Entrectinib, a Pan–TRK, ROS1, and ALK Inhibitor with Activity in Multiple

Published OnlineFirst March 3, 2016; DOI: 10.1158/1535-7163.MCT-15-0758

Small Molecule Therapeutics Molecular Cancer Therapeutics Entrectinib, a Pan–TRK, ROS1, and ALK Inhibitor with Activity in Multiple Molecularly Deﬁned Cancer Indications Elena Ardini1, Maria Menichincheri1, Patrizia Banﬁ1, Roberta Bosotti1, Cristina De Ponti1, Romana Pulci2, Dario Ballinari1, Marina Ciomei1, Gemma Texido1, Anna Degrassi1, Nilla Avanzi1, Nadia Amboldi1, Maria Beatrice Saccardo1, Daniele Casero1, Paolo Orsini1, Tiziano Bandiera1, Luca Mologni3, David Anderson4,GeWei4, Jason Harris4, Jean-Michel Vernier4,GangLi4, Eduard Felder1, Daniele Donati1, Antonella Isacchi1, Enrico Pesenti2, Paola Magnaghi1, and Arturo Galvani1

Abstract

Activated ALK and ROS1 tyrosine kinases, resulting from against lines that are dependent on the drug's pharmacologic chromosomal rearrangements, occur in a subset of non–small targets. Oral administration of entrectinib to tumor-bearing cell lung cancers (NSCLC) as well as other tumor types and their mice induced regression in relevant human xenograft tumors, oncogenic relevance as actionable targets has been demonstrat- including the TRKA-dependent colorectal carcinoma KM12, ed by the efficacy of selective kinase inhibitors such as crizo- ROS1-driven tumors, and several ALK-dependent models of tinib, ceritinib, and alectinib. More recently, low-frequency different tissue origins, including a model of brain-localized rearrangements of TRK kinases have been described in NSCLC, lung cancer metastasis. Entrectinib is currently showing great colorectal carcinoma, glioblastoma, and Spitzoid melanoma. promise in phase I/II clinical trials, including the first docu- Entrectinib, whose discovery and preclinical characterization mented objective responses to a TRK inhibitor in colorectal are reported herein, is a novel, potent inhibitor of ALK, ROS1, carcinoma and in NSCLC. The drug is, thus, potentially suited and, importantly, of TRK family kinases, which shows promise to the therapy of several molecularly defined cancer settings, for therapy of tumors bearing oncogenic forms of these pro- especially that of TRK-dependent tumors, for which no teins. Proliferation profiling against over 200 human tumor cell approved drugs are currently available. Mol Cancer Ther; 15(4); lines revealed that entrectinib is exquisitely potent in vitro 628–39. 2016 AACR.

Introduction niche indication of anaplastic large cell lymphoma (ALCL) had long been established (3) but which had been largely overlooked In 2007, two independent studies reported that the EML4 and as a drug target, suddenly stirred strong interest within the ALK genes were rearranged in a small subset of lung cancers to pharmaceutical industry, spurring the clinical development of produce the EML4–ALK fusion protein, bearing an activated ALK several ALK kinase inhibitors. kinase domain (1,2). Even though the occurrence of this event was A similar story is also unfolding for TRKA, the high-affinity restricted to 3% to 7% of non–small cell lung cancer (NSCLC) receptor for nerve growth factor (NGF) that, together with the patients, this finding was highly significant because it represented highly related TRKB and TRKC, constitutes the TRK subfamily of the first description of a recurring gene translocation leading to the receptor tyrosine kinases (RTK). Whereas the physiologic role of generation of an oncogenic kinase fusion protein in a major TRKA is relatively well elucidated (4), its involvement in neo- epithelial malignancy. Thus ALK, whose oncogenic role in the plastic transformation and tumor progression was until very recently limited to identification of rearrangements in papillary thyroid carcinoma (PTC), a tumor type characterized by a rela- 1Nerviano Medical Sciences srl, Nerviano, Milan, Italy. 2Accelera srl, tively good prognosis and reports of activation due to presence of Nerviano, Milan, Italy. 3Department of Health Sciences, University of Milano-Bicocca, Monza, Italy. 4Ignyta, Inc., San Diego, California. putative autocrine loops in neuroblastoma, prostate, pancreatic, and breast cancer (5–10). However, chromosomal rearrange- Note: Supplementary data for this article are available at Molecular Cancer ments involving the NTRK1 gene, resulting in the expression Therapeutics Online (http://mct.aacrjournals.org/). of different TRKA fusion proteins were recently reported by Current address for T. Bandiera: Italian Institute of Technology, Via Morego 30, Vaishnavi and colleagues (11) in NSCLC, along with robust 16163 Genova, Italy. preclinical demonstration of the oncogenic potential of the pre- Corresponding Author: Elena Ardini, Nerviano Medical Sciences, Viale L. Pas- dicted chimeric proteins. In addition, we very recently reported teur 10, 20014, Nerviano, Milan, Italy. Phone: 39-03-3158-1430; Fax: 39-03-3158- the presence of the recurring rearrangement TPM3-NTRK1 in 1374; E-mail: [email protected] colorectal carcinoma patients, together with preclinical validation doi: 10.1158/1535-7163.MCT-15-0758 data supporting the role of TPM3–TRKA fusion protein as a driver 2016 American Association for Cancer Research. for proliferation and survival of TRKA-positive tumors (12).

