Robust Activity of Avapritinib, Potent and Highly Selective Inhibitor of Mutated KIT, in Patient-Derived Xenograft Models Of

Published OnlineFirst October 1, 2018; DOI: 10.1158/1078-0432.CCR-18-1858

Research Article Clinical Cancer Research Robust Activity of Avapritinib, Potent and Highly Selective Inhibitor of Mutated KIT, in Patient-derived Xenograft Models of Gastrointestinal Stromal Tumors Yemarshet K. Gebreyohannes1, Agnieszka Wozniak1, Madalina-Elena Zhai1, Jasmien Wellens1, Jasmien Cornillie1, Ulla Vanleeuw1, Erica Evans2, Alexandra K. Gardino2, Christoph Lengauer2, Maria Debiec-Rychter3, Raf Sciot4, and Patrick Schoffski€ 1

Abstract

KIT11 Purpose: Gastrointestinal stromal tumors (GIST) are [UZLX-GIST3 ]; and avapritinib (10, 30, 60 mg/kg/once commonly treated with tyrosine kinase inhibitors daily) versus vehicle, imatinib (50, 100 mg/kg/twice daily), KIT9 (TKI). The majority of patients with advanced GIST or sunitinib (40 mg/kg/once daily; UZLX-GIST2B ). ultimately become resistant to TKI due to acquisition of Results: In all models, avapritinib resulted in reduction of secondary KIT mutations, whereas primary resistance is tumor volume, significant inhibition of proliferation, and mainly caused by PDGFRA p.D842V mutation. We tested reduced KIT signaling. In two models, avapritinib led to the activity of avapritinib, a potent and highly selective remarkable histologic responses, increase in apoptosis, and inhibitor of mutated KIT and PDGFRA, in three patient- inhibition of MAPK-phosphorylation. Avapritinib showed KIT 11þ17 KIT 9 derived xenograft (PDX) GIST models carrying differ- superior (UZLX-GIST9 and -GIST2B ) or equal KIT 11 ent KIT mutations, with differential sensitivity to stan- (UZLX-GIST3 ) antitumor activity to the standard dose KIT 11þ17 dard TKI. of imatinib. In UZLX-GIST9 , the antitumor effects of Experimental Design: NMRI nu/nu mice (n ¼ 93) were avapritinib were significantly better than with imatinib or transplanted with human GIST xenografts with KIT exon regorafenib. KIT 11þ17 KIT 11 11þ17 (UZLX-GIST9 ), exon 11 (UZLX-GIST3 ), Conclusions: Avapritinib has significant antitumor KIT 9 or exon 9 (UZLX-GIST2B ) mutations, respectively. activity in GIST PDX models characterized by different We compared avapritinib (10 and 30 mg/kg/once daily) KIT mutations and sensitivity to established TKI. These versus vehicle, imatinib (50 mg/kg/bid) or regorafenib data provide strong support for the ongoing clinical trials KIT 11þ17 (30 mg/kg/once daily; UZLX-GIST9 ); avapritinib with avapritinib in patients with GIST (NCT02508532, (10, 30, 100 mg/kg/once daily) versus vehicle or imatinib NCT03465722). Clin Cancer Res; 1–10. 2018 AACR.

Introduction 1Laboratory of Experimental Oncology, Department of Oncology, KU Leuven, and Department of General Medical Oncology, University Hospi- Gastrointestinal stromal tumors (GIST) are the most com- tals Leuven, Leuven Cancer Institute, Leuven, Belgium. 2Blueprint Med- mon soft tissue sarcomas of the gastrointestinal tract with an 3 icines Corporation, Cambridge, Massachusetts. Department of Human annual incidence of 10 to 15 cases per million people (1). The Genetics, KU Leuven and University Hospitals Leuven, Leuven, Belgium. 4 discovery that the vast majority of GISTs are driven by activat- Department of Pathology, KU Leuven and University Hospitals Leuven, KIT Leuven, Belgium. ing mutations in or platelet-derived growth factor receptor alpha (PDGFRA) has improved our understanding of the Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). molecular pathogenesis of GISTandledtothesuccessful development of targeted therapies for this malignancy (2). Y.K. Gebreyohannes and A. Wozniak contributed equally to this article. Mutations in KIT or PDGFRA lead to a constitutive, ligand- Prior presentation: Part of the results of this study have been presented at the independent activation of kinase activity and their downstream – American Association for Cancer Research Annual Meeting 2017 (April 1 5, 2017 signaling cascades, resulting in increased tumor cell prolifera- Washington DC; abstract 2081). tion and survival (2). Surgical resection is the only available Corresponding Author: Agnieszka Wozniak, KU Leuven, Herestraat 49, post curative treatment for primary, localized, and resectable GIST, 815, Leuven 3000, Belgium. Phone: 321-634-1669; Fax: 321-634-6901; E-mail: yet 40% to 50% of patients will experience recurrent or met- [email protected] astatic disease during follow-up (3). Furthermore, a subset of doi: 10.1158/1078-0432.CCR-18-1858 patients is not eligible for surgical treatment due to anatomic 2018 American Association for Cancer Research. limitations, general condition, or the presence of synchronous

