinase inhibitors in imatinib-resistant gastrointestinal stromal tumors to maximize...
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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
Paper 037
CTOS 18th Annual MeetingOct 30 - Nov 2, 2013
New York
• 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.
Background
Exon 13
Exon 14
Exon 17
Exon 11
Exon 9
SECONDARYMUTATIONS
V654
D816
D820N822
ATP-binding pocket
Activation Loop
Y823
40%
30%
FREQUENCY
Debiec-Rychter M, 2005Antonescu CR, 2005Wardelmann E, 2006Heinrich MC, 2008Liegl B, 2008
Secondary resistance in GIST
Exon 13
Exon 14
Exon 17
Exon 11
Exon 9
SECONDARYMUTATIONS
V654
D816
D820N822
ATP-binding pocket
Activation Loop
Y823
40%
30%
FREQUENCY
Debiec-Rychter M, 2005Antonescu CR, 2005Wardelmann E, 2006Heinrich MC, 2008Liegl B, 2008
Secondary resistance in GIST
Exon 13
Exon 14
Exon 17
Exon 11
Exon 9
SECONDARYMUTATIONS
V654
D816
D820N822Y823
40%
30%
FREQUENCY
Debiec-Rychter M, 2005Antonescu CR, 2005Wardelmann E, 2006Heinrich MC, 2008Liegl B, 2008
Secondary resistance in GISTDRUG
SENSITIVITY
Imatinib Sunitinib
Sensitive
Resistant
Exon 13
Exon 14
Exon 17
Exon 11
Exon 9
SECONDARYMUTATIONS
V654
D816
D820N822Y823
40%
30%
FREQUENCY
Debiec-Rychter M, 2005Antonescu CR, 2005Wardelmann E, 2006Heinrich MC, 2008Liegl B, 2008
Secondary resistance in GISTDRUG
SENSITIVITY
Imatinib Sunitinib
Sensitive
Resistant
• Regorafenib (REGO) has recently obtained FDA-approval 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.
Second- and third-line treatment in GIST
We investigated novel strategies to overcome
heterogeneity of resistant clones in TKI-resistant
GIST patients.
Aims
Imatinib, sunitinib and regorafenib are active against primary KIT exon 11 mutation
nM DMSO
50 100 500
REGO
p-KIT (Y703)
p-S6 (S235/236)
Actin
p-AKT S473
DMSO
50 100 500
IM
DMSO
50 100 500
SU
Imatinib, sunitinib and regorafenib are active against primary KIT exon 11 mutation
nM DMSO
50 100 500
REGO
p-KIT (Y703)
p-S6 (S235/236)
Actin
p-AKT S473
DMSO
50 100 500
IM
DMSO
50 100 500
SU
Imatinib, sunitinib and regorafenib are active against primary KIT exon 11 mutation
nM DMSO
50 100 500
REGO
p-KIT (Y703)
p-S6 (S235/236)
Actin
p-AKT S473
DMSO
50 100 500
IM
DMSO
50 100 500
SU
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
Sunitinib and regorafenib have complementary activity against imatinib-resistant GIST cell lines
*IC50s: Green = predictive of clinical efficacy Red = predictive of clinical resistance
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
Sunitinib and regorafenib have complementary activity against imatinib-resistant GIST cell lines
*IC50s: Green = predictive of clinical efficacy Red = predictive of clinical resistance
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
Sunitinib and regorafenib have complementary activity against imatinib-resistant GIST cell lines
*IC50s: Green = predictive of clinical efficacy Red = predictive of clinical resistance
Progression of KIT Exon 13 imatinib-resistant subclone on regorafenib
Baseline
C12D21
exon 11 + exon 13 (V654A)
KIT exon 13 (V654A). Radiographic and metabolic progression on regorafenib
Resection biopsy
Response of KIT Exon 17imatinib-resistant subclone on regorafenib
Baseline
C4D21
exon 11 + exon 17 (D820Y)
Pre-regorafenib
KIT exon 17 (D820Y). Radiographic and metabolic response on regorafenib
KIT Mutation Activity
Primary Secondary 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
Sunitinib and regorafenib have complementary activity against IM-resistant KIT mutations
ATP-binding pocket
Activation Loop
SUNITINIB
REGORAFENIB
Cycling sunitinib and regorafenib might suppress a
broader spectrum of imatinib-resistant GIST clones
and achieve prolonged long-term disease control
Targeting TKI-resistance heterogeneity in GIST
Phosphorylation of KIT
Phosphorylation of downstream
