1. 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
I would like to thank the organization for allowing us to present the preclinical rationale for cycling multikinase inhibitors in imatinib resistant GIST to maximize disease control, and I would like to thank CTOS as well for KINDLY RECOGNIZING this research with the CTOS YIA
CTOS 18th Annual Meeting
Oct 30 - Nov 2, 2013
New York
Paper 037

2. 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.
KIT and PDGFRA are primary drivers of oncogenic signal in GISTs.
And therefore, KIT inhibition with TKIs improves outcomes in most GIST patients.
However, resistance to imatinib eventually emerges in virtually all GIST patients.
And KIT secondary resistance mutations are the main mechanism of imatinib failure.

3. Secondary resistance in GIST
SECONDARYMUTATIONS
FREQUENCY
ATP-binding pocket
Exon 13
V654
40%
Exon 14
Exon 9
Exon 11
THESE SECONDARY MUTATIONS cluster in two regions, the ATP-BP (encoded by exon 13) and the A-loop (encoded by exon 17).
D816
Activation
Loop
D820
Exon 17
30%
N822
Y823
Debiec-Rychter M, 2005
Antonescu CR, 2005
Wardelmann E, 2006
Heinrich MC, 2008
Liegl B, 2008

4. Secondary resistance in GIST
SECONDARYMUTATIONS
FREQUENCY
ATP-binding pocket
Exon 13
V654
40%
Exon 14
Exon 9
Exon 11
THESE SECONDARY MUTATIONS cluster in two regions, the ATP-BP (encoded by exon 13) and the A-loop (encoded by exon 17).
D816
Activation
Loop
D820
Exon 17
30%
N822
Y823
Debiec-Rychter M, 2005
Antonescu CR, 2005
Wardelmann E, 2006
Heinrich MC, 2008
Liegl B, 2008

5. Secondary resistance in GIST
SECONDARYMUTATIONS
DRUG SENSITIVITY
FREQUENCY
Imatinib
Sunitinib
Exon 13
V654
40%
Exon 14
Exon 9
Exon 11
Both in vitro and clinical evidence show that suntinib, the standard second agent therapy after IM failure, is a very active drug against those KIT secondary mutations in the ATP-binding pocket…
D816
D820
Exon 17
30%
N822
Y823
Sensitive
Debiec-Rychter M, 2005
Antonescu CR, 2005
Wardelmann E, 2006
Heinrich MC, 2008
Liegl B, 2008
Resistant

6. Secondary resistance in GIST
SECONDARYMUTATIONS
DRUG SENSITIVITY
FREQUENCY
Imatinib
Sunitinib
Exon 13
V654
40%
Exon 14
Exon 9
Exon 11
… but is GENERALLY not effective against secondary mutations in the activation loop.
D816
D820
Exon 17
30%
N822
Y823
Sensitive
Debiec-Rychter M, 2005
Antonescu CR, 2005
Wardelmann E, 2006
Heinrich MC, 2008
Liegl B, 2008
Resistant

7. Second- and third-line treatment in GIST
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.
Recently, regorafenib has obtained FDA-approval in GIST patients after failure to IM and SU.
The main challenge after progression to IM is the presence of substantial heterogeneity of secondary resistant mutations between and within metastases from individual patients.
However, due to the presence of these heterogeneous cross-resistant subpopulations, progression-free survival after IM failure is 4 to 6 months irrespective of the second or third line TKI used.

8. Aims
We investigated novel strategies to overcome heterogeneity of resistant clones in TKI-resistant GIST patients.
Based on this, we investigated novel strategies to overcome heterogeneity of resistant clones in TKI-resistant GIST patients.

9. Imatinib, sunitinib and regorafenib are active against primary KIT exon 11 mutation
DMSO
DMSO
DMSO nM 50
100
500 50
100
500 50
100
500
p-KIT (Y703)
p-AKT S473
IM, as well as SU and REGO, are all potent inhibitors of GIST cell line models with KIT exon 11 primary mutations.
p-S6 (S235/236)
Actin

10. Imatinib, sunitinib and regorafenib are active against primary KIT exon 11 mutation
DMSO
DMSO
DMSO nM 50
100
500 50
100
500 50
100
500
p-KIT (Y703)
p-AKT S473
IM, as well as SU and REGO, are all potent inhibitors of GIST cell line models with KIT exon 11 primary mutations.
p-S6 (S235/236)
Actin

11. Imatinib, sunitinib and regorafenib are active against primary KIT exon 11 mutation
DMSO
DMSO
DMSO nM 50
100
500 50
100
500 50
100
500
p-KIT (Y703)
p-AKT S473
IM, as well as SU and REGO, are all potent inhibitors of GIST cell line models with KIT exon 11 primary mutations.
p-S6 (S235/236)
Actin