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the unsatisfactory efficacy of second- and third-line agents, their Translational Relevance broader activity against multiple molecular targets leads to off- Advanced gastrointestinal stromal tumors (GIST) are rou- target toxicity, and many patients do not tolerate sunitinib and tinely treated with tyrosine kinase inhibitors (TKI). However, regorafenib as well as the first-line standard of care. over time, the vast majority of patients develop resistance to Avapritinib (BLU-285, Blueprint Medicines) is an oral, high- TKI, mainly due to the acquisition of a secondary mutation in ly selective, and potent investigational inhibitor with activity the activation loop of KIT. Both imatinib and sunitinib are against KIT exon 17 activation loop mutants, including KIT ineffective in treating GIST with such mutations. Although p.D816V. This mutation is a known driver mutation in syste- regorafenib is active against some of these activation loop mic mastocytosis (9). In vitro, avapritinib disrupts KIT sig- mutants, in the clinic it achieves a median progression-free naling as assessed by inhibition of both KIT phosphorylation survival of only 4.5 months. Avapritinib, a novel, potent, and and activation of downstream proteins such as AKT and STAT3 selective inhibitor of KIT and PDGFRA activation loop muta- in human mast cell and leukemia cell lines (10). In vivo, tions, showed robust in vivo antitumor activity in patient- avapritinib achieves dose-dependent tumor growth inhibition derived GIST xenografts. Our preclinical findings indicate that in a mouse model of systemic mastocytosis (10). Moreover, avapritinib could be a relevant treatment for patients with avapritinib also inhibits PDGFRA p.D842V (11), the mutation GIST with primary or secondary resistance to approved TKI responsible for one out of five primary gastric GIST, for and support investigation in ongoing clinical trials in patients which there is no effective treatment available (12). Avapritinib with GIST. is currently under investigation inpatientswithunresectable, treatment-resistant solid tumors including GIST (ClinicalTrials. gov: NCT02508532 and NCT03465722) and in advanced systemic mastocytosis (NCT02561988). In this work, we assessed the preclinical activity of avapri- metastatic disease (1). The dependence of GIST on mutated tinib in vivo, using three patient-derived xenograft (PDX) mod- receptor tyrosine kinase led to the exploration of tyrosine kinase els of GIST, characterized by diverse KIT mutations and varying inhibitors (TKI) for the systemic treatment of this rare but well- sensitivity to the available standard TKI therapies. characterized malignancy. Imatinib, a small molecule with ATP- mimetic properties, has become the standard first-line treatment for patients with locally advanced, recurrent, inoperable, or met- Materials and Methods astatic disease (4). Imatinib has tremendously improved the Xenograft models survival of patients with GIST with advanced disease and achieves For the current project, we transplanted three PDX models, disease control in approximately 85% of the cases (5). Further- established and fully characterized in the Laboratory of Experi- more, the drug was found to extend relapse-free survival and mental Oncology, KU Leuven (Leuven, Belgium). A total of 93 overall survival when used in the adjuvant setting, after surgery in NMRI (nu/nu) mice (Janvier Laboratories) were bilaterally KIT 11þ17 patients with high risk of relapse (1). In most patients with engrafted with models UZLX-GIST9 (KIT: p.P577del; KIT 11 metastatic GIST, however, the duration of response to imatinib W557LfsX5;D820G), -GIST3 (KIT: p.W557_V559delinsF), KIT 9 is limited. The occurrence of resistance, which is mainly caused by and -GIST2B (KIT: p.A502_Y503dup), which are known to the acquisition of secondary mutations, leads to progression retain morphologic and molecular features of the original tumor during treatment with imatinib or related compounds. Other during passaging. The model characteristics and experimental set small-molecule TKIs, such as sunitinib and regorafenib, are used up are presented in Table 1. Xenografting of human tumors from in patients who are progressing on or are intolerant to imatinib consenting patients with GIST was approved by the Medical Ethics (6). Despite their well-documented clinical activity in imatinib- Committee of the University Hospitals Leuven and the animal refractory GIST and their broader activity against a variety of experiments using PDX were approved by the Ethics Committee molecular targets, progression on these agents typically occurs for Animal Research, KU Leuven, and performed according to its after a median treatment duration of less than a year (6). To date, guidelines and Belgian regulations. there are no established standard treatment alternatives for patients with GIST after failure on all three approved lines of TKI Drugs, reagents, and experimental design treatment, but a number of compounds are currently being tested The dosing solutions of imatinib, sunitinib, and regorafenib in early clinical trials in patients with refractory tumors. Never- (all from Sequoia Research) were prepared as described earlier theless, there is still an unmet medical need for novel treatment (13). Avapritinib, provided by Blueprint Medicines, was dis- approaches, which should be tested first in preclinical settings. solved in 0.5% carboxymethyl cellulose supplemented with 1% Secondary mutation in KIT or PDGFRA is likely the most Tween 80. The resulting suspension was kept at 4Cprotected important event leading to TKI resistance. These mutations can from light; a fresh suspension was prepared every 3 days. occur in KIT exon 13 and 14, encoding the ATP-binding pocket Chemical structures of all drugs used in the study are presented of the receptor, or in exon 17 and 18, in the kinase activation in Supplementary Fig. S1, the structure of avapritinib has been loop. The latter stabilize the receptor in its active conforma- previously published by Evans and colleagues (11). When tion, and the majority of these mutations are known to cause tumor growth had reached a threshold of 500 mm3,micewere resistance to both imatinib and sunitinib, which are widely treated with the oral compounds for 16 days by gavaging. The used as first- and second-line agents in the clinic (4). Although doses of 10 and 30 mg/kg avapritinib, tested in all three regorafenib is active against some of these mutated forms, a models, were chosen based on previous preclinical in vivo work typical patient receiving this third-line treatment progresses in a KIT exon 17 mutant mastocytoma model demonstrating after a median period of only 4–5 months (7, 8). Apart from dose response activity that was well tolerated in vivo (11). The

