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

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.

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

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

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

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

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.

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.

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

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

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

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

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

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

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)

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

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.

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

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.

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

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

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

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

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

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

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

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

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

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

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

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.

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.

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.

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