Cabozantinib Is Active Against Human Gastrointestinal Stromal Tumor Xenografts Carrying Different KIT Mutations Yemarshet K

Published OnlineFirst October 24, 2016; DOI: 10.1158/1535-7163.MCT-16-0224

Small Molecule Therapeutics Molecular Cancer Therapeutics Cabozantinib Is Active against Human Gastrointestinal Stromal Tumor Xenografts Carrying Different KIT Mutations Yemarshet K. Gebreyohannes1, Patrick Schoffski€ 1, Thomas Van Looy1, Jasmien Wellens1, Lise Vreys1, Jasmien Cornillie1, Ulla Vanleeuw1, Dana T. Aftab2, Maria Debiec-Rychter3, Raf Sciot4, and Agnieszka Wozniak1

Abstract

In the majority of gastrointestinal stromal tumors (GIST), (50 mg/kg/bid), and cabozantinib (30 mg/kg/qd) and treated oncogenic signaling is driven by KIT mutations. Advanced orally for 15 days. Cabozantinib resulted in significant tumor GIST is treated with tyrosine kinase inhibitors (TKI) such as regression in UZLX-GIST4 and -GIST2 and delayed tumor imatinib. Acquired resistance to TKI is mainly caused by growth in -GIST9. In all three models, cabozantinib inhibited secondary KIT mutations, but can also be attributed to a switch the proliferative activity, which was completely absent in of KIT dependency to another receptor tyrosine kinase (RTK). UZLX-GIST4 and significantly reduced in -GIST2 and -GIST9. We tested the efficacy of cabozantinib, a novel TKI targeting Increased apoptotic activity was observed only in UZLX-GIST4. KIT, MET, AXL, and vascular endothelial growth factor recep- Cabozantinib inhibited the KIT signaling pathway in UZLX- tors (VEGFR), in patient-derived xenograft (PDX) models of GIST4 and -GIST2. In addition, compared with both control GIST, carrying different KIT mutations. NMRI nu/nu mice (n ¼ and imatinib, cabozantinib significantly reduced microvessel 52) were bilaterally transplanted with human GIST: UZLX- density in all models. In conclusion, cabozantinib showed GIST4 (KIT exon 11 mutation, imatinib sensitive), UZLX- antitumor activity in GIST PDX models through inhibition GIST2 (KIT exon 9, imatinib dose-dependent resistance), of tumor growth, proliferation, and angiogenesis, in both or UZLX-GIST9 (KIT exon 11 and 17 mutations, imatinib imatinib-sensitive and imatinib-resistant models. Mol Cancer resistant). Mice were grouped as control (untreated), imatinib Ther; 15(12); 2845–52. 2016 AACR.

Introduction or in platelet-derived growth factor receptor alpha (PDGFRA) genes (10%–14% of tumors), resulting in a constitutive acti- Gastrointestinal stromal tumor (GIST) is the most common vation of the encoded RTK (1). This leads to continuous mesenchymal tumor of the digestive tract and in some regions stimulation of important downstream signaling pathways, such of Europe constitutes the largest group of sarcomas (1, 2). The as the phosphoinositide 3-kinase (PI3K)/AKT and mitogen- vast majority of GISTs express KIT (CD117), a receptor tyrosine activated protein kinase (MAPK) pathways, ultimately causing kinase (RTK; ref. 1). In about 85% to 90% of cases, GIST is uncontrolled cell proliferation and survival (1). The constitu- driven by activating mutations either in KIT (70–75% of cases) tive activation of these pathways via KIT is the basis for the treatment of GIST patients with tyrosine kinase inhibitors (TKI) targeting the mutated receptor (1). The type of KIT/PDGFRA 1 Laboratory of Experimental Oncology, Department of Oncology, KU mutation is considered to be a prognostic factor as well as to Leuven, and Department of General Medical Oncology, University Hospitals Leuven, Leuven Cancer Institute, Leuven, Belgium. 2Exelixis have a predictive value for response to treatment with a given Inc., South San Francisco, California. 3Department of Human Genetics, TKI (3, 4). KU Leuven and University Hospitals Leuven, Leuven, Belgium. Patients with advanced GIST are commonly treated with 4Department of Pathology, KU Leuven and University Hospitals Leu- ven, Leuven, Belgium. imatinib, a small-molecule TKI that competitively binds to and inhibits the activation of mutated KIT/PDGFRA (5). Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). Despite initial success, withthisdruginthemajorityof patients, with time in nearly all patients, the disease progresses Y.K. Gebreyohannes and T. Van Looy contributed equally to this article. due to emergence of resistance to imatinib, mainly caused by Current address for T. Van Looy: Clinical Pharmacology Unit, Janssen Research heterogeneous secondary mutations in the KIT gene (6, 7), and Development, Antwerp-Merksem, Belgium. which result in structural changes of the receptor that will Corresponding Author: Agnieszka Wozniak, Laboratory of Experimental Oncol- hinder the binding of the TKI (8). Second- and third-line ogy, Department of Oncology, KU Leuven, Herestraat 49 post 815, 3000 Leuven, treatment standards (sunitinib and regorafenib, respectively) Belgium. Phone: 32-16-341-669; Fax: 32-16-346-901; E-mail: are effective but the quality and duration of response tends to [email protected] be worse than with the ﬁrst-line imatinib (9, 10). GIST patients doi: 10.1158/1535-7163.MCT-16-0224 who have failed three lines are left without further approved 2016 American Association for Cancer Research. therapeutic options, while the general condition of many of