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Table 1. Detailed description of xenograft models and experimental set-up Model name UZLX-GIST9KIT 11þ17 UZLX-GIST3KIT 11 UZLX-GIST2BKIT 9 Model characteristics KIT mutation Exon 11: p.P577del;W557LfsX5; exon 17: D820G Exon 11: p.W557_V559delinsF Exon 9: p.A502_Y503dup Sensitivity to imatinib in vivo Resistant Sensitive Dose-dependent sensitivity Treatment groups Control Vehiclea (n ¼ 7) Vehiclea (n ¼ 6) Vehiclea (n ¼ 4) Imatinib 50 mg/kg/bid (n ¼ 7) 50 mg/kg/bid (n ¼ 5) 50 mg/kg/bid (n ¼ 4) n/a n/a 100 mg/kg/bid (n ¼ 4) Sunitinib n/a n/a 40 mg/kg/qd (n ¼ 4) Regorafenib 30 mg/kg/qd (n ¼ 7) n/a n/a Avapritinib 10 mg/kg/qd (n ¼ 6) 10 mg/kg/qd (n ¼ 6) 10 mg/kg/qd (n ¼ 5) 30 mg/kg/qd (n ¼ 6) 30 mg/kg/qd (n ¼ 6) 30 mg/kg/qd (n ¼ 5) n/a n/a 60 mg/kg/qd (n ¼ 5) n/a 100 mg/kg/qd (n ¼ 6) n/a Abbreviations: bid, twice daily; n, number of mice; n/a, not applicable; qd, once daily. a0.5% carbomethyl cellulose with 1% Tween 80. increased doses of avapritinib, used in models with primary The HR was graded by assessing the magnitude of necrosis, KIT 11 KIT 9 mutations (i.e. UZLX-GIST3 and UZLX-GIST2B ), were myxoid degeneration, and/or fibrosis using a previously chosen based on the biochemical evaluation of avapritinib, described grading system: grade 1 (0%–10% of tumor area), which demonstrated IC50 of approximately 0.2–1nmol/Lon grade 2 (>10% and 50%), grade 3 (>50% and 90%), and KIT exon 17 mutants, 1–2 nmol/L against KIT exon 11 mutants, grade 4 (>90%; refs. 14, 15). Moreover, IHC was performed for and >50 nmol/L IC50 against KIT wild-type kinase domain (11) KIT and DOG1, Ki67, and pHH3 staining was used to assess and suggested that potentially higher exposure of avapritinib proliferation, cleaved-PARP to quantify apoptosis and pMAPK might be required for inhibition of GIST tumors driven by to evaluate KIT pathway activity. Proliferation and apoptosis primary KIT exon 9 and 11 mutants. The detailed information were assessed by counting the number of mitotic and apoptotic about treatment groups and doses are presented in Table 1. cells on H&E–stained slides and the IHC analysis was also During the dosing period, tumor volume was measured three based on counting positive cells. Both evaluations were per- times per week using a digital caliper and the body weight and formed in 10 high-power fields (HPF) at 400-fold magnifica- general well-being of the animals was followed up daily. At the tion. The Ki67-labeling index was calculated as an average end of the experiment, mice were sacrificed 2 hours after the last percentage of Ki67-stained nuclei in 5 digital images taken at dose, and tumors were divided with one half snap frozen in 400-fold magnification. KIT pathway inhibition was evaluated liquid nitrogen and one half fixed in 4% buffered formaldehyde by grading the intensity of the pMAPK staining as well as the for further histopathologic and molecular assessments. Antitu- percentage of tumor area showing positivity, as described in mor activity was assessed on the basis of the evolution of tumor Supplementary Table S1. volume expressed as the percentage of the normalized baseline value. Furthermore, histopathology and Western blotting were Western blot analysis performed. For each mouse, the bilateral tumors were counted To determine the effect of the different treatments on the as independent events. KIT signaling pathway, Western blotting was performed Western blotting and IHC were conducted using the fol- as described previously (16). Densitometry was done using lowing antibodies and reagents: KIT from Dako/Agilent; dis- the AIDA software (Raytest) to do semiquantitation of the covered on GIST 1 (DOG1) from Novocastra; phospho-KITY719 phospho-protein levels (17). (pKITY719), phospho-KITY703 (pKITY703), phospho-AKTS473 (pAKTS473), AKT, a-tubulin, p42/44 MAPK, phospho-MAPK Statistical analysis (pMAPK), 4-E binding protein 1 (4EBP1), phospho-4EBP1 The comparison between tumor volumes on day 1 versus day (p4EBP1), histone H3 (HH3), phospho-HH3 (pHH3), and 16 was done using the Wilcoxon matched pair test (WMP). cleaved-PARP all from Cell Signaling Technology; Ki67 Comparisons between different treatment groups were done from Thermo Fisher Scientific; EnVisionþ System-HRP and using the Mann–Whitney U test (MWU). A value of P < 0.05 was 0 3 diaminobenzidine-tetrahydrochloride (DAB), both from defined as statistically significant. STATISTICA version 13 (Dell Dako/Agilent. For Western blotting, the secondary antibodies, Inc.) was used for all calculations. conjugated with horseradish peroxidase, were from Cell Sig- naling Technology and Western Lightning Plus-ECL from PerkinElmer was used for band visualization. Results Tumor volume assessment Histopathology After the 16-day treatment period, vehicle-treated tumors Hematoxylin and eosin (H&E) staining was performed to from all models showed a steady and statistically significant evaluate the general tumor morphology, the histologic increase in relative tumor volume (216% of the baseline KIT 11þ17 KIT 11 response (HR) to treatment, as well as to count mitotic and volume for UZLX-GIST9 , 293% for -GIST3 ,and KIT 9 apoptotic cells. Stained tissue sections were analyzed using an 172% for -GIST2B ; P < 0.05forall,WMP;Fig.1).Consis- Olympus CH-30M microscope (Olympus). Representative pic- tent with previous reports, no significant difference was KIT 11þ17 tures were captured using the Olympus Color View digital observed in the UZLX-GIST9 model between the rel- camera and analyzed with Olympus Cell D imaging software. ative tumor volume of the vehicle- and imatinib-treated tumors

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remarkable and dose-dependent effects on tumor volume in all three models. During the course of this study, the treatment with avapritinib was well tolerated, and mice had a stable body weight within ethically acceptable limits (Supplementary Fig. S2). We did observe a yellowish skin discoloration in all mice treated with 100 mg/kg of avapritinib, although it did not have any impact on the well-being of animals.