12. 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 17 (D816E)
3,111
395
GIST-T1/820
Ex 17 (D820A)
2,599
368
SU and REGO activity against common IM-resistant 2ND mutations is shown.
In GIST cell viability studies, SU but not REGO potently inhibits GIST cells with the Ex 13 IM-resistant mutations. By contrast, REGO, but not SU, potently inhibits cells with common KIT ex 17 IM-resistant mutations.
These in vitro findings were corroborated by clinical data, as shown in the next slide…
*IC50s: Green = predictive of clinical efficacy
Red = predictive of clinical resistance

13. 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 17 (D816E)
3,111
395
GIST-T1/820
Ex 17 (D820A)
2,599
368
SU and REGO activity against common IM-resistant 2ND mutations is shown.
In GIST cell viability studies, SU but not REGO potently inhibits GIST cells with the Ex 13 IM-resistant mutations. By contrast, REGO, but not SU, potently inhibits cells with common KIT ex 17 IM-resistant mutations.
These in vitro findings were corroborated by clinical data, as shown in the next slide…
*IC50s: Green = predictive of clinical efficacy
Red = predictive of clinical resistance

14. 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 17 (D816E)
3,111
395
GIST-T1/820
Ex 17 (D820A)
2,599
368
SU and REGO activity against common IM-resistant 2ND mutations is shown.
In GIST cell viability studies, SU but not REGO potently inhibits GIST cells with the Ex 13 IM-resistant mutations. By contrast, REGO, but not SU, potently inhibits cells with common KIT ex 17 IM-resistant mutations.
These in vitro findings were corroborated by clinical data, as shown in the next slide…
*IC50s: Green = predictive of clinical efficacy
Red = predictive of clinical resistance

15. Progression of KIT Exon 13 imatinib-resistant subclone on regorafenib
KIT exon 13 (V654A). Radiographic and metabolic progression on regorafenib
Baseline
This patient was treated with REGO in the phase II clinical trial, developed a new site of metastatic disease after 12 cycles of treatment. Resection and sequencing of this progressing lesion showed the V654A KIT ATP-BP secondary resistance mutation.
C12D21
Resection biopsy
exon 11 + exon 13
(V654A)

16. imatinib-resistant subclone on regorafenib
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)
By contrast, patients with KIT A-loop IM-resistance mutations showed clinical response to REGO. After 4 cycles of REGO, this patient achieved a partial response and a complete metabolic response.
C4D21

17. Sunitinib and regorafenib have complementary
activity against IM-resistant KIT mutations
KIT Mutation
Activity
Primary
Secondary IM SU
REGO
Ex 11
Sensitive
Ex 13 (V654A)
Resistant
Ex 17 (D816)
Ex 17 (D820)
IN SUMMARY, BOTH LAB MODELS AND CLINICAL CORRELATES SHOW THAT SU AND REGO ARE ACTIVE AGAINST IM-SENSITIVE KIT EX 11 MUTANT GIST CLONES. SU HAS POTENT ACTIVITY AGAINST THE V654A KIT IM-RESISTANCE MUTATION in the exon 13, AND REGO IS ACTIVE AGAINST COMMON KIT Exon 17 IM-RESISTANCE MUTATIONS.

18. Targeting TKI-resistance heterogeneity in GIST
ATP-binding pocket
SUNITINIB
Activation
Loop
REGORAFENIB
Based on this complementary activity of SU and REGO, WE HYPOTHESIZED THAT
Cycling SU and REGO MIGHT suppress a broader spectrum of IM-resistant GIST clones and achieve prolonged long-term disease control
Another option would be to combine both drugs together, but there would be very likely high rate of secondary effects, and would be more difficult to achieve enough drug dose level to target specific resistant subclones.
Cycling sunitinib and regorafenib might suppress a broader spectrum of imatinib-resistant GIST clones and achieve prolonged long-term disease control

19. 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
BECAUSE THIS TKI CYCLING CONCEPT PROVIDES ONLY INTERMITTENT INHIBITORY PRESSURE AGAINST VARIOUS IM-RESISTANT SUBCLONES, WE DETERMINED HOW RAPIDLY KIT SIGNALING AND MITOTIC ACTIVITY ARE RESTORED ONCE TKI-PRESSURE IS RELEASED IN LAB MODELS AND IN GIST PATIENTS in order to establish a time-frame to alternate both drugs.

