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Small Molecule Therapeutics

Entrectinib, a Pan–TRK, ROS1, and ALK Inhibitorwith Activity in Multiple Molecularly DefinedCancer IndicationsElena 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, Ge Wei4, Jason Harris4,Jean-Michel Vernier4, Gang Li4, Eduard Felder1, Daniele Donati1, Antonella Isacchi1,Enrico Pesenti2, Paola Magnaghi1, and Arturo Galvani1

Abstract

Activated ALK and ROS1 tyrosine kinases, resulting fromchromosomal rearrangements, occur in a subset of non–smallcell lung cancers (NSCLC) as well as other tumor types and theironcogenic relevance as actionable targets has been demonstrat-ed by the efficacy of selective kinase inhibitors such as crizo-tinib, ceritinib, and alectinib. More recently, low-frequencyrearrangements of TRK kinases have been described in NSCLC,colorectal carcinoma, glioblastoma, and Spitzoid melanoma.Entrectinib, whose discovery and preclinical characterizationare reported herein, is a novel, potent inhibitor of ALK, ROS1,and, importantly, of TRK family kinases, which shows promisefor therapy of tumors bearing oncogenic forms of these pro-teins. Proliferation profiling against over 200 human tumor celllines revealed that entrectinib is exquisitely potent in vitro

against lines that are dependent on the drug's pharmacologictargets. Oral administration of entrectinib to tumor-bearingmice induced regression in relevant human xenograft tumors,including the TRKA-dependent colorectal carcinoma KM12,ROS1-driven tumors, and several ALK-dependent models ofdifferent tissue origins, including a model of brain-localizedlung cancer metastasis. Entrectinib is currently showing greatpromise in phase I/II clinical trials, including the first docu-mented objective responses to a TRK inhibitor in colorectalcarcinoma and in NSCLC. The drug is, thus, potentially suitedto the therapy of several molecularly defined cancer settings,especially that of TRK-dependent tumors, for which noapproved drugs are currently available. Mol Cancer Ther; 15(4);628–39. �2016 AACR.

IntroductionIn 2007, two independent studies reported that the EML4 and

ALK genes were rearranged in a small subset of lung cancers toproduce the EML4–ALK fusion protein, bearing an activated ALKkinase domain (1,2). Even though the occurrence of this eventwasrestricted to 3% to 7% of non–small cell lung cancer (NSCLC)patients, this finding was highly significant because it representedthefirst description of a recurring gene translocation leading to thegeneration of an oncogenic kinase fusion protein in a majorepithelial malignancy. Thus ALK, whose oncogenic role in the

niche indication of anaplastic large cell lymphoma (ALCL) hadlong been established (3) but which had been largely overlookedas a drug target, suddenly stirred strong interest within thepharmaceutical industry, spurring the clinical development ofseveral ALK kinase inhibitors.

A similar story is also unfolding for TRKA, the high-affinityreceptor for nerve growth factor (NGF) that, together with thehighly related TRKB and TRKC, constitutes the TRK subfamily ofreceptor tyrosine kinases (RTK). Whereas the physiologic role ofTRKA is relatively well elucidated (4), its involvement in neo-plastic transformation and tumor progression was until veryrecently limited to identification of rearrangements in papillarythyroid carcinoma (PTC), a tumor type characterized by a rela-tively good prognosis and reports of activation due to presence ofputative autocrine loops in neuroblastoma, prostate, pancreatic,and breast cancer (5–10). However, chromosomal rearrange-ments involving the NTRK1 gene, resulting in the expressionof different TRKA fusion proteins were recently reported byVaishnavi and colleagues (11) in NSCLC, along with robustpreclinical demonstration of the oncogenic potential of the pre-dicted chimeric proteins. In addition, we very recently reportedthe presence of the recurring rearrangement TPM3-NTRK1 incolorectal carcinoma patients, togetherwith preclinical validationdata supporting the role of TPM3–TRKA fusion protein as a driverfor proliferation and survival of TRKA-positive tumors (12).