Histopathologic evaluation In all three models, vehicle-treated tumors showed spindle cell morphology and diffuse KIT and DOG1 immunopositivity (Sup- plementary Fig. S3). These characteristics resembled features observed in the original patient samples used for xenografting, as well as those found in previous passages, proving stable morphology of the models. In addition, KIT mutational analysis of ex-mouse tumor samples confirmed the presence of mutations as seen in the patient biopsy. HR was assessed on H&E–stained slides by evaluating the magnitude of the necrosis, fibrosis, and myxoid degeneration in the tumor tissue, induced by different treatments (Fig. 2A). Only KIT 11þ17 minimal (grade 1) HR was observed in UZLX-GIST9 tumors treated with imatinib. Regorafenib induced grade 2 HR in 36% of these tumors mainly through the induction of necrosis. As expected, and in line with our prior observations, imatinib KIT 11 caused grade 2 or higher HR in all treated UZLX-GIST3 Figure 1. tumors, with 67% of the xenografts showing grade 3 HR. In the Evolution of tumor volume during the treatment, presented as relative KIT 9 tumor volume (% change compared with normalized, baseline value) SD UZLX-GIST2B model, all tumors treated with imatinib (both in UZLX-GIST9KIT 11þ17 (A), -GIST3KIT 11 (B), and –GIST2BKIT 9 (C). at standard and higher dose) showed grade 1 HR. In two out of three models, avapritinib resulted in remark- KIT 11þ17 able HR. In UZLX-GIST9 , 30 mg/kg avapritinib (13), whereas regorafenib resulted in modest tumor regression induced grade 2 and grade 3 HR in 60% of the tumors. In KIT 11 (82% of baseline, P ¼ 0.02 WMP). As expected, treatment with UZLX-GIST3 , 10 mg/kg avapritinib induced grade 2 HR in imatinib led to a regression in tumor volume (to 32% of the vast majority (80%) of the tumors. Moreover, the higher KIT 11 baseline) in UZLX-GIST3 ,confirming the imatinib sensi- doses (30 and 100 mg/kg) led to grade 3 and grade 4 HR in this KIT 9 tivity described previously (18). In contrast, UZLX-GIST2B model. This effect was slightly more pronounced than tumors treated with the standard dose of imatinib (50 mg/kg) in tumors treated with imatinib. Interestingly, in both KIT 11þ17 KIT 11 grew significantly. In this model, doubling the dose of imatinib UZLX-GIST9 and -GIST3 ,theHRobservedwith led to tumor stabilization owing to the dose-dependent sensi- avapritinib was characterized mainly by the induction of myx- tivity to imatinib previously observed in this model and in line oid degeneration, which is a typical response pattern observed with the known behavior of KIT exon 9–mutated GIST in the in tumors collected from patients with GIST who responded to clinic (1). Sunitinib caused significant tumor shrinkage in this the treatment with imatinib in the clinic (ref. 19; Fig. 2A). model (Fig. 1). In all models, tumors in the vehicle-treated group showed In all three xenograft models, avapritinib (10 mg/kg) resulted high mitotic activity with an average of 45 mitotic figures in in tumor volume stabilization compared with the baseline value. 10 HPF. Compared with vehicle, all doses of avapritinib led to a This effect was comparable to the effects induced by the higher significant reduction of proliferation in all three xenograft KIT 9 dose of imatinib in UZLX-GIST2B (100 mg/kg) or to regor- models (Fig. 2B; Table 2). Remarkably, in the imatinib-resistant KIT 11þ17 KIT 11þ17 afenib in UZLX-GIST9 (Fig. 1). Remarkably, at the dose of model UZLX-GIST9 , both 10 and 30 mg/kg avapritinib 30 mg/kg, avapritinib treatment resulted in substantial tumor inhibited tumor proliferation significantly better than the regression as compared with baseline in two of the tested models, other tested agents. The antiproliferative effect of avapritinib KIT 11þ17 to 27% in UZLX-GIST9 (P ¼ 0.005) and to 26% in was comparable with imatinib in the imatinib-sensitive model, KIT 11 KIT 11 KIT 9 -GIST3 (P ¼ 0.008, both WMP), and tumor volume stabi- UZLX-GIST3 .InUZLX-GIST2B , higher doses (30 and KIT 9 lization (90%) in UZLX-GIST2B (P ¼ 0.08, WMP). Similarly, 60 mg/kg) of avapritinib inhibited the proliferation significant- KIT 11 in UZLX-GIST3 , higher dose of avapritinib (100 mg/kg), led ly better than imatinib. Moreover, avapritinib at 60 mg/kg to a significant tumor regression to 26% of baseline value (P ¼ had the same antiproliferative effect as sunitinib. These find- 0.005, WMP), which was similar to the effect of imatinib in this ings were confirmed by Western blotting of pHH3 and by KIT 9 model (Fig. 1B). In addition, in UZLX-GIST2B , avapritinib at a IHC for pHH3 and Ki67 (Fig. 2B; Supplementary Fig. S4A; dose of 60 mg/kg led to tumor shrinkage, which was signifi- Table 2). The expression level of pHH3 showed a near-com- cantly better than imatinib (at both doses) and comparable plete absence in avapritinib-treated tumors at both 10 and 30 KIT 11þ17 with sunitinib (Fig. 1C). Taken together, avapritinib induced mg/kg doses in UZLX-GIST9 , a marked decrease in

