CYCLING MULTI-KINASE INHIBITORS IN IMATINIB-RESISTANT GASTROINTESTINAL STROMAL TUMORS TO MAXIMIZE...

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

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

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



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


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



Regorafenib treatment

DAY 0

DAY 1

DAY 3

DAY 7

REGO 500nM



pAKT S473

pKIT Y703

pRB S795

Cyclin A

Actin



DAY 0

DAY 1

DAY 3

DAY 7

REGO 500nM

pAKT S473

pKIT Y703

pRB S795

Cyclin A

Actin





Untreated REGO 100nM REGO 500nM

Day 0

Day 1

Day 3

Day 7

Regorafenib washout: reactivation of proliferation



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

CYCLING MULTI-KINASE INHIBITORS IN IMATINIB-RESISTANT GASTROINTESTINAL STROMAL TUMORS TO MAXIMIZE...

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