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Preclinical Characterization of Entrectinib

Although an accurate estimation of the frequency of translocation Here, we disclose the structure and preclinical activity profile of events involving TRKA in either NSCLC or colorectal carcinoma is entrectinib (RXDX-101, also formerly known as NMS-E628, not yet available due to the low number of cases reported to date, NMS-01191372), a potent, orally available inhibitor of the TRK these findings pave the way for targeted therapy with selective kinases invented and initially developed at NMS, and report TRKA inhibitors in these patients (13). Moreover, in a rapidly the characterization of its biologic activity in TRKA-driven evolving landscape, large-scale sequencing efforts have led tumor models. The studies described herein provide the scientific to identification of additional chromosomal rearrangements rationale supporting the enrollment of patients harboring involving NTRK1 as well as NTRK2 and NTRK3, the genes which TRKA-dependent tumors in clinical trials of entrectinib, which respectively encode the TRKA, TRKB, and TRKC proteins, in are currently ongoing (EudraCT Number:2012-000148-88, further tumor types (14–17). These findings provide a strong NCT02097810; refs. 29–31). Being also a potent ROS1 and ALK rationale for the development of TRK kinase inhibitors in several inhibitor, entrectinib additionally possesses excellent in vitro and distinct selected patient populations. in vivo antitumor activity in models derived from different tumor An additional RTK recently identified to be involved in recur- types that are variously dependent upon ROS1 or ALK fusion ring gene rearrangements in NSCLC is ROS1, an orphan receptor proteins, supporting the rationale for exploring its clinical activity whose physiologic functions are still poorly understood (18). in ROS1- and ALK-dependent tumor settings. ROS1-positive patients, representing approximately 1% of NSCLC cases, tend to possess typical clinicopathologic features Materials and Methods similar to those described for ALK-positive NSCLC patients Compound (18,19). Entrectinib was synthesized at Nerviano Medical Sciences srl as The identification of recurring chromosomal rearrangements previously reported (WO2009/013126). Crizotinib was pur- that result in the constitutive activation of ALK, TRKA, and ROS1 chased from Selleck Chemicals. kinases in different clinical indications, together with the preclinical validation of the driving role of these oncogenes in the Kinase biochemical profiling tumors, have led to the clinical exploration of inhibitors of these Evaluation of the selectivity of inhibitors (IC s) against a panel kinases as novel therapeutic opportunities. The clinical proof of 50 of kinases was performed using a radiometric assay format as concept that the EML4–ALK fusion protein found in NSCLC is previously described (32). All assays were performed in house, indeed a driver oncogene in this disease was achieved in a with the exception of TRKB and TRKC, for which IC swere very short time after the initial description of this aberration. A 50 extrapolated from percentage inhibition values at 10 and 100 phase I/II study of crizotinib in selected ALK-positive NSCLC nmol/L of inhibitor in duplicate obtained using the SelectScreen patients demonstrated striking clinical efficacy of the drug in this Kinase ProfilingServicesfromLifeTechnologiesusing the equation: subset, with a 60% objective response rate, resulting in accelerated IC ¼ ([I] 100/% inhibition) [I], with the assumption approval of crizotinib in 2011 by the FDA for the treatment of 50 extrap that Hill Slope ¼ 1. The IC value reported in Table 1 is the average ALK-positive NSCLC patients (20). However, the observation that 50 of the IC s calculated at each concentration. about one third of ALK-positive NSCLC patients progresses after 50 initial clinical response to the drug due to the acquisition of Cell culture and cell proliferation analysis secondary mutations (21–23), together with the apparently poor Human cancer cell lines were obtained from the ATCC, ECACC, ability of crizotinib to reach effective concentrations beyond the Interlab Cell Line Collection (ICLC) and from NCI tumor cell line blood–brain barrier (BBB; ref. 24), have prompted the clinical repository (see Supplementary Table S1). Cells were maintained development of several "second-generation" ALK inhibitors, such in the media recommended by the suppliers, in a humidified 37C as ceritinib (25) and alectinib (26). incubator with 5% CO . The identity of all cell lines used in this Interestingly, some of the ALK inhibitors mentioned above, 2 study was verified using DNA fingerprinting technology most notably crizotinib itself, are also active against ROS1 (27). (AmpFlSTR Identifiler Plus PCR Amplification kit, Applied Bio- On the basis of this cross-reactivity and supported by the pre- systems; ref. 33). Oncogene-driven Ba/F3 cells were generated as clinical activity observed in ROS1-driven tumor models, a total of detailed in Supplementary Data. The antiproliferative activity of 50 ROS1-positive NSCLC patients were enrolled in an expansion test compounds and the effect of entrectinib on cell cycle were cohort of the phase I study of crizotinib. The results of this study evaluated as previously described (12). showed marked antitumor activity in this patient population and prompted the FDA to assign breakthrough therapy designation fi for crizotinib to support its intensive clinical development in Table 1. Enzymatic pro le of entrectinib against a panel of selected kinases ROS1-positive NSCLC (19,28). IC50 (nmol/L) TRKA 1 IGF1R 122 Although it has long been known that TRKA is of oncogenic TRKB 3 FAK 140 relevance in PTC (5), there are as of today very few selective TRKC 5 FLT3 164 inhibitors of this kinase in clinical development. The recent ROS1 7 BRK 195 description of recurring rearrangements of the NTRK1 gene, which ALK 12 IR 209 encodes this kinase, in subsets of major indications such as JAK2 40 AUR2 215 NSCLC, colorectal carcinoma together with the identification of ACK1 70 JAK3 349 JAK1 112 RET 393 NTRK2 and NTRK3 rearrangements in glial tumors and inflam- NOTE: IC 1 mol/L: FGFR1, VEGFR2, VEGFR3, LCK, KIT, AUR1, ABL, PKCß, matory breast cancer, respectively, and perhaps additional tumor 50 > m CDK2/CycA, SYK. types (11,12,14,16,17), has highlighted the relevance of these IC50 > 10 mmol/L: AKT1, CDC7, CHK1, CK2, eEF2K, EGFR1, ERK2, GSK3ß, IKK2, kinases as pharmacologic targets and the clinical need for effective MAPKAPK2, MELK, MET, MPS1, MST4, NEK6, NIM, p38, PAK4, PDGFRß, PDK1, inhibitors. PERK, PIM1, PKAa, PLK1, SULU1, ZAP70.