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A 100% 80% 60% 40% 20% 0% Vehicle Vehicle Vehicle Imatinib Sunitinib Imatinib Regorafenib Imatinib (50 mg/kg) Imatinib (100 mg/kg) Avapritinib (10 mg/kg) Avapritinib Avapritinib (10 mg/kg) Avapritinib (30 mg/kg) Avapritinib Avapritinib (10 mg/kg) Avapritinib (30 mg/kg) Avapritinib (60 mg/kg) Avapritinib Avapritinib (100 mg/kg) Avapritinib Avapritinib (30 mg/kg) Avapritinib UZLX-GIST9KIT 11+17 UZLX-GIST3KIT 11 UZLX-GIST2BKIT 9 Garde 1 Garde 2 Garde 3 Garde 4 B KIT 11+17

UZLX-GIST9 Vehicle Imatinib Regorafenib Avapritinib (10 mg/kg) Avapritinib (30 mg/kg) KIT 11

UZLX-GIST3 Vehicle Imatinib Avapritinib (10 mg/kg) Avapritinib (30 mg/kg) Avapritinib (100 mg/kg) KIT 9

UZLX-GIST2B Vehicle Imatinib (50 mg/kg) Imatinib (100 mg/kg) Sunitinib Avapritinib (10 mg/kg) Avapritinib (30 mg/kg) Avapritinib (60 mg/kg) C

76 80 69 71 61 60 47 40 35 19 23 20 8 3 0 1 110 0 2 0 Vehicle Vehicle Vehicle Imatinib Imatinib Sunitinib Regorafenib Mean number of mitoses per 10 HPF Imatinib (50 mg/kg) Avapritinib (30 mg/kg) Avapritinib Imatinib (100 mg/kg) Avapritinib (10 mg/kg) Avapritinib Avapritinib (10 mg/kg) Avapritinib Avapritinib (30 mg/kg) Avapritinib (10 mg/kg) Avapritinib (30 mg/kg) Avapritinib (60 mg/kg) Avapritinib Avapritinib (100 mg/kg) Avapritinib KIT 11+17 KIT 9 UZLX-GIST9 UZLX-GIST3KIT 11 UZLX-GIST2B

D Avapritinib (mg/kg) Avapritinib (mg/kg) Imatinib (mg/kg) Avapritinib (mg/kg) 10 30 10 30 100 50 100 Sunitinib 10 30 60 Vehicle ImatinibRegorafenib Vehicle Imatinib Vehicle pHH3

HH3

Figure 2. A, Assessment of HR. B, Representative images of H&E staining after treatment (200 magniﬁcation). C, Mean number of mitotic cells in 10 HPF grouped by treatment. D, Assessment of pHH3 expression by Western blotting.

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Table 2. Assessment of proliferation and apoptotic activity in GIST UZLX-GIST9KIT 11þ17 UZLX-GIST3KIT 11 UZLX-GIST2BKIT 9 pHH3 Ki67 pHH3 Ki67 pHH3 Ki67 Proliferative activity Imatinib (50 mg/kg) ¼ ¼ ###a ###a ¼¼ Imatinib (100 mg/kg) n/a n/a n/a n/a #2.5a #2.1a Sunitinib n/a n/a n/a n/a ###a ###a Regorafenib ¼¼n/a n/a n/a n/a Avapritinib (10 mg/kg) #14.4a ###a ###a ###a #1.3a #1.9a Avapritinib (30 mg/kg) ###a ###a ###a ###a #3.4a #3.9a Avapritinib (60 mg/kg) n/a n/a n/a n/a #13.9a #7.9a Avapritinib (100 mg/kg) n/a n/a ###a ###a n/a n/a

H&E Cleaved PARP H&E Cleaved PARP H&E Cleaved PARP Apoptotic activity Imatinib (50 mg/kg) "2.1a ¼"6.5a "6.8a ¼¼ Imatinib (100 mg/kg) n/a n/a n/a n/a #6.8a #2.4a Sunitinib n/a n/a n/a n/a #3.7 #1.3a Regorafenib "3.0 "2.6 n/a n/a n/a n/a Avapritinib (10 mg/kg) ¼#2.6a "4.5a "2.3a ¼¼ Avapritinib (30 mg/kg) "3.4a "3.0a "5.7a "17.1a #2.3a #1.9a Avapritinib (60 mg/kg) n/a n/a n/a n/a #2.6a #1.9a Avapritinib (100 mg/kg) n/a n/a "8.7a "18.1a n/a n/a NOTE: Values are presented as fold change in comparison with the vehicle-treated tumors. Abbreviations: pHH3, phospho-histone H3. aP < 0.05 compared with the vehicle-treated by Mann–Whitney U test. #Decrease. ###>50-fold decrease. "Increase. ¼No signiﬁcant difference.