20. Sunitinib washout: reactivation of
KIT, downstream pathways, and cell cycle
GIST430/654
exon 11 + exon 13
Sunitinib treatment
SU 500nM
Days of drug withdrawal
DAY 0
DAY 1
DAY 3
DAY 7
pKIT Y703
pAKT S473
pRB S795
AFTER DISCONTINUING SU TX FOR V654A IM-RESISTANT GIST, KIT AND DOWNSTREAM KINASES ARE REACTIVATED IN 1 TO 3 DAYS, WHEREAS REACTIVATION OF CELL CYCLE BIOMARKERS, INCLUDING HYPERPHOSPHORYLATED RB and Cyclin A expression, takes 3 to 7 days.
THESE RESPONSES ARE DOSE-DEPENDENT.
Cyclin A
Actin

21. Sunitinib washout: reactivation of
KIT, downstream pathways, and cell cycle
GIST430/654
exon 11 + exon 13
Sunitinib treatment
SU 500nM
Days of drug withdrawal
DAY 0
DAY 1
DAY 3
DAY 7
pKIT Y703
pAKT S473
pRB S795
AFTER DISCONTINUING SU TX FOR V654A IM-RESISTANT GIST, KIT AND DOWNSTREAM KINASES ARE REACTIVATED IN 1 TO 3 DAYS, WHEREAS REACTIVATION OF CELL CYCLE BIOMARKERS, INCLUDING HYPERPHOSPHORYLATED RB and Cyclin A expression, takes 3 to 7 days.
THESE RESPONSES ARE DOSE-DEPENDENT.
Cyclin A
Actin

22. Sunitinib washout: reactivation of
KIT, downstream pathways, and cell cycle
GIST430/654
exon 11 + exon 13
Sunitinib treatment
SU 500nM
Days of drug withdrawal
DAY 0
DAY 1
DAY 3
DAY 7
pKIT Y703
pAKT S473
pRB S795
AFTER DISCONTINUING SU TX FOR V654A IM-RESISTANT GIST, KIT AND DOWNSTREAM KINASES ARE REACTIVATED IN 1 TO 3 DAYS, WHEREAS REACTIVATION OF CELL CYCLE BIOMARKERS, INCLUDING HYPERPHOSPHORYLATED RB and Cyclin A expression, takes 3 to 7 days.
THESE RESPONSES ARE DOSE-DEPENDENT.
Cyclin A
Actin

23. reactivation of proliferation
Sunitinib washout:
reactivation of proliferation
GIST430/654
exon 11 + exon 13
Sunitinib treatment
Untreated
SU 100nM
SU 500nM
Day 0
KI-67 expression
Day 1
After withdrawal of low dose SU (100nM), KI67 proliferation marker and mitotic activity were restored in 3 days, whereas after wd of high dose of SU (500nM) they were restored in 7 days.
Mitotic Count (per 5 mm2) UT
100nM
500nM
Day 0 62 4 1
Day 1 60 3
Day 3 63 45
Day 7 68 65 11
Day 3
Day 7

24. reactivation of proliferation
Sunitinib washout:
reactivation of proliferation
GIST430/654
exon 11 + exon 13
Sunitinib treatment
Untreated
SU 100nM
SU 500nM
Day 0
KI-67 expression
Day 1
After withdrawal of low dose SU (100nM), KI67 proliferation marker and mitotic activity were restored in 3 days, whereas after wd of high dose of SU (500nM) they were restored in 7 days.
Mitotic Count (per 5 mm2) UT
100nM
500nM
Day 0 62 4 1
Day 1 60 3
Day 3 63 45
Day 7 68 65 11
Day 3
Day 7

25. Regorafenib washout: reactivation of
KIT, downstream pathways, and cell cycle
GIST48/820
exon 11 + exon 17
Regorafenib treatment
REGO 500nM
Days of drug withdrawal
DAY 0
DAY 1
DAY 3
DAY 7
pKIT Y703
pAKT S473
pRB S795
The same pattern of recovery was seen after discontinuing REGO against exon 17 resistant mutation.
KIT AND DOWNSTREAM KINASES ARE REACTIVATED IN 1 TO 3 DAYS, WHEREAS REACTIVATION OF CELL CYCLE BIOMARKERS, takes at least 3 days.
Cyclin A
Actin

26. Regorafenib washout: reactivation of
KIT, downstream pathways, and cell cycle
GIST48/820
exon 11 + exon 17
Regorafenib treatment
REGO 500nM
Days of drug withdrawal
DAY 0
DAY 1
DAY 3
DAY 7
pKIT Y703
pAKT S473
pRB S795
The same pattern of recovery was seen after discontinuing REGO against exon 17 resistant mutation.
KIT AND DOWNSTREAM KINASES ARE REACTIVATED IN 1 TO 3 DAYS, WHEREAS REACTIVATION OF CELL CYCLE BIOMARKERS, takes at least 3 days.
Cyclin A
Actin