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these patients would still allow for further systemic therapy. The treatment was administered orally by gavage and lasted 15 Hence, there is a need for developing and testing novel treat- days, and tumor volumes were measured three times per week. ment strategies for these patients. The relative values to the baseline tumor volume expressed as Cabozantinib is a multitargeted TKI, with activity against percentages were used for each time point. The general wellbeing several receptor tyrosine kinases, including KIT, MET, VEGFR2 of mice and their body weight was followed up daily. On day 15, (vascular endothelial growth factor receptor 2), and AXL (11, 12). all mice were euthanized 2 hours after the last treatment and In preclinical studies, cabozantinib has been shown to have tumors were partly snap-frozen and partly fixed in formalin for potent antitumor and antiangiogenic activity in several in vivo further histological and molecular assessments. and in vitro models (13). Currently, cabozantinib is approved for Xenografting of tumor samples collected from consenting the treatment of progressive, metastatic medullary thyroid cancer patients was approved by the Medical Ethics Committee, Univer- (14). In a recent randomized phase III trial, cabozantinib resulted sity Hospitals Leuven, Belgium. In vivo experiments were in improved progression-free survival and objective response rate approved by the Ethics Committee for Laboratory Animal in patients with advanced renal cell carcinoma as compared with Research, KU Leuven and performed according to their guidelines everolimus (15). and Belgian regulations. In the current study, we investigated the in vivo efficacy of cabozantinib in GIST using patient-derived xenografts carrying Histologic assessment heterogeneous KIT mutations. Formalin-fixed tumor specimens were embedded in paraffin, and 4-mm sections were cut for hematoxylin and eosin (H&E) Materials and Methods and IHC stainings. Stained tumor fragments were used for the assessment of histologic response (HR) as previously reported Drugs and reagents (19). Mitotic and apoptotic activity was evaluated by counting Imatinib mesylate was purchased from Sequoia Research mitotic and apoptotic cells on H&E-stained slides in 10 high Products Ltd. and was dissolved in sterile water. Cabozantinib power fields (HPF) at 400-fold magnification (400). Phos- was provided by Exelixis Inc. and was prepared as a suspension pho-histone H3 (pHH3) and cleaved PARP immunostainings in sterile water. The following primary antibodies were were used to assess the proliferative and apoptotic activity by used to perform Western blotting (WB) and immunohis- counting immunopositive cells in 10 HPF at 400.TheKi67 tochemistry (IHC): phospho-KITY719 (pKITY719), phospho- labeling index was calculated as the average percentage of Ki67- KITY703 (pKITY703), phospho-AKTS473(pAKTS473), AKT, alpha- stained tumor cells in 5 pictures (400), to assess proliferation. tubulin, phospho-MAPKT202/Y204 (pMAPK), p42/44 MAPK The antiangiogenic activity was assessed by measuring the (MAPK), 4E-binding protein 1 (4EBP1), phospho-4EBP1 tumor vessel density in ex-mouse tumors using CD31 immu- (p4EBP1), phospho-histone H3 (pHH3), and cleaved PARP nostaining, defined as the number of vessels counted in 5 HPF (poly ADP ribose polymerase) from Cell Signaling Technology, at 200-fold magnification (200). KIT and DOG1 immunos- Ki67 from Thermo Scientific, KIT from Dako, DOG1 from taining was used to confirm the GIST diagnosis of the ex-mouse Novocastra, beta-actin from Sigma-Aldrich, CD31 from Dia- tumors and to follow KIT expression. For all evaluations we nova. For WB secondary antibodies, conjugated with horse used a CH30 microscope equipped with Color View digital radish peroxidase (HRP) from Santa Cruz Biotech and Western camera (Olympus), and images were analyzed using Cell D Lightning Plus-ECL (PerkinElmer) were applied for band visual- imaging software (Olympus). ization. For IHC anti-rabbit EnVisionþ System-HRP and 30diaminobenzidine-tetrahydrochloride (DAB) both from Dako Western blotting were used. For WB, tumor lysates were prepared from the snap-frozen tumor specimens as described previously (16). Levels of chemi- Generation of xenografts and study design luminescence were captured with the LAS mini 3000 system Human GIST xenografts were established by subcutaneous (Fuji). transplantation of clinical GIST specimens from consenting patients undergoing surgical interventions or biopsies to Statistical analysis female adult NMRI (nu/nu) mice (Janvier Laboratories) as Comparisons between the tumor volumes on day 1 versus day previously described (16). Three PDX models, namely UZLX- 15 and between treatment groups were done using Wilcoxon- GIST4 [passage (p) 21, KIT p.K558_G565delinsR; imatinib- matched pair (WMP) and the Mann–Whitney U (MWU) tests, sensitive model], -GIST2 (p. 17, KIT p.A502_Y503dup; dose- respectively. Statistica 12.0 (StatSoft) was used for all statistical dependent resistance to imatinib), and -GIST9 (p. 6, KIT analyses, and a P value of <0.05 was considered statistically p.P577del;W557LfsX5;D820G; imatinib- and sunitinib-resis- significant. tant) were used in the study. These models are characterized by human origin and stable morphological features and molecular characteristics similar to the original tumor as previously Results described (16–18). Tumor volume A total of 52 mice bearing tumors, engrafted on both sides, were After two weeks, untreated tumors from all models showed a grouped as follows: control (untreated; UZLX-GIST4, n ¼ 7 mice; significant increase in tumor volume from baseline (P < 0.05 for UZLX-GIST9, n ¼ 6; UZLX-GIST2, n ¼ 4); imatinib 50 mg/kg/bid all models; WMP; Fig. 1). (UZLX-GIST4, n ¼ 7; UZLX-GIST9, n ¼ 6; UZLX-GIST2, n ¼ 5) As expected, imatinib caused significant tumor regression and cabozantinib 30 mg/kg/qd (UZLX-GIST4, n ¼ 6; UZLX-GIST9, compared with baseline in UZLX-GIST4 (to 37% of the base- n ¼ 6; UZLX-GIST2, n ¼ 5). line volume; P ¼ 0.005, WMP). In the two other models,