signal intermediates (AKT and ERK)
Increase of Cyclin A expression
Increase of Ki-67 expression
Mitotic activity
TKI withdrawal
Time-frame for restoration of kinase signaling and proliferation after TKI withdrawal
pAKT S473
pKIT Y703
DAY 0
DAY 1
DAY 3
DAY 7
SU 500nM
pRB S795
Cyclin A
Actin
Sunitinib washout: reactivation of KIT, downstream pathways, and cell cycle
GIST430/654exon 11 + exon 13
Sunitinib treatment
Days of drug withdrawal
pAKT S473
pKIT Y703
DAY 0
DAY 1
DAY 3
DAY 7
SU 500nM
pRB S795
Cyclin A
Actin
Sunitinib washout: reactivation of KIT, downstream pathways, and cell cycle
Days of drug withdrawal
GIST430/654exon 11 + exon 13
Sunitinib treatment
pAKT S473
pKIT Y703
DAY 0
DAY 1
DAY 3
DAY 7
SU 500nM
pRB S795
Cyclin A
Actin
Sunitinib washout: reactivation of KIT, downstream pathways, and cell cycle
Days of drug withdrawal
GIST430/654exon 11 + exon 13
Sunitinib treatment
Untreated SU 100nM SU 500nM
Day 0
Day 1
Day 3
Day 7
KI-67 expression
Sunitinib washout: reactivation of proliferation
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
GIST430/654exon 11 + exon 13
Sunitinib treatment
Untreated SU 100nM SU 500nM
Day 0
Day 1
Day 3
Day 7
KI-67 expression
Sunitinib washout: reactivation of proliferation
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
GIST430/654exon 11 + exon 13
Sunitinib treatment
DAY 0
DAY 1
DAY 3
DAY 7
REGO 500nM
pAKT S473
pKIT Y703
pRB S795
Cyclin A
Actin
Regorafenib washout: reactivation of KIT, downstream pathways, and cell cycle
Days of drug withdrawal
GIST48/820exon 11 + exon 17
Regorafenib treatment
DAY 0
DAY 1
DAY 3
DAY 7
REGO 500nM
GIST48/820exon 11 + exon 17
Regorafenib treatment
pAKT S473
pKIT Y703
pRB S795
Cyclin A
Actin
Days of drug withdrawal
Regorafenib washout: reactivation of KIT, downstream pathways, and cell cycle
DAY 0
DAY 1
DAY 3
DAY 7
REGO 500nM
pAKT S473
pKIT Y703
pRB S795
Cyclin A
Actin
Days of drug withdrawal
GIST48/820exon 11 + exon 17
Regorafenib treatment
Regorafenib washout: reactivation of KIT, downstream pathways, and cell cycle
Untreated REGO 100nM REGO 500nM
Day 0
Day 1
Day 3
Day 7
Regorafenib washout: reactivation of proliferation
GIST48/820exon 11 + exon 17
Regorafenib treatment
KI-67 expression
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
Untreated REGO 100nM REGO 500nM
Day 0
Day 1
Day 3
Day 7
Regorafenib washout: reactivation of proliferation
GIST48/820exon 11 + exon 17
Regorafenib treatment
KI-67 expression
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
Once a KIT inhibitor is withdrawn:
Phosphorylation of KIT
Phosphorylation of downstream
signal intermediates (AKT and ERK)
Increase of Cyclin A expression
Increase of Ki-67 expression
Mitotic activity
2 days
4 days
7 days
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.
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.
Conclusions
1. Sunitinib and regorafenib have complementary activity against secondary KIT mutations.
Brigham and Women’s HospitalJonathan Fletcher LabJonathan A. FletcherGrant EilersAlbert HaAdrián Mariño-EnríquezAnna QuattroneGloria RavegniniInga-Marie SchaeferDerrick TaoYue-Xiang WangMei-Jun Zhu
Pathology DepartmentChristopher D.M. FletcherLeona A. DoyleJason Hornick
Division of Surgical OncologyChandrajit P. Raut
Co-authors / AcknowledgmentsLudwig Center at Dana-Farber Cancer Institute
George D. DemetriJames E. ButrynskiDavid R. D’Adamo Suzanne GeorgeJeffrey A. MorganAndrew J. Wagner
Lerner Research Institute and Cleveland ClinicAnu GuptaBrian P. Rubin
West German Cancer CenterSebastian Bauer
ASCO Young Investigator AwardSpanish Society of Medical Oncology Translational Award
Faculty from the 2013 Flims WorkshopGIST Cancer Research Fund, The LifeRaft Group
Virginia and Daniel K. Ludwig Trust for Cancer Research
Vall d’Hebron University HospitalJoan Carles Galcerán