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Western blot analysis of 1 mL every minute. Hole was closed using bone wax and the Cellular mechanism of action of entrectinib was investigated wound with sterile autoclips. After the end of surgery, mice were as described (12). The following antibodies were used for monitored for recovery until complete wakening. Two weeks after Western blot analysis: Phospho-TRKA-Tyr490 (#625725) and cell implantation, all mice were examined by MRI as described in TRKA (#PC31) from Calbiochem, ALK (#35-4300) from Invi- Supplementary Data and randomized into four groups (n ¼ 6 trogen, Phospho-ALK-Tyr1604 (#3341), Phospho-ROS1- controls; n ¼ 7 Entrectinib 120 mg/kg; n ¼ 7 Entrectinib 60 mg/kg; Tyr2274 (#3078), ROS1 (#3266), Phospho-PLCg1-Tyr783 n ¼ 7 Crizotinib 100 mg/kg). At the end of treatment (day 11) all (#2821), PLCg1 (#2822), Phospho-STAT3-Tyr705 (#9131), mice were reexamined by MRI. Phospho-AKT-Ser473 (#9271), AKT (#9272), Phospho- MAPK-Thr202/Tyr204 (#9101), MAPK (#9102), caspase-3 Results (#9662), cleaved caspase-3 (Asp175; #9661), and PARP (#9542) from Cell Signaling Technology, STAT3 (#610189), Entrectinib is a potent inhibitor of TRK, ROS1, and ALK kinase p21 (#556431) and p27 (#610242) from BD Biosciences, actin activities (#A4700) from SIGMA, GAPDH (#sc-25778) from Santa Cruz A high-throughput screening (HTS) campaign of the Nerviano Biotechnology. Medical Sciences (NMS) collection of kinase-targeted chemical libraries performed against a series of potentially cancer-related Efficacy studies and ex vivo target modulation in xenograft tyrosine kinases, including ALK and TRKA, resulted in the iden- models tification of several series of hit compounds belonging to different All procedures adopted for housing and handling of animals chemical classes with intriguing activity on both ALK and TRKA were in strict compliance with Italian and European guidelines for tyrosine kinases. Among these, subsequent medicinal chemistry Laboratory Animal Welfare. For epithelial models, a total of 107 efforts within the optimization program of a 3-amino-5-substi- NCI-H2228 NSCLC cells or 5 106 KM-12 colorectal carcinoma tuted-indazole chemical class led to the identification of entrecti- cells were transplanted subcutaneously in athymic nu/nu mice nib (Fig. 1A), which was found to be a highly potent inhibitor of (Harlan). For the ALCL model, a total of 107 Karpas-299 cells were all three members of the TRK subfamily of tyrosine kinases TRKA, transplanted subcutaneously in SCID mice (Harlan) previously TRKB, and TRKC and of ALK, as well as of ROS1 kinases and pre-treated with total body irradiation of 2 Gy. For the ROS1 which was endowed with favorable administration/metabolism model, 5 105 Ba/F3-TEL-ROS1 cells were transplanted subcu- (ADME) and toxicologic properties. taneously in SCID mice (Harlan). Mice bearing a minimal tumor In biochemical assay, entrectinib inhibits the kinase activity 3 mass (150–250 mm ) were randomized into vehicle and treated of TRK family members with IC50 values of 1, 3, and 5 nmol/L groups made of 6 to 7 animals each. Oral treatments started the for TRKA, TRKB, and TRKC, and of ALK and ROS1 with IC50 day after randomization, with different doses as described. Tumor values of 12 and 7 nmol/L, respectively. Steady-state kinetic dimensions were measured regularly using Vernier calipers and experiments performed on the ALK kinase enzyme demonstrat- tumor volume was calculated according to the following formula: ed that entrectinib is a competitive inhibitor with respect to length (mm) width2 (mm)/2. The percentage of tumor inhi- ATP and noncompetitive with respect to peptide substrate, bition (%TI) was calculated as follows: %TI ¼ 100 (mean tumor thus behaving as a pure ATP competitor (Fig. 1B). When volume of treated group/mean tumor volume of control group) profiled against the Kinase Selectivity Screen (KSS), a panel of 100. kinases representative of the human kinome that is used Analysis of statistical significance was performed using the internally within NMS to profile inhibitor selectivity (34), Student t test (P values of less than 0.05 were considered to be entrectinib showed limited kinase cross-reactivity with other statistically significant). tested kinases (Table 1). To evaluate the cellular potency and Toxicity was evaluated on the basis of body weight reduction. At selectivity of entrectinib, the in vitro antiproliferative activity of the end of the experiment, mice were sacrificed and gross autopsy the compound was profiled against a collection of more than findings were reported. 200 human tumor cell lines following 72 hours of continuous Generation of the NPM-ALK transgenic mouse model is exposure (Fig. 1C and Supplementary Table S1). Entrectinib described in Supplementary Data. was found to be exquisitely active in inhibiting the prolifera- For ex vivo target modulation analysis xenograft tumor samples tion of a limited number of cell lines: although mean IC50 were snap frozen in liquid nitrogen immediately after excision of the compound across the entire panel was approximately and stored at 80 C until analyzed. The frozen samples were 2 mmol/L, only seven cell lines were inhibited with IC50slower homogenized using an Ultra Turrax T25 potter (Janke & Kunkel) than 100 nmol/L. These were: the TRKA-driven colorectal at a 5:1 ratio (v/w) in a lysis buffer containing 100 mmol/L Tris- carcinoma cell line KM12 with an IC50 of 17 nmol/L, the HCl pH 7.4, 2% SDS, 1 mmol/L EDTA, 1 mmol/L sodium ALK-dependent ALCL cell lines SU-DHL-1, Karpas-299, SUP- orthovanadate, 1 mmol/L DTT and immediately boiled. Tumor M2 and SR-786 which exhibited IC50s of 20, 31, 41, and 81 lysates were briefly sonicated, clarified by centrifugation, assayed nmol/L respectively, the ALK-dependent NSCLC cell line NCI- for protein content and used for Western blot analysis. H2228withanIC50 of 68 nmol/L and the FLT3-dependent AML cell line MV-4-11, with an IC50 of 81 nmol/L. Efficacy study in an intracranial growth model Because we also wished to explore the biologic activity of Twenty Balb/c male nude mice, ages 6 to 8 weeks, were used for entrectinib in cellular settings dependent upon TRKB, TRKC, or the experiment. For intracranial injection, mice were anesthetized ROS1 kinases, we used engineered variants of the murine pro-B and stereotactically implanted in the caudate nuclei region with 2 cell line Ba/F3, which is normally dependent on interleukin-3 105 NCI-H2228 cells in 2 mL. With a microdrill, a small hole was (IL3) for growth, but can be transformed to growth factor done at the chosen coordinates and cells were injected at the speed independence by a wide range of oncogenic kinases, and