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Imatinib Cabozantinib

B 400% Control UZLX-GIST4 Imatinib Cabozantinib 300%

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Figure 1. Assessment of the tumor volume 0% evolution as a percentage of baseline. 02468101214 200% Chemical structures of imatinib and Control cabozantinib (A). Tumor volume Imatinib UZLX-GIST2 evolution in the three xenograft Cabozantinib models (B). 150%

100%

50% Relative tumor volume (%) Relative tumor volume 0% 1 3 6 8 10 13 15 300% Control UZLX-GIST9 Imatinib Cabozantinib 200%

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0% 0 2468101214 Day of treatment despite treatment with imatinib, the tumor volume increased, resistant model, cabozantinib slowed tumor growth (141%), reaching 139% in UZLX-GIST2 and 199% in UZLX-GIST9, and the volume of the xenograft was signiﬁcantly lower than which was similar to the tumor growth seen in untreated the tumor volume of the control group (P < 0.001, MWU) and controls (Fig. 1). imatinib (P ¼ 0.01, MWU), on day 15 (Fig. 1). Of note, no In contrast, treatment with cabozantinib decreased the tumor relevant treatment-related toxicity was observed during the two volume in all three models. In the imatinib-sensitive model, weeks of treatment. cabozantinib treatment resulted in a signiﬁcant regression of the tumor volume on day 15 (to 30% of baseline; P ¼ 0.002, Histopathology WMP). In the KIT exon 9-mutant model, cabozantinib signif- Control tumors from all models showed stable morphological icantly reduced the tumor volume to 51% of baseline (P ¼ and immunohistochemical characteristics in terms of KIT and 0.005, WMP) and the effect was more pronounced than in the DOG1 immunopositivity (Supplementary Fig. S1), resembling imatinib-treated tumors (P < 0.001, MWU). In the imatinib- the features observed in the original patient sample used for

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100%

80%

60%

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20% Percentage of tumors Percentage

0% Control Imatinib Cabozantinib Control Imatinib Cabozantinib Control Imatinib Cabozantinib UZLX-GIST4 UZLX-GIST2 UZLX-GIST9

Grade 1 Grade 2 Grade 3 Grade 4

Figure 2. Assessment of histologic response (HR). HR was graded on H&E using a system originally described previously by Antonescu et al. (19).