1Nerviano Medical Sciences srl, Nerviano, Milan, Italy. 2Accelera srl,Nerviano, Milan, Italy. 3Department of Health Sciences, University ofMilano-Bicocca, Monza, Italy. 4Ignyta, Inc., San Diego, California.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Current address for T. Bandiera: Italian Institute of Technology, Via Morego 30,16163 Genova, Italy.

Corresponding Author: Elena Ardini, Nerviano Medical Sciences, Viale L. Pas-teur 10, 20014, Nerviano, Milan, Italy. Phone: 39-03-3158-1430; Fax: 39-03-3158-1374; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-15-0758

�2016 American Association for Cancer Research.

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Although an accurate estimation of the frequency of translocationevents involving TRKA in either NSCLC or colorectal carcinoma isnot yet available due to the low number of cases reported to date,these findings pave the way for targeted therapy with selectiveTRKA inhibitors in these patients (13). Moreover, in a rapidlyevolving landscape, large-scale sequencing efforts have ledto identification of additional chromosomal rearrangementsinvolving NTRK1 as well as NTRK2 and NTRK3, the genes whichrespectively encode the TRKA, TRKB, and TRKC proteins, infurther tumor types (14–17). These findings provide a strongrationale for the development of TRK kinase inhibitors in severaldistinct selected patient populations.

An additional RTK recently identified to be involved in recur-ring gene rearrangements in NSCLC is ROS1, an orphan receptorwhose physiologic functions are still poorly understood (18).ROS1-positive patients, representing approximately 1% ofNSCLC cases, tend to possess typical clinicopathologic featuressimilar to those described for ALK-positive NSCLC patients(18,19).

The identification of recurring chromosomal rearrangementsthat result in the constitutive activation of ALK, TRKA, and ROS1kinases in different clinical indications, together with the preclin-ical validation of the driving role of these oncogenes in thetumors, have led to the clinical exploration of inhibitors of thesekinases as novel therapeutic opportunities. The clinical proof ofconcept that the EML4–ALK fusion protein found in NSCLC isindeed a driver oncogene in this disease was achieved in avery short time after the initial description of this aberration. Aphase I/II study of crizotinib in selected ALK-positive NSCLCpatients demonstrated striking clinical efficacy of the drug in thissubset, with a 60%objective response rate, resulting in acceleratedapproval of crizotinib in 2011 by the FDA for the treatment ofALK-positiveNSCLCpatients (20).However, the observation thatabout one third of ALK-positive NSCLC patients progresses afterinitial clinical response to the drug due to the acquisition ofsecondary mutations (21–23), together with the apparently poorability of crizotinib to reach effective concentrations beyond theblood–brain barrier (BBB; ref. 24), have prompted the clinicaldevelopment of several "second-generation" ALK inhibitors, suchas ceritinib (25) and alectinib (26).

Interestingly, some of the ALK inhibitors mentioned above,most notably crizotinib itself, are also active against ROS1 (27).On the basis of this cross-reactivity and supported by the pre-clinical activity observed in ROS1-driven tumormodels, a total of50 ROS1-positive NSCLC patients were enrolled in an expansioncohort of the phase I study of crizotinib. The results of this studyshowedmarked antitumor activity in this patient population andprompted the FDA to assign breakthrough therapy designationfor crizotinib to support its intensive clinical development inROS1-positive NSCLC (19,28).

Although it has long been known that TRKA is of oncogenicrelevance in PTC (5), there are as of today very few selectiveinhibitors of this kinase in clinical development. The recentdescriptionof recurring rearrangements of theNTRK1 gene,whichencodes this kinase, in subsets of major indications such asNSCLC, colorectal carcinoma together with the identification ofNTRK2 and NTRK3 rearrangements in glial tumors and inflam-matory breast cancer, respectively, and perhaps additional tumortypes (11,12,14,16,17), has highlighted the relevance of thesekinases as pharmacologic targets and the clinical need for effectiveinhibitors.