KIT 11 KIT 9 -GIST3 , and a dose-dependent inhibition in -GIST2B phorylation of KITY703 and downstream proteins was inhib- (Fig. 2B). ited by all treatments, with remarkable inhibition resulting In two out of three models, avapritinib had significant from treatment with sunitinib and avapritinib at dose of KIT 11þ17 proapoptotic activity. In UZLX-GIST9 , avapritinib 60 mg/kg (Fig. 3A and B). Even though avapritinib did not (30 mg/kg) induced a significant increase in apoptosis (3.4-fold inhibit phospho-KIT completely in this model (Fig. 3A), the increase compared with vehicle-treated tumors, P < 0.001, phospho-KIT expression, normalized against the total form of MWU). This was comparable with the effects of regorafenib KIT, is significantly lower when compared with the vehicle- KIT 11 KIT 9 (3-fold increase; Table 2). In UZLX-GIST3 , all doses of treated tumors (Fig. 3B). In UZLX-GIST2B , sunitinib avapritinib led to a significant and dose-dependent increase of showed increased ability to decrease phospho-KIT, however apoptotic activity compared with the vehicle-treated tumors. its downstream inhibitory effect was mainly through the inhi- However, the difference in the induction of apoptosis between bition of phospho-AKT and not phospho-MAPK pathway, as the 30 and 100 mg/kg avapritinib or in comparison with imati- is seen with other effective GIST agents (Fig. 3). nib was not statistically significant. Of note, in this model, the Subsequently, we also performed histopathologic assess- majority of the tumor cells in actively treated tumors were re- ment of KIT signaling using pMAPK immunostaining. The placed by myxoid matrix, therefore counting apoptotic cells evaluation was based on both the staining intensity and per- could only be done in areas with remaining viable cells, which centage of tumor area showing positivity (Supplementary Table KIT 11þ17 KIT 11 may have had an impact on the reliability of the analysis. S1). In the two models (UZLX-GIST9 and -GIST3 ), we observed a strong to very strong MAPK phosphorylation Evaluation of KIT signaling in the majority of vehicle-treated tumors, whereas in KIT 9 Western blot analysis showedthatKITanditsdownstream UZLX-GIST2B , staining was variable with focal positivity. KIT 11þ17 signaling proteins were expressed and activated in vehicle- In UZLX-GIST9 , treatment with imatinib or regorafenib treated tumors from all three models, as expected (Fig. 3A). did not affect MAPK phosphorylation, but in the majority of KIT 11þ17 In UZLX-GIST9 , avapritinib (30 mg/kg) inhibited tumors treated with avapritinib, there was almost complete phosphorylation of KITY703 as well as its downstream compo- inhibition of phosphorylation, independent of a dose (Fig. 4). KIT 11 nents, AKT and MAPK, and to a lesser extent, the phosphory- In UZLX-GIST3 , all active treatments reduced the MAPK KIT 11 KIT 9 lation of 4EBP1. Moreover, in UZLX-GIST3 , all treatments phosphorylation substantially. In UZLX-GIST2B ,however, inhibited the phosphorylation of KIT as well as the downstream only a slight inhibition of phosphorylation was observed in signaling proteins. In addition, the expression of total forms of avapritinib-treated tumors in comparison with the vehicle- the proteins was lower in imatinib- and avapritinib-treated tu- treated group. mors (30 and 100 mg/kg) in comparison with vehicle-treated, which was most likely related to the substantial decrease of the cellularity in the response to treatment. Similarly, expression Discussion KIT 11þ17 ofKITwasfoundtobelowerinUZLX-GIST9 tumors Primary and acquired resistance to treatment with established treated with 30 mg/kg avapritinib as compared with the TKI represents the ultimate challenge in the clinical setting of KIT 9 vehicle-treated group (Fig. 3A). In UZLX-GIST2B ,phos- GIST, as there is no approved therapy for those who progress

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Activity of Avapritinib in GIST Xenografts

A UZLX-GIST9KIT 11+17 UZLX-GIST3KIT 11 UZLX-GIST2BKIT 9

Avapritinib (mg/kg) Avapritinib (mg/kg) Imatinib (mg/kg) Avapritinib (mg/kg) 10 30 10 30100 50100 10 30 60 Vehicle ImatinibRegorafenib Vehicle Imatinib Vehicle Sunitinib KIT pKITY719 pKITY703 AKT pAKTS473 MAPK pMAPK 4EBP1 p4EBP1 Tubulin

50% 50% 50%

0% 0% 0%

−50% −50% −50%

− −100% −100% 100% pKITY703 pKITY719 pAKT pMAPK p4EBP1 pKITY703 pKITY719 pAKT pMAPK p4EBP1 pKITY703 pKITY719 pAKT pMAPK p4EBP1 Imatinib (50 mg/kg) Imatinib (100 mg/kg) Sunitinib Imatinib Regorafenib Avapritinib (10 mg/kg) Avapritinib (30 mg/kg) Imatinib Avapritinib (10 mg/kg) Avapritinib (30 mg/kg) Avapritinib (100 mg/kg) Avapritinib (10 mg/kg) Avapritinib (30 mg/kg) Avapritinib (60 mg/kg) Relative intensity values

Figure 3. KIT signaling pathway. A, Assessment of the effect of treatments in different xenograft models. B, Densitometric assessment of phospho-protein forms in KIT signaling pathway.

on agents with regulatory approval for treatment of patients Avapritinib was designed to potently and selectively inhibit with GIST. In this study, we were able to document a robust KIT exon 17 mutations (11). In vitro biochemical activity for antitumor activity of avapritinib, a potent and selective KIT the KIT p.D816V–mutated receptor was achieved with an inhibitor, in three GIST PDX models characterized by different IC50 ¼ 0.27 nmol/L (11). This mutation is known to be KIT mutations and varying sensitivity to standard TKI. All three causative for systemic mastocytosis, and is found in the vast xenografts utilized in this study were previously validated for majority of patients with this systemic disorder (10). Further- preclinical testing of novel anti-GIST therapies (17, 20). more, avapritinib inhibits proliferation both in vitro and in vivo In the presented experiments, imatinib led to a significant in leukemia models that harbor the KIT exon 17 p.N822K KIT 11 tumor regression in UZLX-GIST3 and a dose-dependent mutation (23). As expected, in our PDX model with KIT KIT 9 KIT 11þ17 effect on tumor volume in UZLX-GIST2B , as already observed exon 11 and 17 mutations (UZLX-GIST9 with KIT: in multiple studies, confirming once again the stable biological p.P577del; W557LfsX5; D820G), avapritinib showed a benefi- behavior of this model (13, 17, 18). These findings are also in line cial effect on tumor volume as well as on proliferation, which with the behavior of such tumors in the clinic; patients with most likely was caused by inhibition of KIT signaling. Of KIT exon 9 mutations respond better to higher dose of imatinib, note, the secondary p.D820G exon 17 KIT mutation, present KIT 11þ17 as confirmed by a clinical meta-analysis (21). Furthermore, in in our UZLX-GIST9 model, has been reported in several KIT 9 UZLX-GIST2B , sunitinib caused tumor regression, which is TKI-resistant GIST cases with the incidence similar to other consistent with others' observations that KIT exon 9 mutations mutations affecting kinase activation loop domain of the KIT 11þ17 benefit more from the therapy with sunitinib than with other receptor (24–27). In our UZLX-GIST9 –resistant model, agents (22). Taken together, these data demonstrate that our the avapritinib dose of 10 mg/kg affected pathway activation, xenograft models exactly mirror the clinical situation and can which resulted in a remarkable decrease of proliferation, how- provide further useful guidance for clinical evaluation of novel ever this dose led only to tumor stabilization and limited HR. therapeutic approaches for GIST. On the other hand, the higher dose of the investigational agent