xenografting and previous passages. Moreover, ex-mouse tumor Microvessel density (MVD) samples maintained the same KIT mutations as previously found. Assessment of MVD using CD31 staining showed imatinib In all models, the HR was mainly characterized by the induc- treatment did not have a significant impact on the vessel count tion of necrosis, as assessed on H&E. In the UZLX-GIST9 model, in any of the three models. In contrast, compared with both cabozantinib resulted in strong (grade 2 and 3) HR, observed in control and imatinib, cabozantinib significantly reduced the about 50% of tumors, while only a minimal HR was found in average number of CD31-positive vessels in all models, regardless UZLX-GIST4 and UZLX-GIST2 (Fig. 2). of their molecular background (Table 1; Fig. 3). Subsequently, we evaluated the effect of the different treatments on the proliferative and apoptotic activity of the tumors. Assessment of KIT signaling in response to the treatment Regardless of the model, control tumors showed high mitotic The WB analysis showed that KIT and its main downstream activity with the average of 36 mitotic figures per 10 HPF. Imatinib intermediate signaling proteins were expressed and activated in all treatment resulted in a significant reduction of mitotic activity three models (Fig. 4). As expected, in UZLX-GIST4, imatinib only in UZLX-GIST4 (>50-fold reduction compared with control, inhibited phosphorylation of pKITY719 and pKITY703 as well as P ¼ 0.004; MWU). In contrast, cabozantinib significantly reduced pAKTS473 compared with control. Cabozantinib inhibited the the mitotic activity in all three models compared with control (P ¼ phosphorylation of KIT at both phosphorylation sites (pKITY719 0.002 in UZLX-GIST4, P < 0.001 in UZLX-GIST2, and P ¼ 0.01 in and pKITY703), with a more pronounced inhibition of the latter. In UZLX-GIST9; MWU), with a more pronounced effect in the first the UZLX-GIST2 model, cabozantinib resulted in strong inhibi- model (Table 1). In the other two models, -GIST2 and -GIST9, tion of KIT, AKT, MAPK, and 4EBP1 phosphorylation when cabozantinib significantly reduced the mitotic activity also when compared with the untreated controls. On the other hand, no comparing with imatinib treatment. The assessment of apoptotic remarkable inhibition of KIT or its downstream intermediates was activity showed that cabozantinib significantly increased the observed in UZLX-GIST9 (Fig. 4). apoptosis only in the UZLX-GIST4 model, when compared with control (>50-fold, P ¼ 0.002; MWU) and imatinib (15-fold, P ¼ 0.004; MWU). These results were confirmed using immunohis- Discussion tochemical markers (pHH3 and Ki67) for proliferation and In spite of the tremendous advances in the treatment of met- cleaved PARP for apoptotic activity (Table 1). astatic GIST with the development of several TKI therapies, the

Table 1. Histopathologic assessment of the proliferative and apoptotic activity and effects on microvessel density in tumors treated with imatinib and cabozantinib Proliferation Apoptosis Microvessel density Model, genotype treatment H&E pHH3 Ki67 H&E Cleaved PARP CD31 UZLX-GIST4 (KIT exon 11) Imatinib ###a ###a ###a "3.6a " 1.4 "1.1 Cabozantinib ###a ###a ###a """a,c """a,c #1.8b,c UZLX-GIST2 (KIT exon 9) Imatinib #1.6b ¼#1.4 #1.6b #1.5 ¼ Cabozantinib #12.5a,c #3.7a,c #6.3a,c # 2.6b,c #2.1 #1.5a,c UZLX-GIST9 (KIT exon 11 and 17) Imatinib ¼#1.1 #1,1 ¼"1.4b #1.2 Cabozantinib #1.5b #1.2 #1.6b,c "1.3 "1.8b,c #1.9a,c NOTE: Values are presented as a fold change in comparison with the control (untreated) tumors. Abbreviation: pHH3, phospho-histone H3. aP < 0.005, compared with control (MWU). bP < 0.05 compared with control (MWU). cP < 0.05 compared with imatinib (MWU). ### >50-fold decrease; # decrease; """ >50-fold increase; " increase.

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Figure 3. Representative images of CD31 immunostaining of the different treatment groups in the three models.