Here, we disclose the structure and preclinical activity profile ofentrectinib (RXDX-101, also formerly known as NMS-E628,NMS-01191372), a potent, orally available inhibitor of the TRKkinases invented and initially developed at NMS, and reportthe characterization of its biologic activity in TRKA-driventumor models. The studies described herein provide the scientificrationale supporting the enrollment of patients harboringTRKA-dependent tumors in clinical trials of entrectinib, whichare currently ongoing (EudraCT Number:2012-000148-88,NCT02097810; refs. 29–31). Being also a potent ROS1 and ALKinhibitor, entrectinib additionally possesses excellent in vitro andin vivo antitumor activity in models derived from different tumortypes that are variously dependent upon ROS1 or ALK fusionproteins, supporting the rationale for exploring its clinical activityin ROS1- and ALK-dependent tumor settings.

Materials and MethodsCompound

Entrectinib was synthesized at NervianoMedical Sciences srl aspreviously reported (WO2009/013126). Crizotinib was pur-chased from Selleck Chemicals.

Kinase biochemical profilingEvaluation of the selectivity of inhibitors (IC50s) against a panel

of kinases was performed using a radiometric assay format aspreviously described (32). All assays were performed in house,with the exception of TRKB and TRKC, for which IC50s wereextrapolated from percentage inhibition values at 10 and 100nmol/L of inhibitor in duplicate obtained using the SelectScreenKinaseProfilingServices fromLifeTechnologiesusing theequation:IC50 extrap ¼ ([I] � 100/% inhibition) � [I], with the assumptionthatHill Slope¼ 1. The IC50 value reported in Table 1 is the averageof the IC50s calculated at each concentration.

Cell culture and cell proliferation analysisHuman cancer cell lineswere obtained from theATCC, ECACC,

Interlab Cell Line Collection (ICLC) and fromNCI tumor cell linerepository (see Supplementary Table S1). Cells were maintainedin themedia recommendedby the suppliers, in a humidified37�Cincubator with 5% CO2. The identity of all cell lines used in thisstudy was verified using DNA fingerprinting technology(AmpFlSTR Identifiler Plus PCR Amplification kit, Applied Bio-systems; ref. 33). Oncogene-driven Ba/F3 cells were generated asdetailed in Supplementary Data. The antiproliferative activity oftest compounds and the effect of entrectinib on cell cycle wereevaluated as previously described (12).

Table 1. Enzymatic profile of entrectinib against a panel of selected kinases

IC50 (nmol/L)TRKA 1 IGF1R 122TRKB 3 FAK 140TRKC 5 FLT3 164ROS1 7 BRK 195ALK 12 IR 209JAK2 40 AUR2 215ACK1 70 JAK3 349JAK1 112 RET 393

NOTE: IC50 > 1 mmol/L: FGFR1, VEGFR2, VEGFR3, LCK, KIT, AUR1, ABL, PKCß,CDK2/CycA, SYK.IC50 > 10 mmol/L: AKT1, CDC7, CHK1, CK2, eEF2K, EGFR1, ERK2, GSK3ß, IKK2,MAPKAPK2, MELK, MET, MPS1, MST4, NEK6, NIM, p38, PAK4, PDGFRß, PDK1,PERK, PIM1, PKAa, PLK1, SULU1, ZAP70.

Preclinical Characterization of Entrectinib

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Western blot analysisCellular mechanism of action of entrectinib was investigated

as described (12). The following antibodies were used forWestern blot analysis: Phospho-TRKA-Tyr490 (#625725) andTRKA (#PC31) from Calbiochem, ALK (#35-4300) from Invi-trogen, Phospho-ALK-Tyr1604 (#3341), Phospho-ROS1-Tyr2274 (#3078), ROS1 (#3266), Phospho-PLCg1-Tyr783(#2821), PLCg1 (#2822), Phospho-STAT3-Tyr705 (#9131),Phospho-AKT-Ser473 (#9271), AKT (#9272), Phospho-MAPK-Thr202/Tyr204 (#9101), MAPK (#9102), caspase-3(#9662), cleaved caspase-3 (Asp175; #9661), and PARP(#9542) from Cell Signaling Technology, STAT3 (#610189),p21 (#556431) and p27 (#610242) from BD Biosciences, actin(#A4700) from SIGMA, GAPDH (#sc-25778) from Santa CruzBiotechnology.