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Figure 1. Biochemical and cellular profile of entrectinib. A, chemical structure of the indazole entrectinib. B, the reciprocal plot of ALK kinase enzymatic activity at different substrate and ATP concentrations in presence of 0 to 180 nmol/L entrectinib. The reciprocal plot 1/V0 versus 1/ATP intersects on the 1/V0 axis whereas 1/V0 versus 1/substrate intersects on the 1/substrate axis. This behavior demonstrated that entrectinib behaves as a competitive inhibitor with respect to ATP and noncompetitive with respect to subtrate. C, antiproliferative activity of entrectinib against a panel of more than 200 tumor cell lines, expressed as 1/IC50 in micromoles/liter (mmol/L). therefore represents an excellent model system for the cellular somal rearrangement that leads to the expression of oncogenic assessment of kinase inhibitor potency and selectivity (35). We fusion protein TPM3–TRKA (12) was selected as an in vitro and in generated diverse Ba/F3 cell lines exogenously expressing the vivo model for characterization of entrectinib preclinical activity in ALK, TRKA, TRKB, TRKC, or ROS1 intracellular domains as a TRKA-driven setting. To confirm that the high antiproliferative ETV6/TEL or EML4 fusions. Entrectinib potently blocked pro- activity observed for entrectinib in KM12 (IC50, 17 nmol/L) is liferation of Ba/F3-TEL-TRKB (IC50: 2.9 nmol/L), Ba/F3-TEL- related to its ability to inhibit signaling activity of TRKA, we TRKC (IC50: 3.3 nmol/L), and Ba/F3-TEL-ROS1 (IC50:5.3 treated cells with a range of drug concentrations and performed nmol/L) cells, with a high degree of selectivity versus parental Western Blot analysis to assess levels of kinase domain autopho- Ba/F3 cells or those transformed by nontargeted kinases such sphorylation and activation status of TRKA downstream signaling as ABL and RET, which were inhibited with IC50s in the range of components. Entrectinib abolishes autophosphorylation of 2to3mmol/L. As expected from the high antiproliferative TPM3–TRKA after 2 hours treatment, concomitant with complete activity of the compound previously observed against human inhibition of the phosphorylation of PLCg1, AKT, and MAPK tumor cell lines dependent upon TRKA (KM12 cells) or ALK (Fig. 2A). Evaluation of the cell-cycle effects of entrectinib in this (Karpas-299, SU-DHL-1, SUP-M2, SR-786, and NCI-H2228 tumor cell line showed that a dose as low as 10 nmol/L is able to cells), both TEL–TRKA- and EML4–ALK-transformed Ba/F3 induce accumulation of cells in the G1 phase of the cell cycle at 24 cells (IC50, 2.8 and 28 nmol/L respectively) were also selectively hours treatment, followed by apoptosis induction at 48 hours, as sensitive to entrectinib compared with controls. assessed by subG1 DNA content and PARP cleavage (Fig. 2B and C). Efficacy of entrectinib against a TRKA-driven human colorectal The preliminary pharmacokinetic characteristics of entrectinib carcinoma tumor in the mouse (Supplementary Table S2) suggested that the drug is The colorectal carcinoma cell line KM12, which is strictly highly suited to oral administration and that by this route sus- dependent on activated TRKA due to the presence of a chromo- tained plasma levels at concentrations compatible with predicted

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Preclinical Characterization of Entrectinib

Table 2. Antiproliferative activity of entrectinib and crizotinib on Ba/F3 parental cells or Ba/F3 cells stably transfected with TEL-ROS1

IC50 (mmol/L); 72 h Ba/F3 Ba/F3-TEL-ROS1 þIL3 þIL3 Entrectinib 2.08 1.05 0.005 Crizotinib 2.10 1.45 0.180

Additional characterization of the pharmacologic activity of entrectinib against TRKA was performed in a TRKA-driven Ba/F3 model. Consistent with that observed for KM12 cells, entrectinib inhibits proliferation and completely suppresses TRKA phosphorylation in Ba/F3-TEL-TRKA cells with low nanomolar potency (Fig. 2F) whereas in vivo, the treatment of mice bearing Ba/F3-TEL- TRKA tumors with 30 mg/kg of drug per os twice daily for 10 days induced complete tumor regression (Fig. 2G). As mentioned above, entrectinib resulted to be well tolerated, with no signs of overt toxicity at any of the doses and regimens Figure 3. tested in efficacy studies. Within the context of an exploratory Activity of entrectinib in ROS1-transformed Ba/F3 cells. A, Ba/F3 cells repeated-dose toxicity study in the mouse, the compound was expressing TEL-ROS1 were treated with the indicated concentrations of administered to animals by the oral route for 14 consecutive days entrectinib or crizotinib for 2 hours. Levels of ROS1 and phospho-ROS1 were up to 240 mg/kg/d, which was found to be a NOAEL (No detected by immunoblot analysis using specific antibodies. B, mice bearing Ba/F3-TEL-ROS1 tumors were administered vehicle or entrectinib Observed Adverse Effect Level). In this study, all animals showed orally twice daily at the dose of 60 mg/kg for 10 consecutive days. Tumor good general condition at the end of the treatment period at all the volume for each group was measured. Data are expressed as mean doses tested. No changes in blood counts or in pathology of all the SD (n ¼ 7). P < 0.0001 for treated versus control group at day 28. organs examined were reported: these included liver, lungs, kidneys, and brain. Interestingly, in this study no neurologic effects or abnormal behavior were observed up to maximal tested efficacy (based on in vitro activity) can readily be achieved. dose of 240 mg/kg/d (corresponding to an entrectinib level in the Thus, nude mice bearing KM12 tumor xenografts were treated brain of 1.94 mmol/L). twice daily by oral administration (per os twice daily) of entrectinib at the doses of 15, 30, and 60 mg/kg for 21 Efficacy of entrectinib in a ROS1-driven model consecutive days. In this study, potent tumor growth inhibition To evaluate the activity of entrectinib against ROS1-driven was observed (Fig. 2D), with no body weight loss or other overt tumors, we generated IL3-independent Ba/F3 cells through the signs of toxicity. Ex vivo target modulation was evaluated after 3 expression of TEL–ROS1 fusion protein, which bears constitu- days of treatment at the dose of 15 mg/kg, with tumors excised tively active ROS1 kinase. Entrectinib potently blocks prolifera- at different time points after the last administration of entrec- tion of these Ba/F3-TEL-ROS1 cells with an IC50 of 5 nmol/L, tinib. Complete inhibition of TPM3-TRKA phosphorylation whereas crizotinib, tested in parallel, was found to be 40-fold less and of the downstream transducer PLCg1 was observed at this potent (Table 2). As expected, the inclusion of IL3 in culture dose (Fig. 2E). Interestingly, inhibition was maintained for a medium, which allows cells to bypass reliance on TEL-ROS1, very long time after administration and prompted us to explore abrogates the growth inhibitory activity of entrectinib, confirming intermittent scheduling in this model. Comparably strong that its antiproliferative activity against this line is selectively efficacy was also observed after three repeated cycles of admin- dependent on the ability of the drug to inhibit ROS1 (Table istration of the compound at the same doses with an intermit- 2). The higher potency of entrectinib against ROS1 with respect to tent 4 days on/3 days off scheduling (Supplementary Fig. S1), crizotinib was also confirmed by Western blot analysis of target which is one of the dosing schedules currently being adopted in modulation in lysates of drug-treated cells (Fig. 3A). As further the entrectinib clinical development program (29). validation, entrectinib and crizotinib were tested in parallel also