problem of resistance to these agents presents a considerable with induction of necrosis which was not observed in the other clinical challenge. To date, many patients with advanced GIST models. The presence of necrosis may sometimes lead to an are left without any approved therapy options within a relatively underestimation of the antitumor effect of the treatment as the short period of time after starting systemic treatment, mainly due necrotic tissue may replace the tumor and result in a temporary to the emergence of resistance to the approved agents imatinib, increase in tumor volume instead of tumor regression due to sunitinib, and regorafenib. The development of novel therapeutic osmotic changes within the necrotic lesions (21). In refractory options that can target possible resistance mechanisms is a key disease, tumor growth delay resulting from treatment can be research priority. considered a beneficial effect and hence prolong time to pro- Our current study reveals that cabozantinib is active in PDX gression and potentially improve survival (22, 23). models of GIST carrying heterogeneous KIT mutations and dif- Cabozantinib induced apoptotic activity in UZLX-GIST4, ferent levels of sensitivity to imatinib. These models have already which was much more pronounced than the effect of imatinib. proven their utility for preclinical drug testing and supported the It has been suggested that imatinib as a cytostatic drug is not able initiation of clinical trials (16, 17). to eradicate all GIST cells and that a subset of tumor cells remain Cabozantinib is a novel compound, with an inhibitory effect viable and enter into a state of reversible cell quiescence rather on KIT as well as on other receptors such as MET, VEGFR2, and than going into apoptosis (24, 25). The remarkable proapoptotic AXL (12), some of which have been implicated in resistance to effect of cabozantinib can possibly be linked its antiangiogenic imatinib (11). Using several cellular assays and in vivo experi- effects. Using PDX models of colorectal cancer, Song and collea- ments, Yakes and colleagues have shown the kinase inhibition gues showed that cabozantinib inhibited tumor angiogenesis and antiproliferative effects of cabozantinib in several preclin- which was accompanied by increased apoptotic activity (26). ical solid tumor models (13). In GIST models, cabozantinib Interestingly, in all xenografts tested, we observed a significant demonstrated profound antitumor effects in vivo (20). In our reduction in MVD in cabozantinib treated-tumors compared with experiments, treatment with cabozantinib resulted in substan- the control and imatinib. Moreover, in previous studies published tialtumorregressionintwomodelswithprimaryKIT muta- by our group, sunitinib, another multitarget TKI with anti-angio- tions, and tumor growth delay in an imatinib-resistant model. genic properties, also resulted in a prominent increase in apo- The reduction in tumor burden was accompanied by a signif- ptotic activity in the UZLX-GIST4 model as compared with icant reduction in proliferation of the tumor cells; the mitotic imatinib (27, 28). In the UZLX-GIST2 and -GIST9 models, how- activity was completely suppressed in UZLX-GIST4 and signif- ever, the antiangiogenic effect was not accompanied by a significantly decreased in the other two models. Furthermore, cabo- icant increase in apoptotic activity. This could be due to the zantinib substantially decreased KIT and AKT activation in two presence of a high rate of spontaneous apoptosis among the out of three models. In UZLX-GIST4, the efficacy of cabozanti- untreated control mice; therefore, the treatment effect could not nib on proliferation and KIT signaling was similar to that of be observed. Moreover, in experiments performed earlier by our imatinib, suggesting that cabozantinib has a direct effect on group, treatment of UZLX-GIST9 or -GIST2 models with the GIST cells through inhibition of KIT. On the other hand, the angiogenesis inhibitor sunitinib showed an effect similar to that tumor growth delay in the UZLX-GIST9 model was associated of cabozantinib on tumor volume and MVD but did not result in

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A UZLX-GIST4 UZLX-GIST2 UZLX-GIST9 Control Imatinib Cabozantinib Control Imatinib Cabozantinib Control Imatinib Cabozantinib

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4EBP1 Figure 4. p4EBP1 KIT signaling pathway. A, Assessment of the effect of treatments in different Tubulin xenograft models. B, Densitometric assessment of phospho-protein forms in the KIT signaling pathway. B 300%

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UZLX-GIST4 UZLX-GIXT2 UZLX-GIST9

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an increase of apoptotic activity, suggesting the antiangiogenic an important advantage of this compound, as other drugs used in effects in these models may not induce apoptosis but rather this setting tend to work genotype specific. necrosis and/or inhibition of proliferation (18, 27). These obser- As expected, cabozantinib strongly inhibited the KIT signaling vations support the investigation of cabozantinib in patients with pathway in two models with primary mutations, while in the GIST and suggest a potential advantage over imatinib. double-mutant, imatinib-resistant model, the inhibition was only Cabozantinib is known to be a potent antiangiogenic com- marginal. Nevertheless, also in the latter model, cabozantinib led pound, which is likely a result of its inhibitory effect on kinases, to a delay in tumor growth and strong inhibition of proliferation, including VEGFR2 MET, and AXL (12, 13). In GIST, a high MVD is which was more pronounced than in the imatinib-treated group. correlated with tumor burden, VEGF expression, and cell prolif- We postulate that in this case cabozantinib's activity is mainly eration and was shown to be a poor prognostic factor (29). In our attributable to its effects on the tumor vasculature rather than models, cabozantinib led to a significant reduction in microvas- directly affecting the GIST cells. Since cancer cell survival and cularization, as assessed by CD31 immunostaining, and the effect proliferation requires nutrient and oxygen supply through angio- was more pronounced than that in imatinib-treated tumors. genesis, the blockade of neovascularization could lead to the V588del/ Similar results were shown previously in Kit , which is a inhibition of proliferation (30). Recently, a similar observation genetically modified GIST mouse model (20), but to our knowl- was reported by our group in PDX models of liposarcoma when edge this is the first report of cabozantinib's activity in PDX treated with the antiangiogenic multikinase inhibitor pazopanib, models of GIST established by immediate transplantation of which showed antitumor activity mainly through inhibition of tumor material. The level of antiangiogenic activity was similar angiogenesis and proliferation (31). in all three models in our study, suggesting that this effect may be As a multitargeted kinase inhibitor, cabozantinib can act independent of the tumor's molecular background, which may be through blocking several RTKs, including KIT, but also other