Efficacy studies and ex vivo target modulation in xenograftmodels

All procedures adopted for housing and handling of animalswere in strict compliancewith Italian and European guidelines forLaboratory Animal Welfare. For epithelial models, a total of 107

NCI-H2228 NSCLC cells or 5 � 106 KM-12 colorectal carcinomacells were transplanted subcutaneously in athymic nu/nu mice(Harlan). For the ALCLmodel, a total of 107Karpas-299 cells weretransplanted subcutaneously in SCID mice (Harlan) previouslypre-treated with total body irradiation of 2 Gy. For the ROS1model, 5 � 105 Ba/F3-TEL-ROS1 cells were transplanted subcu-taneously in SCID mice (Harlan). Mice bearing a minimal tumormass (150–250 mm3) were randomized into vehicle and treatedgroups made of 6 to 7 animals each. Oral treatments started theday after randomization, with different doses as described. Tumordimensions were measured regularly using Vernier calipers andtumor volumewas calculated according to the following formula:length (mm) � width2 (mm)/2. The percentage of tumor inhi-bition (%TI)was calculated as follows:%TI¼100� (mean tumorvolumeof treated group/mean tumor volumeof control group)�100.

Analysis of statistical significance was performed using theStudent t test (P values of less than 0.05 were considered to bestatistically significant).

Toxicitywas evaluatedon thebasis of bodyweight reduction. Atthe end of the experiment, mice were sacrificed and gross autopsyfindings were reported.

Generation of the NPM-ALK transgenic mouse model isdescribed in Supplementary Data.

For ex vivo target modulation analysis xenograft tumor sampleswere snap frozen in liquid nitrogen immediately after excisionand stored at �80�C until analyzed. The frozen samples werehomogenized using an Ultra Turrax T25 potter (Janke & Kunkel)at a 5:1 ratio (v/w) in a lysis buffer containing 100 mmol/L Tris-HCl pH 7.4, 2% SDS, 1 mmol/L EDTA, 1 mmol/L sodiumorthovanadate, 1 mmol/L DTT and immediately boiled. Tumorlysates were briefly sonicated, clarified by centrifugation, assayedfor protein content and used for Western blot analysis.

Efficacy study in an intracranial growth modelTwenty Balb/cmale nudemice, ages 6 to 8weeks, were used for

the experiment. For intracranial injection, mice were anesthetizedand stereotactically implanted in the caudate nuclei region with 2�105NCI-H2228 cells in 2mL.With amicrodrill, a small holewasdone at the chosen coordinates and cellswere injected at the speed

of 1 mL every minute. Hole was closed using bone wax and thewound with sterile autoclips. After the end of surgery, mice weremonitored for recovery until complete wakening. Twoweeks aftercell implantation, all mice were examined by MRI as described inSupplementary Data and randomized into four groups (n ¼ 6controls; n¼7Entrectinib 120mg/kg; n¼7Entrectinib 60mg/kg;n¼ 7 Crizotinib 100 mg/kg). At the end of treatment (day 11) allmice were reexamined by MRI.

ResultsEntrectinib is a potent inhibitor of TRK, ROS1, and ALK kinaseactivities

A high-throughput screening (HTS) campaign of the NervianoMedical Sciences (NMS) collection of kinase-targeted chemicallibraries performed against a series of potentially cancer-relatedtyrosine kinases, including ALK and TRKA, resulted in the iden-tification of several series of hit compounds belonging to differentchemical classes with intriguing activity on both ALK and TRKAtyrosine kinases. Among these, subsequent medicinal chemistryefforts within the optimization program of a 3-amino-5-substi-tuted-indazole chemical class led to the identification of entrecti-nib (Fig. 1A), which was found to be a highly potent inhibitor ofall three members of the TRK subfamily of tyrosine kinases TRKA,TRKB, and TRKC and of ALK, as well as of ROS1 kinases andwhich was endowed with favorable administration/metabolism(ADME) and toxicologic properties.