27. Regorafenib washout: reactivation of
KIT, downstream pathways, and cell cycle
GIST48/820
exon 11 + exon 17
Regorafenib treatment
REGO 500nM
Days of drug withdrawal
DAY 0
DAY 1
DAY 3
DAY 7
pKIT Y703
pAKT S473
pRB S795
The same pattern of recovery was seen after discontinuing REGO against exon 17 resistant mutation.
KIT AND DOWNSTREAM KINASES ARE REACTIVATED IN 1 TO 3 DAYS, WHEREAS REACTIVATION OF CELL CYCLE BIOMARKERS, takes 3 to 7 days.
Cyclin A
Actin

28. reactivation of proliferation
Regorafenib washout:
reactivation of proliferation
GIST48/820
exon 11 + exon 17
Regorafenib treatment
Untreated
REGO 100nM
REGO 500nM
Day 0
KI-67 expression
Day 1
The same dose-response pattern with the low dose (3 days) and the high dose (7 days) of REGO was observed.
Mitotic Count (per 5 mm2) UT
100nM
500nM
Day 0 42 15 1
Day 1 44 13
Day 3 54 63 2
Day 7 35
Day 3
Day 7

29. reactivation of proliferation
Regorafenib washout:
reactivation of proliferation
GIST48/820
exon 11 + exon 17
Regorafenib treatment
Untreated
REGO 100nM
REGO 500nM
Day 0
KI-67 expression
Day 1
The same dose-response pattern with the low dose (3 days) and the high dose (7 days) of REGO was observed.
Mitotic Count (per 5 mm2) UT
100nM
500nM
Day 0 42 15 1
Day 1 44 13
Day 3 54 63 2
Day 7 35
Day 3
Day 7

30. 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
Therefore, we have shown that, once a KIT inhibitor is withdrawn (either sunitinib or regorafenib), there is a time-dependent recovery from phosphorylation, to the activation of the cell cycle, to the presence of overt mitotic figures, and in all cases, we start to see mitotic figures after the day 3 and before the day 7 after the drug withdrawal.
7 days

31. 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 1b
Exon 13 (V654A) 2a
REGO 9
Exon 17 (Y823D) 13 2b
Exon 17 (D820Y) 7
A biopsy series from GIST patients responding to KIT inhibitors supported the laboratory evidence for recovery time after withdrawal of TKIs against susceptible mutations.
In patient 1, mitotic activity have not recovered after 3 days of wd of SU
By contrast, in patient 2 the mitotic activity in responding lesions had substantially recovered after 9 days of REGO withdrawal.

32. 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.
Our studies characterize a novel strategy to target heterogeneous drug-resistant subclones in cancer, and provide a biological rational for a forthcoming clinical trial in which we plan to test sequential rapid-alternation of sunitinib and regorafenib in advanced GIST patients after progression to imatinib. We have observed that there is not recovery of proliferation after 3 to 4 days of drug withdrawal, but there is substantial in 7 days, leading to an alternation regimen of 3 days of SU followed by 4 days of REGO.
This rapid alternation regimen might minimize toxic effects while broadening the spectrum of inhibition of IM-resistant clones.

33. Conclusions
Sunitinib and regorafenib have complementary activity against secondary KIT mutations.
After withdrawal of an effective KIT inhibitor, target re-activation occurs in 1 to 3 days.
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.
In conclusion:
SU and REGO have complementary activity against secondary KIT mutations.
Our results demonstrate that after withdrawal of an effective KIT inhibitor, target re-activation occurs in 1 to 3 days.
Proliferation markers are re-activated between 3 to 7 days, and mitotic re-activation is observed in vitro and in clinical correlates between 4 to 7 days.
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.
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.

34. Co-authors / Acknowledgments
Brigham and Women’s Hospital
Ludwig Center at Dana-Farber Cancer Institute
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
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
And finally, I would like to thank MY LAB MENTOR, JONATHAN FLETCHER, ALL MY COLLEAGUES IN THE FLETCHER LABORATORY, and ALL MY CLINICAL MENTORS AT DANA-FARBER, OF WHOM SUZANNE GEORGE AND GEORGE DEMETRI HAVE PLAYED KEY ROLES IN SUPPORTING ME IN DEVELOPING THE RAPID ALTERNATING TKI CLINICAL TRIAL, AND I WOULD ALSO LIKE TO THANK THE FACULTY OF FLIMS 2013 WORKSHOP FOR THEIR INVALUABLE EFFORTS IN HELPING ME IMPROVE THE TRIAL DESIGN. And all of you for your attention. Thanks so much.
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