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Efﬁcacy of Cabozantinib in GIST Xenografts

kinases some authors postulate a potential role in GIST as shown Disclosure of Potential Conflicts of Interest in several preclinical models and patient samples (20, 32, 33). For all conflicts related to institutional activities, P. Schoffski's€ institution was One of cabozantinib's targets is MET, the tyrosine kinase receptor compensated for some of his activities but not him personally. T. Van Looy is for hepatocyte growth factor which is implicated in the resistance currently a clinical research coordinator at Janssen Pharmaceutical companies of Johnson and Johnson. D.T. Aftab is EVP, Business Operations at Exelixis and has to different TKIs or monoclonal antibodies in several preclinical fl – ownership interest (including patents) in the same. No potential con icts of models and human tumors (34 36). In GIST, Cohen and col- interest were disclosed by the other authors. leagues showed that tumor samples from imatinib-resistant tumors can have acquired MET activation. In their study, treatment with MET inhibitors led to enhanced efficacy compared with Authors' Contributions imatinib in both sensitive and resistant models (20). AXL is Conception and design: P. Schoffski,€ T. Van Looy, D.T. Aftab, R. Sciot, another target inhibited by cabozantinib, which is possibly over- A. Wozniak € expressed in imatinib-resistant tumors, taking over the activity of Development of methodology: Y.K. Gebreyohannes, P. Schoffski, T. Van Looy, J. Wellens, L. Vreys, M. Debiec-Rychter, R. Sciot, A. Wozniak KIT when inhibited by imatinib ("tyrosine kinase switch" hypoth- Acquisition of data (provided animals, acquired and managed patients, esis; refs. 37, 38). In our study, however, we did not observe MET provided facilities, etc.): Y.K. Gebreyohannes, P. Schoffski,€ T. Van Looy, or AXL expression on both transcript and protein levels in the ex- J. Wellens, L. Vreys, D.T. Aftab, M. Debiec-Rychter, A. Wozniak mouse tumors (data not shown); therefore, we could not attribute Analysis and interpretation of data (e.g., statistical analysis, biostatistics, € the antitumor effect of cabozantinib to the inhibition of these computational analysis): Y.K. Gebreyohannes, P. Schoffski, T. Van Looy, alternative RTKs in the GIST cells. We postulate that in our GIST R. Sciot, A. Wozniak Writing, review, and/or revision of the manuscript: Y.K. Gebreyohannes, PDX models, cabozantinib is acting mainly through the direct P. Schoffski,€ T. Van Looy, J. Cornillie, D.T. Aftab, M. Debiec-Rychter, R. Sciot, inhibition of KIT and its profound antiangiogenic effects on the A. Wozniak tumor vasculature. Administrative, technical, or material support (i.e., reporting or organizing In conclusion, cabozantinib demonstrated antitumor efficacy data, constructing databases): P. Schoffski,€ T. Van Looy, J. Wellens, L. Vreys, in patient-derived GIST xenograft models characterized by differ- U. Vanleeuw € ent KIT genotypes and different sensitivities to imatinib. The Study supervision: P. Schoffski, T. Van Looy, A. Wozniak Other (development of the xenograft platform that was the basis for this kinase inhibitor decreased the tumor burden and reduced tumor research): P. Schoffski€ growth mainly through a significant decrease in cell proliferation and antiangiogenic effects, and to some extent through inhibition of KIT/AKT signaling. In imatinib-resistant models, cabozantinib Grant Support was more active than the standard treatment. This observation Exelixis Inc., South San Francisco, California provided cabozantinib and warrants further clinical assessment of the compound in GIST financial support for the presented studies. P. Schoffski€ received honoraria for patients refractory to imatinib. Based on our xenograft findings, educational and advisory functions from Exelixis. The costs of publication of this article were defrayed in part by the payment of the European Organization for Research and Treatment of Cancer page charges. This article must therefore be hereby marked advertisement in (EORTC) is now activating an early phase II trial testing cabo- accordance with 18 U.S.C. Section 1734 solely to indicate this fact. zantinib in patients with metastatic GIST, who progressed during treatment with imatinib and sunitinib (EORTC 1317 "CABO- Received April 11, 2016; revised August 11, 2016; accepted August 17, 2016; GIST," NCT00216578). published OnlineFirst October 24, 2016.