In biochemical assay, entrectinib inhibits the kinase activityof TRK family members with IC50 values of 1, 3, and 5 nmol/Lfor TRKA, TRKB, and TRKC, and of ALK and ROS1 with IC50

values of 12 and 7 nmol/L, respectively. Steady-state kineticexperiments performed on the ALK kinase enzyme demonstrat-ed that entrectinib is a competitive inhibitor with respect toATP and noncompetitive with respect to peptide substrate,thus behaving as a pure ATP competitor (Fig. 1B). Whenprofiled against the Kinase Selectivity Screen (KSS), a panel ofkinases representative of the human kinome that is usedinternally within NMS to profile inhibitor selectivity (34),entrectinib showed limited kinase cross-reactivity with othertested kinases (Table 1). To evaluate the cellular potency andselectivity of entrectinib, the in vitro antiproliferative activity ofthe compound was profiled against a collection of more than200 human tumor cell lines following 72 hours of continuousexposure (Fig. 1C and Supplementary Table S1). Entrectinibwas found to be exquisitely active in inhibiting the prolifera-tion of a limited number of cell lines: although mean IC50

of the compound across the entire panel was approximately2 mmol/L, only seven cell lines were inhibited with IC50s lowerthan 100 nmol/L. These were: the TRKA-driven colorectalcarcinoma cell line KM12 with an IC50 of 17 nmol/L, theALK-dependent ALCL cell lines SU-DHL-1, Karpas-299, SUP-M2 and SR-786 which exhibited IC50s of 20, 31, 41, and 81nmol/L respectively, the ALK-dependent NSCLC cell line NCI-H2228 with an IC50 of 68 nmol/L and the FLT3-dependentAML cell line MV-4-11, with an IC50 of 81 nmol/L.

Because we also wished to explore the biologic activity ofentrectinib in cellular settings dependent upon TRKB, TRKC, orROS1 kinases, we used engineered variants of the murine pro-Bcell line Ba/F3, which is normally dependent on interleukin-3(IL3) for growth, but can be transformed to growth factorindependence by a wide range of oncogenic kinases, and

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therefore represents an excellent model system for the cellularassessment of kinase inhibitor potency and selectivity (35). Wegenerated diverse Ba/F3 cell lines exogenously expressing theALK, TRKA, TRKB, TRKC, or ROS1 intracellular domains asETV6/TEL or EML4 fusions. Entrectinib potently blocked pro-liferation of Ba/F3-TEL-TRKB (IC50: 2.9 nmol/L), Ba/F3-TEL-TRKC (IC50: 3.3 nmol/L), and Ba/F3-TEL-ROS1 (IC50: 5.3nmol/L) cells, with a high degree of selectivity versus parentalBa/F3 cells or those transformed by nontargeted kinases suchas ABL and RET, which were inhibited with IC50s in the range of2 to 3 mmol/L. As expected from the high antiproliferativeactivity of the compound previously observed against humantumor cell lines dependent upon TRKA (KM12 cells) or ALK(Karpas-299, SU-DHL-1, SUP-M2, SR-786, and NCI-H2228cells), both TEL–TRKA- and EML4–ALK-transformed Ba/F3cells (IC50, 2.8 and 28 nmol/L respectively) were also selectivelysensitive to entrectinib compared with controls.

Efficacy of entrectinib against a TRKA-driven human colorectalcarcinoma tumor

The colorectal carcinoma cell line KM12, which is strictlydependent on activated TRKA due to the presence of a chromo-

somal rearrangement that leads to the expression of oncogenicfusion protein TPM3–TRKA (12) was selected as an in vitro and invivomodel for characterization of entrectinib preclinical activity ina TRKA-driven setting. To confirm that the high antiproliferativeactivity observed for entrectinib in KM12 (IC50, 17 nmol/L) isrelated to its ability to inhibit signaling activity of TRKA, wetreated cells with a range of drug concentrations and performedWestern Blot analysis to assess levels of kinase domain autopho-sphorylation and activation status of TRKA downstream signalingcomponents. Entrectinib abolishes autophosphorylation ofTPM3–TRKA after 2 hours treatment, concomitant with completeinhibition of the phosphorylation of PLCg1, AKT, and MAPK(Fig. 2A). Evaluation of the cell-cycle effects of entrectinib in thistumor cell line showed that a dose as low as 10 nmol/L is able toinduce accumulation of cells in the G1 phase of the cell cycle at 24hours treatment, followed by apoptosis induction at 48 hours, asassessed by subG1 DNA content and PARP cleavage (Fig. 2Band C).