Figure 2. In vitro and in vivo activity of entrectinib in TRKA-driven models. A, KM12 cells were treated with entrectinib for 2 hours at the indicated concentrations. Levels of phosphorylated and total TPM3-TRKA, PLCg1, AKT, and MAPK were evaluated by Western Blot analysis using specific antibodies. B, cell-cycle distribution was evaluated by PI staining and cytofluorimetric analysis after treatment of KM12 cells with entrectinib for 24 or 48 hours. C, KM12 cells were treated for 24 hours with the indicated concentrations of entrectinib. Cell lysates were analyzed by Western blot analysis with specific antibodies to detect caspase-3 and PARP cleavage. D, nu/nu mice bearing established KM12 xenografts were administered entrectinib per os twice daily at the indicated doses or vehicle daily for 21 consecutive days. Tumor volume for each group was measured. Data are expressed as mean SD (n ¼ 7). P < 0.0001 for treated versus control group at day 20. See also Supplementary Fig. S1. E, KM12 tumor–bearing mice were treated with the drug per os twice a day for 3 consecutive days at 15 mg/kg. Tumors were harvested at the indicated time points after the last administration and phosphorylation of TRKA and PLCg1 in tumor lysates were evaluated by Western blot analysis. F, TRKA-dependent Ba/F3 cells were treated for 2 hours with different concentrations of entrectinib and analyzed by Western blot usingthe indicated antibodies. G, SCID mice bearing Ba/F3-TEL-TRKA tumors were orally administered vehicle or entrectinib at 30 mg/kg twice daily for 10 consecutive days. Tumor volume for each group was measured. Data are expressed as mean SD (n ¼ 7). P < 0.0001 for treated versus control group at day 19.

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Figure 4. Mechanism of action and in vivo activity of entrectinib in ALK-driven ALCL cell lines and xenograft models. Karpas-299 cells (A) and SR-786 cells (B) were treated with entrectinib at different concentrations for 2 hours. Levels of proteins and phospho-proteins were detected by immunoblot analysis using speciﬁc antibodies. See also Supplementary Fig. S4. Mice bearing Karpas-299 (C) or SR-786 (D) tumors were orally administered vehicle or entrectinib at the dose of 30 or 60 mg/kg twice daily for 10 consecutive days. Tumor volume for each group was measured. Data are expressed as mean SD (n ¼ 7). P < 0.0001 for treated versus control group at day 24 (C) or at day 27 (D). E, mice bearing Karpas-299 tumors were orally treated with single administration of entrectinib at the doses of 30 or 60 mg/kg and the tumors were collected at the indicated time points after dosing. The levels of ALK, phospho-ALK, STAT3, and phospho-STAT3 were detected by immunoblot analysis using speciﬁc antibodies.

on TEL–ALK-driven Ba/F3 cells confirming the higher potency of Western Blot analysis, as well as cell-cycle analysis by flow cyto- entrectinib also on this target (Supplementary Fig. S2). The ROS1- metry (Fig. 4 and Supplementary Fig. S3). In the several cellular driven engineered model was also used for efficacy studies: in models tested, inhibition of in vitro growth correlated with mod- SCID mice bearing tumors generated by s.c. injection of Ba/F3- ulation of driver kinase activation. In Karpas-299 cells (Fig. 4A), TEL-ROS1 cells, treatment with entrectinib at 60 mg/kg per os for example, almost complete inhibition of NPM-ALK phosphor- twice daily for 10 consecutive days resulted in complete tumor ylation can be observed at 2 hours after treatment starting from the regression in all animals (Fig. 3B). dose of 10 nmol/L, with concomitant modulation of phospho- STAT3, which is an ALK transducer in ALCL (36). At the same In vitro and in vivo activity of entrectinib in ALK-dependent doses, the drug also induced a concomitant block of the cell cycle tumors in G1 phase, as judged by flow cytometric evaluation of DNA To confirm that the high antiproliferative activity of entrectinib content (Supplementary Fig. S3A). Similar results were obtained in ALK-dependent tumors is related to its ability to inhibit for the other ALK-dependent ALCL cell lines examined, SR-786 signaling activity of this kinase, we treated various ALCL cell lines (Fig. 4B and Supplementary Fig. S3B), SU-DHL-1, and SUP-M2 for 2 hours with a range of drug concentrations and performed (Supplementary Fig. S4A and S4B).

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Figure 5. In vivo activity of entrectinib in an NPM-ALK transgenic model. Magnetic resonance images of the thymic mass in a representative animal before and following 2 days of treatment with entrectinib at the dose of 60 mg/kg twice daily.