References 1. Corless CL, Barnett CM, Heinrich MC. Gastrointestinal stromal tumours: Leukemia Group B and Southwest Oncology Group. J Clin Oncol origin and molecular oncology. Nat Rev Cancer 2011;11:865–78. 2008;26:5360–7. 2. Ducimetiere F, Lurkin A, Ranchere-Vince D, Decouvelaere AV, peoc'h M, 8. Nishida T, Kanda T, Nishitani A, Takahashi T, Nakajima K, Ishikawa T, et al. Istier L, et al. Incidence of sarcoma histotypes and molecular in a prospec- Secondary mutations in the kinase domain of the KIT gene are predom- tive epidemiological study with central pathology review and molecular inant in imatinib-resistant gastrointestinal stromal tumor. Cancer Sci testing. PLoS One 2011;6:1–12. 2008;99:799–804. 3. Wozniak A, Rutkowski P, Schoffski€ P, Ray-Coquard I, Hostein I, Schild- 9. Gajiwala KS, Wu JC, Christensen J, Deshmukh GD, Diehl W, DiNitto JP, haus HU, et al. Tumor genotype is an independent prognostic factor in et al. KIT kinase mutants show unique mechanisms of drug resistance to primary gastrointestinal stromal tumors of gastric origin: a European imatinib and sunitinib in gastrointestinal stromal tumor patients. Proc multicenter analysis based on ConticaGIST. Clin Cancer Res 2014; Natl Acad U S A 2009;106:1542–7. 20:6105–16. 10. Demetri GD, Reichardt P, Kang YK, Blay JY, Rutkowski P, Gelderblom H, 4. Debiec-Rychter M, Sciot R, Le Cesne A, Schlemmer M, Hohenberger P, van et al. Efﬁcacy and safety of regorafenib for advanced gastrointestinal Oosterom AT, et al. KIT mutations and dose selection for imatinib in stromal tumours after failure of imatinib and sunitinib (GRID): an inter- patients with advanced gastrointestinal stromal tumours. Eur J Cancer national, multicentre, randomised, placebo-controlled, phase 3 trial. Lan- 2006;42:1093–103. cet 2013;381:295–302. 5. Demetri GD, von Mehren M, Blanke CD, Van den Abbeele AD, Eisenberg B, 11. Grullich€ C. Cabozantinib: a MET, RET, and VEGFR2 tyrosine kinase Roberts PJ, et al. Efﬁcacy and safety of imatinib mesylate in advanced inhibitor. Recent Results Cancer Res 2014;201:207–14. gastrointestinal stromal tumors. N Engl J Med 2002;347:472–80. 12. Yakes FM, Chen J, Tan J, Yamaguchi K, Shi Y, Yu P, et al. Cabozantinib 6. Liegl B, Kepten I, Le C, Zhu M, Demetri GD, Heinrich MC, et al. Hetero- (XL184), a novel MET and VEGFR2 inhibitor, simultaneously suppresses geneity of kinase inhibitor resistance mechanisms in GIST. J Pathol metastasis, angiogenesis, and tumor growth. Mol Cancer Ther 2011;10: 2008;216:64–74. 2298–308. 7. Heinrich MC, Owzar K, Corless CL, Hollis D, Borden EC, Fletcher CD, et al. 13. Roy S, Narang BK, Rastogi SK, Rawal RK. A novel multiple tyrosine-kinase Correlation of kinase genotype and clinical outcome in the North American targeted agent to explore the future perspectives of anti-angiogenic therapy Intergroup Phase III Trial of imatinib mesylate for treatment of advanced for the treatment of multiple solid tumors: cabozantinib. Anticancer gastrointestinal stromal tumor: CALGB 150105 Study by Cancer and Agents Med Chem 2014;15:37–47.

www.aacrjournals.org Mol Cancer Ther; 15(12) December 2016 2851

Gebreyohannes et al.