The preliminary pharmacokinetic characteristics of entrectinibin themouse (Supplementary Table S2) suggested that the drug ishighly suited to oral administration and that by this route sus-tained plasma levels at concentrations compatible with predicted

Figure 1.Biochemical and cellular profile of entrectinib. A, chemical structure of the indazole entrectinib. B, the reciprocal plot of ALK kinase enzymatic activity atdifferent 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 whereas1/V0 versus 1/substrate intersects on the 1/substrate axis. This behavior demonstrated that entrectinib behaves as a competitive inhibitor with respect to ATP andnoncompetitive with respect to subtrate. C, antiproliferative activity of entrectinib against a panel of more than 200 tumor cell lines, expressed as 1/IC50 inmicromoles/liter (mmol/L).






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efficacy (based on in vitro activity) can readily be achieved.Thus, nude mice bearing KM12 tumor xenografts were treatedtwice daily by oral administration (per os twice daily) ofentrectinib at the doses of 15, 30, and 60 mg/kg for 21consecutive days. In this study, potent tumor growth inhibitionwas observed (Fig. 2D), with no body weight loss or other overtsigns of toxicity. Ex vivo target modulation was evaluated after 3days of treatment at the dose of 15 mg/kg, with tumors excisedat different time points after the last administration of entrec-tinib. Complete inhibition of TPM3-TRKA phosphorylationand of the downstream transducer PLCg1 was observed at thisdose (Fig. 2E). Interestingly, inhibition was maintained for avery long time after administration and prompted us to exploreintermittent scheduling in this model. Comparably strongefficacy was also observed after three repeated cycles of admin-istration of the compound at the same doses with an intermit-tent 4 days on/3 days off scheduling (Supplementary Fig. S1),which is one of the dosing schedules currently being adopted inthe entrectinib clinical development program (29).

Additional characterization of the pharmacologic activity ofentrectinib against TRKA was performed in a TRKA-driven Ba/F3model. Consistent with that observed for KM12 cells, entrectinibinhibits proliferation and completely suppresses TRKAphosphor-ylation in Ba/F3-TEL-TRKA cells with low nanomolar potency(Fig. 2F)whereas in vivo, the treatment ofmice bearing Ba/F3-TEL-TRKA tumors with 30mg/kg of drug per os twice daily for 10 daysinduced 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 regimenstested in efficacy studies. Within the context of an exploratoryrepeated-dose toxicity study in the mouse, the compound wasadministered to animals by the oral route for 14 consecutive daysup to 240 mg/kg/d, which was found to be a NOAEL (NoObserved Adverse Effect Level). In this study, all animals showedgood general condition at the endof the treatment period at all thedoses tested.No changes in blood counts or in pathology of all theorgans examined were reported: these included liver, lungs,kidneys, and brain. Interestingly, in this study no neurologiceffects or abnormal behavior were observed up tomaximal testeddose of 240mg/kg/d (corresponding to an entrectinib level in thebrain of 1.94 mmol/L).

Efficacy of entrectinib in a ROS1-driven modelTo evaluate the activity of entrectinib against ROS1-driven

tumors, we generated IL3-independent Ba/F3 cells through theexpression of TEL–ROS1 fusion protein, which bears constitu-tively active ROS1 kinase. Entrectinib potently blocks prolifera-tion of these Ba/F3-TEL-ROS1 cells with an IC50 of 5 nmol/L,whereas crizotinib, tested in parallel, was found to be 40-fold lesspotent (Table 2). As expected, the inclusion of IL3 in culturemedium, which allows cells to bypass reliance on TEL-ROS1,abrogates the growth inhibitory activity of entrectinib, confirmingthat its antiproliferative activity against this line is selectivelydependent on the ability of the drug to inhibit ROS1 (Table2). The higher potency of entrectinib against ROS1with respect tocrizotinib was also confirmed by Western blot analysis of targetmodulation in lysates of drug-treated cells (Fig. 3A). As furthervalidation, entrectinib and crizotinib were tested in parallel also

Figure 3.Activity of entrectinib in ROS1-transformed Ba/F3 cells. A, Ba/F3 cellsexpressing TEL-ROS1 were treated with the indicated concentrations ofentrectinib or crizotinib for 2 hours. Levels of ROS1 and phospho-ROS1 weredetected by immunoblot analysis using specific antibodies. B, micebearing Ba/F3-TEL-ROS1 tumors were administered vehicle or entrectiniborally twice daily at the dose of 60 mg/kg for 10 consecutive days. Tumorvolume for each group was measured. Data are expressed as mean� SD (n ¼ 7). P < 0.0001 for treated versus control group at day 28.