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A 100%

80%

60%

40%

20%

Percent of tumors 0% Vehicle Vehicle Vehicle Imatinib Imatinib Sunitinib Regorafenib Imatinib (50 mg/kg) Imatinib (100 mg/kg) Avapritinib (30 mg/kg) Avapritinib (60 mg/kg) Avapritinib Avapritinib (10 mg/kg) Avapritinib Avapritinib (30 mg/kg) Avapritinib Avapritinib (10 mg/kg) Avapritinib Avapritinib (30 mg/kg) Avapritinib Avapritinib (10 mg/kg) Avapritinib Avapritinib (100 mg/kg) Avapritinib UZLX-GIST9KIT 11+17 UZLX-GIST3KIT 11 UZLX-GIST2BKIT 9 Negative (-) Very weak (±) Weak (+) Strong (++) Very strong (+++) B KIT 11+17

Vehicle Imatinib Regorafenib Avapritinib (10 mg/kg) Avapritinib (30 mg/kg) UZLX-GIST9 KIT 11

UZLX-GIST3 Vehicle Imatinib Avapritinib (10 mg/kg) Avapritinib (30 mg/kg) Avapritinib (100 mg/kg) KIT 9

UZLX-GIST2B Vehicle Imatinib (50 mg/kg) Imatinib (100 mg/kg)Sunitinib Avapritinib (10 mg/kg) Avapritinib (30 mg/kg) Avapritinib (60 mg/kg)

Figure 4. A, Evaluation of pMAPK positivity based on the intensity and percent tumor area showing pMAPK positivity. Grading system is presented in Supplementary Table S1. B, Representative images of pMAPK immunostaining of the different treatment groups at 200.

(30 mg/kg) caused a striking tumor volume shrinkage (to 27% than what was achieved with 10 mg/kg, both treatment groups of baseline) and an impressive HR with complete absence of exposed to this novel TKI had better responses than imatinib- proliferative activity. This observation is similar to findings by or regorafenib-treated tumors. The antitumor activity of ava- Evans and colleagues where avapritinib potently inhibited the pritinib is currently being evaluated in GIST (NCT02508532 mouse equivalent of KIT p.D816Y–driven tumor growth in vivo and NCT03465722; ref. 28). Of note, the maximum tolerated in a dose-dependent manner (11). Moreover, in a GIST PDX dose of avapritinib in patients is 400 mg/day whereas the dose model derived from a refractory GIST tumor that harbors of 300 mg/day is being evaluated in the currently ongoing KIT exon 11/17 (p.K557_K558del; Y823D), avapritinib again phase III trial (NCT03465722). The animal equivalent dose, led to tumor regression from the baseline value (11). Interest- calculated on the basis of the body surface (29), is respectively ingly, in our experiments, the pattern of response observed in 82 and 61.5 mg/kg, which falls within the range of doses tested KIT 11þ17 UZLX-GIST9 tumors treated with 30 mg/kg was char- in this study. acterized by myxoid degeneration; a phenomenon where Subsequently, we evaluated avapritinib in two additional viable tumor cells are replaced by an amorphous collagenous PDX models with a primary mutation in KIT exon 11 KIT 11 matrix containing only a few scattered cells. Myxoid degener- (UZLX-GIST3 , imatinib sensitive) or exon 9 (UZLX- KIT 9 ation is described frequently as a feature characteristic for GIST2B , dose-dependent sensitivity to imatinib, sensitive GIST responding to the treatment with imatinib, both in to sunitinib). We observed a significant inhibition of prolifer- preclinical and clinical settings (18, 19). Although in this ation in these models at all doses tested, suggesting broader resistant model, the effect of 30 mg/kg avapritinib on the inhibitory capacity of avapritinib against different KIT muta- tumor volume and histologic features was significantly better tions outside of the activation loop region (11). In the UZLX-

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Activity of Avapritinib in GIST Xenografts