In vivo activity of entrectinib was next evaluated against Karpas- additional animals, confirming that this ALK-driven tumor is 299 xenograft tumors. Tumor-bearing animals were treated with highly sensitive to treatment with entrectinib. twice daily oral administration (per os twice daily) of entrectinib Because ALK is a clinically validated target in a subset of NSCLC at the doses of 30 and 60 mg/kg for 10 consecutive days (Fig. 4C). patients whose tumors harbor EML4–ALK, the in vitro and in vivo Both dose levels were efficacious, inducing regression of all activity of entrectinib was then evaluated in NCI-H2228, a human tumors to nonpalpable dimensions. During the observation cell line derived from a lung adenocarcinoma patient bearing this period following cessation of treatment at 60 mg/kg, tumor rearrangement (Fig. 6). As observed for the ALCL lines described eradication persisted in 4 of 7 mice at day 80 after end of above, strong inhibition of ALK phosphorylation is observed treatment, whereas tumor regrowth was observed in all animals following 2 hours of drug exposure, with concomitant inhibition treated at the dose of 30 mg/kg. Excellent efficacy was also of phosphorylation of downstream signaling components AKT observed in SR-786 ALCL tumor xenografts, where the dose of and p42/44MAPK (Fig. 6A). Analysis of cell cycle distribution in 30 mg/kg was sufficient to induce tumor eradication in 6 out of 7 cells treated with entrectinib showed an accumulation of cells in mice at the end of the observation period (Fig. 4D). G1 phase at 24 hours after treatment and increased subG1 fraction, To confirm in vivo mechanism of action of entrectinib against an indicator of apoptosis induction, at 96 hours (Fig. 6B). Con- ALK, a study was performed in which Karpas-299 tumor-bear- sistent with this observation, Western blot analysis performed in ing animals were treated with a single oral administration of parallel with flow cytometry showed increased levels of p21 and entrectinib at the doses of 30 and 60 mg/kg, followed by tumor p27 and of PARP cleavage. collection at 6, 12, 18, or 24 hours following treatment (Fig. We then tested the in vivo efficacy of entrectinib in NCI-H2228 4E). Six hours after administration of 30 mg/kg entrectinib, tumor xenografts. NCI-H2228 cells were injected s.c. in left flanks complete inhibition of ALK and STAT3 phosphorylation was of nude mice and, when tumors were established (volume ca. 200 achieved, but recovery of both signals was observed within 12 mm3), entrectinib was administered orally at the dose of 60 mg/kg hours, whereas at 60 mg/kg complete inhibition of signaling is twice daily for 10 consecutive days. Tumors began to regress maintained for at least 12 hours, consistent with the greater within a few days of compound administration, and complete degree of activity seen at this dose over extended observation regression was observed in all animals at the end of the treatment period. period. At 97 days after the end of treatment, the last observation To further characterize efficacy of entrectinib in the setting of made for the study, 3 of 7 mice remained tumor free (Fig. 6C). ALCL, we tested the compound against a transgenic mouse model Analogously, crizotinib, used as reference in the same experiment in which the NPM-ALK oncogene (a common form of oncogenic and administered for 10 consecutive days at the dose of 100 mg/ ALK in human ALCL) is expressed in T cells under the control of kg, induced complete tumor regression but tumor regrowth was the T-cell–specific Lck tyrosine kinase gene promoter. This in vivo observed in all treated mice few weeks after the end of treatment. model faithfully recapitulates the human disease, with the devel- To confirm in vivo mechanism of action of entrectinib in this opment of thymic lymphomas as well as tumor masses at the level model, biomarker analysis by Western Blot of tumor lysates was of peripheral lymph nodes appearing in all transgenic animals performed on tumors excised at different time points after a single with a latency of about 20 weeks. For the study, animals were oral administration of entrectinib. This analysis confirmed strong monitored by MRI and treated with entrectinib when the tumors dose-dependent modulation of ALK phosphorylation as well as of were well established. Figure 5 depicts the MRI scans of a repre- the downstream signaling component AKT consistent with the sentative animal bearing an extensive thymic lymphoma that efficacy observed (Fig. 6D). Additional efficacy studies were invades the thoracic cavity. Treatment with entrectinib at the dose performed in NCI-H2228 tumor-bearing animals to explore of 60 mg/kg twice daily for 2 days induced complete tumor dose/scheduling of the drug: Similarly, high efficacy was main- regression by day 2. Similar results were achieved in a series of tained at the doses of 15 and 30 mg/kg per os twice daily for 10