Table 2. Antiproliferative activity of entrectinib and crizotinib on Ba/F3parental cells or Ba/F3 cells stably transfected with TEL-ROS1

IC50 (mmol/L); 72 hBa/F3 Ba/F3-TEL-ROS1

þIL3 þIL3 �Entrectinib 2.08 1.05 0.005Crizotinib 2.10 1.45 0.180

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 ofphosphorylated and total TPM3-TRKA, PLCg1, AKT, and MAPK were evaluated by Western Blot analysis using specific antibodies. B, cell-cycle distributionwas evaluated by PI staining and cytofluorimetric analysis after treatment of KM12 cells with entrectinib for 24 or 48 hours. C, KM12 cells were treated for24 hourswith the indicated concentrations of entrectinib. Cell lysateswere analyzed byWestern blot analysiswith specific antibodies to detect caspase-3 and PARPcleavage. D, nu/nu mice bearing established KM12 xenografts were administered entrectinib per os twice daily at the indicated doses or vehicle daily for 21consecutive 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 day20. 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. Tumorswere harvested at the indicated time points after the last administration and phosphorylation of TRKA and PLCg1 in tumor lysates were evaluated by Westernblot analysis. F, TRKA-dependent Ba/F3 cells were treated for 2 hours with different concentrations of entrectinib and analyzed by Western blot using theindicated antibodies. G, SCID mice bearing Ba/F3-TEL-TRKA tumors were orally administered vehicle or entrectinib at 30 mg/kg twice daily for 10 consecutivedays. 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.






on TEL–ALK-driven Ba/F3 cells confirming the higher potency ofentrectinib also on this target (Supplementary Fig. S2). The ROS1-driven engineered model was also used for efficacy studies: inSCID mice bearing tumors generated by s.c. injection of Ba/F3-TEL-ROS1 cells, treatment with entrectinib at 60 mg/kg per ostwice daily for 10 consecutive days resulted in complete tumorregression in all animals (Fig. 3B).

In vitro and in vivo activity of entrectinib in ALK-dependenttumors

To confirm that the high antiproliferative activity of entrectinibin ALK-dependent tumors is related to its ability to inhibitsignaling activity of this kinase, we treated various ALCL cell linesfor 2 hours with a range of drug concentrations and performed

Western Blot analysis, as well as cell-cycle analysis by flow cyto-metry (Fig. 4 and Supplementary Fig. S3). In the several cellularmodels tested, inhibition of in vitro growth correlated with mod-ulation of driver kinase activation. In Karpas-299 cells (Fig. 4A),for example, almost complete inhibition of NPM-ALK phosphor-ylation canbeobserved at 2hours after treatment starting from thedose of 10 nmol/L, with concomitant modulation of phospho-STAT3, which is an ALK transducer in ALCL (36). At the samedoses, the drug also induced a concomitant block of the cell cyclein G1 phase, as judged by flow cytometric evaluation of DNAcontent (Supplementary Fig. S3A). Similar results were obtainedfor the other ALK-dependent ALCL cell lines examined, SR-786(Fig. 4B and Supplementary Fig. S3B), SU-DHL-1, and SUP-M2(Supplementary Fig. S4A and S4B).

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 treatedwith entrectinib at different concentrations for 2 hours. Levels of proteins and phospho-proteins were detected by immunoblot analysis using specificantibodies. See also Supplementary Fig. S4. Mice bearing Karpas-299 (C) or SR-786 (D) tumorswere orally administered vehicle or entrectinib at the dose of 30 or 60mg/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 treatedversus 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 thedoses 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-STAT3were detected by immunoblot analysis using specific antibodies.