KIT 11 GIST3 model, we found a pronounced effect on tumor multitargeted agents currently being used in the clinical setting. volume and histologic responses at 30 and 100 mg/kg, and this Agents such as sunitinib, regorafenib, dasatinib, or ponatinib observation was similar to the effect caused by imatinib. On the show effectiveness in some genotypes of resistant GIST, but KIT 9 other hand, avapritinib in UZLX-GIST2B was better than dose-limiting toxicities, arising from the simultaneous block- imatinib, but only the dose of 60 mg/kg led to a similar efficacy age of several kinases, translates into higher toxicity and limit as achieved with sunitinib. This observation suggests that the clinical usefulness of some of these agents (36, 37). avapritinib could also be effective in GIST with primary muta- Although very preliminary data from the ongoing phase I tions, however higher in vivo concentrations are required for clinical trial suggests a favorable safety profile (28), the high exon 9 antitumor activity. Our studies in mice suggest avapri- specificity of avapritinib can in theory increase the risk of rapid tinib is well tolerated at these higher concentrations, but development of secondary resistance to this compound. GIST is clinical data will be required to fully assess the activity of known for its clinical inter- and intratumoral heterogeneity in avapritinib in patients with KIT exon 9–driven tumors The terms of the mutational profile, and it is likely that some clones ongoing phase I clinical trial with avapritinib accepts patients of this disease may lead to further progression on treatment whohavefailedtwoormoreagentsandlikelyhaveaccumu- with the novel agent. In this context, it is noteworthy that we lated secondary resistance mutations, and patients with saw broad activity in a variety of GIST genotype in our mice. PDGFRA p.D842 mutant–driven GIST independent of prior In conclusion, we provide in vivo evidence that the novel TKI lines of treatment. Interestingly,itisalreadyknownthatava- avapritinib has significant antitumor activity in GIST PDX pritinib inhibits the activity of PDGFRA p.D842V–mutated models. Our results demonstrate that in KIT exon 11þ17 receptor in vitro (11); the in vivo evaluation is hampered by double mutated GIST, this inhibitor is more active than estab- the lack of relevant models with this mutation. lished standard treatments. Moreover, in imatinib-sensitive In our study, we found only a moderate proapoptotic effect models with primary KIT mutations, avapritinib shows a sim- KIT 11 KIT 11þ17 in UZLX-GIST3 and -GIST9 (at a dose of 30 mg/kg), ilar or even higher level of activity in comparison with imatinib. in part explained by the presence of myxoid degeneration as a In all models tested, the pharmacologic antiproliferative effect response to avapritinib in these models. The reduced cellularity on tumor volume was mainly achieved through a marked potentially leads to an underestimation of the proapoptotic inhibition of KIT signaling. Our data strongly support the effects of this treatment in our models. This speculation is sup- scientific rationale of the ongoing exploration of avapritinib ported by the observation of increased apoptosis in tumors in GIST and provide arguments for exploration of the novel harvested after 8 days of treatment with avapritinib, when the compound in the clinic in both imatinib-sensitive and TKI- histologic response was not as pronounced as on day 16 (11). The resistant genotypes of this mesenchymal malignancy. The proapoptotic effect of avapritinib is likely induced by inhibition results seen in our mouse PDXs and the early findings reported of KIT signaling. This observation matches previous findings from the ongoing clinical trial in patients with GIST suggest that reported by our group, where potent KIT signaling inhibition avapritinib has the potential to become an important treatment resulted in an increased apoptosis (16, 17). Moreover, Evans and option for this orphan malignancy. colleagues showed proapoptotic activity of avapritinib in a mouse mastocytoma cell line with KIT exon 17 substitution p.D814Y, the Disclosure of Potential Conflicts of Interest equivalent of human p.D816Y mutant (10, 23). On the other E. Evans, A.K. Gardino, and C. Lengauer have ownership interests (including KIT 9 hand, in the UZLX-GIST2B , neither of the experimental patents) in Blueprint Medicines. No potential conflicts of interest were disclosed treatments led to an increase of apoptotic activity after 16 days by the other authors. of treatment. A trend toward a decline of apoptosis was observed with increasing dose of avapritinib. Similar observations were Authors' Contributions previously reported in this model, where treatments that led to Conception and design: A. Wozniak, E. Evans, C. Lengauer, P. Schoffski€ inhibition of KIT signaling resulted in a decrease of apoptotic Development of methodology: Y.K Gebreyohannes, A. Wozniak, J. Wellens, € activity (17, 20). This effect could be a consequence of the variable M. Debiec-Rychter, P. Schoffski expression of signaling molecules due to specific KIT genotype as Acquisition of data (provided animals, acquired and managed patients, – provided facilities, etc.): Y.K Gebreyohannes, A. Wozniak, M.-E. Zhai, reported by several groups (30 32). J.Wellens,J.Cornillie,R.Sciot,P.Schoffski€ Treatment with avapritinib was well tolerated in our nude Analysis and interpretation of data (e.g., statistical analysis, biostati- mice. Although we observed yellow skin discoloration in mice stics, computational analysis): Y.K Gebreyohannes, A. Wozniak, M.-E. Zhai, treated with the highest avapritinib dose tested (100 mg/kg), it E. Evans, C. Lengauer, R. Sciot, P. Schoffski€ did not impact the animals' well-being. We also did not find Writing, review, and/or revision of the manuscript: Y.K Gebreyohannes, any macroscopic or microscopic changes in their organs, A. Wozniak, M.-E. Zhai, J. Cornillie, E. Evans, A.K. Gardino, C. Lengauer, M. Debiec-Rychter, R. Sciot, P. Schoffski€ including the liver. The skin color change may be due to a Administrative, technical, or material support (i.e., reporting or organiz- stronger systemic inhibition of KIT, disrupting its physiologic ing data, constructing databases): J. Wellens, U. Vanleeuw, M. Debiec-Rychter, function in follicular melanocytes and or impairing melano- P. Schoffski€ genesis (33). Skin depigmentation as a result of strong KIT Study supervision: E. Evans, C. Lengauer, P. Schoffski€ inhibition was previously reported by Kim and colleagues in the evaluation of another potent KIT inhibitor, PLX3397 and The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked was reported in metastatic renal cell carcinoma, treated with advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate TKI sorafenib (34, 35). this fact. As a selective inhibitor of KIT and PDGFRA p.D842V mutations, avapritinib is less likely to cause significant off target Received June 13, 2018; revised August 9, 2018; accepted September 25, 2018; treatment-related toxicity at efficacious doses, in contrast to published first October 1, 2018.

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Robust Activity of Avapritinib, Potent and Highly Selective Inhibitor of Mutated KIT, in Patient-derived Xenograft Models of Gastrointestinal Stromal Tumors

Yemarshet K. Gebreyohannes, Agnieszka Wozniak, Madalina-Elena Zhai, et al.

Clin Cancer Res Published OnlineFirst October 1, 2018.

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