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consecutive days (data not shown). Interestingly, also in this Table 3. Antiproliferative activity of entrectinib on Ba/F3 cells expressing model strong efficacy was observed after repeated cycles of different EML4–ALK mutants m administration with the 4 days on/3 days off scheduling currently Cell lines IC50 ( mol/L) þ used in the clinical development of entrectinib (Supplementary Ba/F3 IL3 2.650 Ba/F3-EML4-ALK-wt - 0.028 Fig. S5). þ IL3 1.490 As mentioned above, entrectinib resulted to be well tolerated, Ba/F3-EML4-ALK-C1156Y - 0.029 with no signs of overt toxicity at any of the doses tested in þ IL3 1.070 efficacy studies. Interestingly, toxicologic studies revealed that Ba/F3-EML4-ALK-L1196M - 0.067 entrectinib efficiently crosses the BBB (i.e., with drug levels þ IL3 1.380 within the brain comparable with or exceeding plasma levels) Ba/F3-EML4-ALK-G1202R - 0.897 þ IL3 2.780 in all three preclinical species tested (data not shown). Because Ba/F3-EML4-ALK-G1269A - 0.390 metastasis to the brain represents a major problem in the þ IL3 1.150 clinical care of NSCLC and is the cause of relapse in many patients treated with the ALK inhibitor crizotinib (24), this finding prompted us to evaluate the efficacy of entrectinib in reported for ROS1-positive patients treated with crizotinib and we a murine xenograft model of brain metastasis, in which human cannot exclude, based on previous experience on ALK that addi- NCI-H2228 cells were injected intracranially in nude mice, with tional, entrectinib-sensitive ROS1 mutations will be identified. animals subsequently monitored by MRI until tumor masses could be clearly detected in the brain. Animals were then treated Discussion with entrectinib per os twice daily for 10 consecutive days. Because in mouse the drug distributes to brain at approximately In the present study, we disclose for the first time the 50% of concurrent plasma concentrations (Supplementary Table structure of entrectinib and describe the preclinical character- S2), a dosing of 120 mg/kg/d was initially selected for this study ization of this novel, orally available, small-molecule inhibitor, and crizotinib was used as comparator. Treatment with entrectinib which is currently being investigated in two clinical trials in resulted in a favorable disease control rate compared with vehicle adult patients with TRK-positive tumors (29–31). Its preclinical treated animals (Fig. 6E and Supplementary Fig. S6A and S6B), activity on ROS1- and ALK-positive tumor models additionally that translated into a significant increase in overall survival supports the enrollment of ROS1- and ALK-positive patients in (Fig. 6F). In addition a lower dose of entrectinib corresponding the clinical trial. In the initial characterization of the molecule, to 60 mg/kg was also tested with partial but still significant activity the high biochemical potency observed against TRKA, ROS1, achieved (Supplementary Fig. S6C). As expected due to its well- and ALK was reflected by remarkable activity and selectivity in known low brain penetration, crizotinib was poorly active in this cellular models. When profiled on a panel of more than 200 model (Supplementary Fig. S6D). human tumor cell lines, entrectinib selectively inhibited pro- Clinical experience with crizotinib as well as with second- liferation of tumor cells dependent upon endogenous expres- generation ALK inhibitors reveals that a frequent cause of relapse sion of these kinases with sub 100 nmol/L activity, with in patients after initial clinical efficacy is the acquired resistance negligible activity against unrelated cell lines. Subsequent stud- associated with the introduction of single point mutations that ies were conducted to confirm mechanism of action of entrec- impair the binding of the inhibitor to the active site of ALK kinase. tinib in TRK-, ROS1- and ALK-dependent models, and to Interestingly, clinical data demonstrated that patients who explore in vivo antitumor activity. relapsed due to appearance of such mutations might still benefit With regard to these distinct oncogenic events, it is fair to say from treatment with an ALK inhibitor with a different binding that until recently, TRKA was not considered by the pharmaceu- mode (22,26,37). tical industry as a high priority therapeutic target in the cancer With the aim of testing the activity of entrectinib on different field, because the best characterized oncogenic rearrangements of crizotinib or ceritinib-resistant mutated forms of ALK, Ba/F3 cells its gene, NTRK1, were primarily associated with a subset of PTC, a were made ALK-dependent upon the expression of activated tumor type that has a good prognosis, with thyroidectomy gen- EML4–ALK wt or harboring the most relevant mutations erally being a curative surgical procedure. No TRKA inhibitors described to date. Entrectinib showed good antiproliferative were explored in clinical trials with the possible exception of activity on ALK-driven Ba/F3 cells (Table 3), which, despite a lestaurtinib (CEP-701), a staurosporine-derivative multikinase slight loss of potency with respect to wt ALK for the latter one, is inhibitor that was investigated in clinical settings with reported maintained in the presence of C1156Y and L1196M mutations. TRKA overexpression, without evidence of efficacy (7,38). How- More than 10-fold lower activity was found when Ba/F3 cells ever, recent studies that have identified recurring oncogenic driven by G1269A-mutated form of ALK were tested. As expected, NTRK1 rearrangements in subsets of NSCLC and colorectal car- on the basis of predicted binding mode of entrectinib, very low cinoma patients (11,12), as well as other studies suggesting a activity was found on G1202R mutant, a feature that is in potential role of activated TRKA in other tumor types, including common with most of the second-generation ALK inhibitors such glioblastoma and Spitz melanoma (14–16,39), have created as ceritinib and alectinib. This finding is particularly relevant as much renewed interest in the identification and clinical investi- the G1202R mutation in ALK is predicted to correspond to the gation of effective TRKA inhibitors (13). In a rapidly evolving G2032R mutation in ROS1 kinase, the most important acquired scenario, these findings indicate that TRKA inhibition may rep- resistance mechanism that has been described to date in ROS1- resent an innovative opportunity for the therapy of selected positive NSCLC, and so based on these data we would not predict patients whose tumors harbor NTRK1 gene rearrangements and significant activity of entrectinib in such clinical cases. Neverthe- this has supported the enrollment of TRKA-positive patients in less, G2032 is, as of today, the only acquired resistance mutant ongoing clinical trials of entrectinib. Here, we report that

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Figure 6. Activity of entrectinib against NCI-H2228 NSCLC tumors. A, NCI-H2228 cells were treated with entrectinib at different concentrations for 2 or 96 hours. Levels of proteins and phospho-proteins were detected by immunoblot analysis using specific antibodies. B, NCI-H2228 cells were treated with 50 nmol/L entrectinib for 24 and 96 hours and cell-cycle distribution was evaluated by PI staining and cytofluorimetric analysis (top). Induction of apoptosis was confirmed by Western Blot analysis of PARP cleavage in treated cells (bottom). C, nu/nu mice bearing established NCI-H2228 xenografts were administered entrectinib per os twice daily at 60 mg/kg or crizotinib per os die at 100 mg/kg or vehicle for 10 consecutive days. Tumor volume for each group was measured. Data are expressed as mean SD (n ¼ 7). P < 0.0001 for treated versus control group at day 70. See also Supplementary Fig. S5. D, mice bearing established NCI-H2228 xenografts were administered orally a single dose of entrectinib per os and animals were sacrificed 6, 12, or 24 hours after treatment. Tumors were resected, snap frozen, and protein lysates were analyzed by Western Blot analysis with the indicated antibodies. E, mice bearing intracranial tumors were treated with vehicle or with entrectinib orally at the dose of 120 mg/kg twice daily for 10 consecutive days. Representative magnetic resonance images of vehicle or entrectinib-treated mice are shown. To gain insight into viability and perfusion of tumors, a Dynamic Contrast Enhanced (DCE)-MRI experiment was also performed by acquiring images before and after i.v. injection of a Gd- based contrast agent (see also Supplementary Figure S6). F, survival curves of vehicle- or entrectinib-treated mice used in the experiment (E) are reported (n ¼ 6).