14. Viola D, Cappagli V, Elisei R. Cabozantinib (XL184) for the treatment of inhibitor, in a colorectal cancer patient-derived tumor explant model. Int J locally advanced or metastatic progressive medullary thyroid cancer. Cancer 2015;136:1967–75. Future Oncol 2013;9:1083–92. 27. Floris G, Sciot R, Wozniak A, Van Looy T, Wellens J, Faa G, et al. The Novel 15. Choueiri TK, Escudier B, Powles T, Mainwaring PN, Rini BI, Donskov F, HSP90 inhibitor, IPI-493, is highly effective in human gastrostrointestinal et al. Cabozantinib versus everolimus in advanced renal-cell carcinoma. N stromal tumor xenografts carrying heterogeneous KIT mutations. Clin Engl J Med 2015;373:1814–23. Cancer Res 2011;17:5604–14. 16. Floris G, Wozniak A, Sciot R, Li H, Friedman L, Van Looy T, et al. A potent 28. Van Looy T, Wozniak A, Floris G, Li H, Wellens J, Vanleeuw U, et al. combination of the novel PI3K inhibitor, GDC-0941, with imatinib in Therapeutic efficacy assessment of CK6, a monoclonal KIT antibody, in a gastrointestinal stromal tumor xenografts: long-lasting responses after panel of gastrointestinal stromal tumor xenograft models. Transl Oncol treatment withdrawal. Clin Cancer Res 2013;19:620–30. 2015;8:112–8. 17. Van Looy T, Wozniak A, Floris G, Sciot R, Li H, Wellens J, et al. Phosphoi- 29. Imamura M, Yamamoto H, Nakamura N, Oda Y, Yao T, Kakeji Y, et al. nositide 3-kinase inhibitors combined with imatinib in patient-derived Prognostic significance of angiogenesis in gastrointestinal stromal tumor. xenograft models of gastrointestinal stromal tumors: rationale and efficacy. Mod Pathol 2007;20:529–37. Clin Cancer Res 2014;20:6071–82. 30. Leite de Oliveira R, Hamm A, Mazzone M. Growing tumor vessels: more 18. Van Looy T, Gebreyohannes YK, Wozniak A, Cornillie J, Wellens J, Li H, than one way to skin a cat – implications for angiogenesis targeted cancer et al. Characterization and assessment of the sensitivity and resistance of a therapies. Mol Aspects Med 2011;32:71–87. newly established human gastrointestinal stromal tumour xenograft mod- 31. Li H, Wozniak A, Sciot R, Cornillie J, Wellens J, Van Looy T, et al. et al. el to treatment with tyrosine kinase inhibitors. Clin Sarcoma Res Pazopanib, a receptor tyrosine kinase inhibitor, suppresses tumor growth 2014a;4:10. through angiogenesis in dedifferentiated liposarcoma xenograft models. 19. Antonescu CR, Besmer P, Guo T, Arkun K, Hom G, Koryotowski B, Transl Oncol 2014;7:665–71. et al. Acquired resistance to imatinib in gastrointestinal stromal 32. Navis AC, Bourgonje A, Wesseling P, Wright A, Hendriks W, Verrijp K, et al. tumor occurs through secondary gene mutation. Clin Cancer Res Effects of dual targeting of tumor cells and stroma in human glioblastoma 2005;11:4182–90. xenografts with a tyrosine kinase inhibitor against c-MET and VEGFR. PLoS 20. Cohen NA, Zeng S, Seifert AM, Kim TS, Sorenson EC, Greer JB, et al. One 2013;8:e58262. Pharmacological inhibition of KIT activates MET signaling in gastrointes- 33. Sameni M, Tovar EA, Essenburg CJ, Chalasani A, Linklater ES, Borgman A, tinal stromal tumors. Cancer Res 2015;75:2061–70. et al. Cabozantinib (XL184) inhibits growth and invasion of preclinical 21. Hong X, Choi H, Loyer EM, Benjamin RS, Trent JC, Charnsangavej C. TNBC models. Clin Cancer Res 2016;22:923–34. Gastrointestinal stromal tumor: role of CT in diagnosis and in response 34. Bardelli A, Corso S, Bertotti A, Hobor S, Valtorta E, Siravegna G, et al. evaluation and surveillance after treatment with imatinib. Radiographics Amplification of the MET receptor drives resistance to anti-EGFR therapies 2006;26:481–95 in colorectal cancer. Cancer Discov 2013;3:658–73. 22. Casali PG, Fumagalli E, Gronchi A. Adjuvant therapy of gastrointestinal 35. Engelman JA, Zejnullahu K, Mitsudomi T, Song Y, Hyland C, Park JO, et al. stromal tumors (GIST). Curr Treat Options Oncol 2012;13:277–84. MET amplification leads to gefitinib resistance in lung cancer by activating 23. Casali PG. Successes and limitations of targeted cancer therapy in gastro- ERBB3 signaling. Science 2007;316:1039–43. intestinal stromal tumors. Prog Tumor Res 2014;41:51–61. 36. Shattuck DL, Miller JK, Carraway KL3rd, Sweeney C. Met receptor con- 24. Liu Y, Perdreau SA, Chatterjee P, Wang L, Kuan SF, Duensing A. Imatinib tributes to trastuzumab resistance of Her2-overexpressing breast cancer mesylate induces quiescence in gastrointestinal stromal tumor cells cells. Cancer Res 2008;68:1471–7. through the CDH1-SKP2-p27Kip1 signaling axis. Cancer Res 2008;68: 37. Mahadevan D, Cooke L, Riley C, Swart R, Simons B, Della Croce K, et al. A 9015–23. novel tyrosine kinase switch is a mechanism of imatinib resistance in 25. DeCaprio JA, Duensing A. The DREAM complex in antitumor activity of gastrointestinal stromal tumors. Oncogene 2007;26:3909–19. imatinib mesylate in gastrointestinal stromal tumors. Curr Opin Oncol 38. Mahadevan D, Theiss N, Morales C, Stejskal AE, Cooke LS, Zhu M, et al. 2014;26:415–21. Novel receptor tyrosine kinase targeted combination therapies for 26. Song EK, Tai WM, Messersmith WA, Bagby S, Purkey A, Quackenbush KS, imatinib-resistant gastrointestinal stromal tumors (GIST). Oncotarget et al. Potent antitumor activity of cabozantinib, a c-MET and VEGFR2 2015;6:1954–66.

2852 Mol Cancer Ther; 15(12) December 2016 Molecular Cancer Therapeutics

Cabozantinib Is Active against Human Gastrointestinal Stromal Tumor Xenografts Carrying Different KIT Mutations

Yemarshet K. Gebreyohannes, Patrick Schöffski, Thomas Van Looy, et al.

Mol Cancer Ther 2016;15:2845-2852. Published OnlineFirst October 24, 2016.

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