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entrectinib is extremely effective against the human TRKA-depen- ROS1-, and ALK-dependent tumor settings. Clinical trials with dent colorectal carcinoma cell line KM12, with efficacy studies the compound are currently ongoing, with promising early signs showing that oral administration yields potent tumor growth of activity (29–31). Interestingly, the active recruitment of TRKA- inhibition at well-tolerated doses. Ex vivo target modulation positive patients already in phase I trial resulted in successful analysis showed strong correlation between in vivo antitumor treatment of a colorectal carcinoma patient and a NSCLC patient activity and target inhibition, as assessed by the phosphorylation harboring TRKA-rearranged tumors, with remarkable activity status of TRKA, as well as of the downstream signaling transducer observed after the first cycle of administration, including com- PLCg1, thus providing strong evidence of on-target mechanism of plete regression of a brain metastasis in the latter case (30,31). In action. These data support the rationale for clinical development addition, durable objective responses were observed in several of entrectinib in TRKA-dependent indications. As entrectinib was NSCLC patients whose tumors harbored ROS1 or ALK fusions and found to be also a potent inhibitor of the other members of the in ALK-driven neuroblastoma (29). These exciting preliminary TRK family, the drug is currently being explored in TRKB- and clinical data are fully coherent with the preclinical studies TRKC-dependent clinical settings. described herein, and strongly support further investigation of While being an extremely potent TRK inhibitor, we found that the drug in the patient subsets described above. entrectinib also possesses excellent biochemical activity against ROS1 kinase, which translates into potent in vitro and in vivo Disclosure of Potential Conflicts of Interest fi ef cacy in a ROS1-driven tumor model. Although detailed data D. Anderson has ownership interest (including patents) in and is a consul- on remarkable clinical benefit observed in ROS1-positive patient tant/advisory board member for Ignyta. G. Li has ownership interest (including population enrolled in an expansion cohort of the phase I clinical patents) in Ignyta. No potential conflicts of interest were disclosed by the other trial with crizotinib have been recently reported (28), we believe authors. that our report of the high in vitro and in vivo activity of entrectinib against ROS1-driven tumor models is an additional significant Authors' Contributions finding. In particular, cellular proliferation and mechanism of Conception and design: E. Ardini, M. Menichincheri, M.B. Saccardo, action data indicate that entrectinib is in the range of 10- to 100- D. Anderson, J. Harris, G. Li, A. Isacchi, P. Magnaghi, A. Galvani fold more potent against ROS1 than crizotinib, and we thus Development of methodology: C. De Ponti, D. Ballinari, G. Texido, N. Amboldi, M.B. Saccardo, P. Orsini, D. Anderson, J. Harris, E. Felder, D. Donati propose that it holds potential for exciting clinical activity in Acquisition of data (provided animals, acquired and managed patients, specific subsets of NSCLC, as well as additional settings such as provided facilities, etc.): P. Banfi, D. Ballinari, M. Ciomei, G. Texido, cholangiocarcinoma and inflammatory myofibroblastic tumors, A. Degrassi, N. Avanzi, N. Amboldi, M.B. Saccardo, D. Casero, L. Mologni, which harbor genetic aberrancies in ROS1 (40). G. Wei, G. Li Regarding the ALK-dependent tumors examined in this study, Analysis and interpretation of data (e.g., statistical analysis, biostatistics, fi the correlation between in vitro and in vivo antitumor activity and computational analysis): E. Ardini, P. Ban , D. Ballinari, M. Ciomei, A. Degrassi, N. Avanzi, N. Amboldi, D. Anderson, G. Wei, G. Li, E. Felder, target inhibition, as assessed by the phosphorylation status of D. Donati, A. Isacchi, E. Pesenti NPM-ALK in ALCL cell lines and of EML4-ALK in the NSCLC cell Writing, review, and/or revision of the manuscript: E. Ardini, R. Bosotti, line NCI-H2228, as well as detailed pharmacokinetic/pharmaco- R. Pulci, G. Texido, A. Degrassi, M.B. Saccardo, D. Anderson, G. Wei, dynamic studies provide confirmation of target-dependent activ- J.-M. Vernier, G. Li, D. Donati, A. Isacchi, A. Galvani ity of entrectinib in these models. In ALK-driven xenograft mod- Administrative, technical, or material support (i.e., reporting or organizing els, oral treatment with entrectinib at extremely well-tolerated data, constructing databases): R. Bosotti, G. Wei Study supervision: E. Ardini, A. Isacchi, E. Pesenti, P. Magnaghi, A. Galvani doses induced remarkable tumor regression with all treated Other (participated in the chemistry program that led to entrectinib): animals being tumor free by the end of a 10 days treatment T. Bandiera period and again with persistent complete tumor eradication observed in the majority of animals. Acknowledgments Because the development of brain metastases is a major cause of The authors thank the NMS Biochemical Screening and Experimental Ther- relapse after initial clinical benefit with crizotinib (24), we believe apy Groups, as well as our colleagues in various support units for their that a highly significant feature of entrectinib is its favorable BBB contribution to the experimental activities. We are indebted to Ettore Perrone for skilled synthetic chemistry support and to Valter Croci, colleague and friend, penetration in all preclinical species tested. To explore this feature for his dedicated work. Finally, the authors thank Zachary Hornby and Robert fi of entrectinib's pro le, the drug was tested against an intracranial Wild for critical reading of the article. growth model of NSCLC in the mouse, showing an excellent tumor growth control rate, and suggesting that the drug may be of Grant Support fi particular clinical bene t in patients with brain metastases. This This work was financially supported by Nerviano Medical Sciences srl. finding is particularly relevant in the perspective of potential The costs of publication of this article were defrayed in part by the payment of clinical development in indications such as TRKA-positive glio- page charges. This article must therefore be hereby marked advertisement in blastoma and in clinical settings where brain metastases are accordance with 18 U.S.C. Section 1734 solely to indicate this fact. commonly encountered, as occurs in fusion-driven NSCLC. In conclusion, the studies described herein provide a scientific Received September 11, 2015; revised January 20, 2016; accepted January 21, rationale supporting the development of entrectinib in TRK-, 2016; published OnlineFirst March 3, 2016.

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Entrectinib, a Pan−TRK, ROS1, and ALK Inhibitor with Activity in Multiple Molecularly Defined Cancer Indications

Elena Ardini, Maria Menichincheri, Patrizia Banfi, et al.

Mol Cancer Ther 2016;15:628-639. Published OnlineFirst March 3, 2016.

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