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In vivo activity of entrectinib was next evaluated against Karpas-299 xenograft tumors. Tumor-bearing animals were treated withtwice daily oral administration (per os twice daily) of entrectinibat the doses of 30 and 60mg/kg for 10 consecutive days (Fig. 4C).Both dose levels were efficacious, inducing regression of alltumors to nonpalpable dimensions. During the observationperiod following cessation of treatment at 60 mg/kg, tumoreradication persisted in 4 of 7 mice at day 80 after end oftreatment, whereas tumor regrowth was observed in all animalstreated at the dose of 30 mg/kg. Excellent efficacy was alsoobserved in SR-786 ALCL tumor xenografts, where the dose of30 mg/kg was sufficient to induce tumor eradication in 6 out of 7mice at the end of the observation period (Fig. 4D).

To confirm in vivomechanism of action of entrectinib againstALK, a study was performed in which Karpas-299 tumor-bear-ing animals were treated with a single oral administration ofentrectinib at the doses of 30 and 60 mg/kg, followed by tumorcollection at 6, 12, 18, or 24 hours following treatment (Fig.4E). Six hours after administration of 30 mg/kg entrectinib,complete inhibition of ALK and STAT3 phosphorylation wasachieved, but recovery of both signals was observed within 12hours, whereas at 60 mg/kg complete inhibition of signaling ismaintained for at least 12 hours, consistent with the greaterdegree of activity seen at this dose over extended observationperiod.

To further characterize efficacy of entrectinib in the setting ofALCL, we tested the compound against a transgenicmousemodelin which the NPM-ALK oncogene (a common form of oncogenicALK in human ALCL) is expressed in T cells under the control ofthe T-cell–specific Lck tyrosine kinase gene promoter. This in vivomodel faithfully recapitulates the human disease, with the devel-opment of thymic lymphomas aswell as tumormasses at the levelof peripheral lymph nodes appearing in all transgenic animalswith a latency of about 20 weeks. For the study, animals weremonitored by MRI and treated with entrectinib when the tumorswere well established. Figure 5 depicts the MRI scans of a repre-sentative animal bearing an extensive thymic lymphoma thatinvades the thoracic cavity. Treatment with entrectinib at the doseof 60 mg/kg twice daily for 2 days induced complete tumorregression by day 2. Similar results were achieved in a series of

additional animals, confirming that this ALK-driven tumor ishighly sensitive to treatment with entrectinib.

Because ALK is a clinically validated target in a subset of NSCLCpatients whose tumors harbor EML4–ALK, the in vitro and in vivoactivity of entrectinibwas then evaluated inNCI-H2228, a humancell line derived from a lung adenocarcinoma patient bearing thisrearrangement (Fig. 6). As observed for the ALCL lines describedabove, strong inhibition of ALK phosphorylation is observedfollowing 2 hours of drug exposure, with concomitant inhibitionof phosphorylation of downstream signaling components AKTand p42/44MAPK (Fig. 6A). Analysis of cell cycle distribution incells treated with entrectinib showed an accumulation of cells inG1 phase at 24hours after treatment and increased subG1 fraction,an indicator of apoptosis induction, at 96 hours (Fig. 6B). Con-sistent with this observation, Western blot analysis performed inparallel with flow cytometry showed increased levels of p21 andp27 and of PARP cleavage.

We then tested the in vivo efficacy of entrectinib in NCI-H2228tumor xenografts. NCI-H2228 cells were injected s.c. in left flanksof nudemice and, when tumors were established (volume ca. 200mm3), entrectinibwas administered orally at thedose of 60mg/kgtwice daily for 10 consecutive days. Tumors began to regresswithin a few days of compound administration, and completeregression was observed in all animals at the end of the treatmentperiod. At 97 days after the end of treatment, the last observationmade for the study, 3 of 7 mice remained tumor free (Fig. 6C).Analogously, crizotinib, used as reference in the same experimentand administered for 10 consecutive days at the dose of 100 mg/kg, induced complete tumor regression but tumor regrowth wasobserved in all treated mice few weeks after the end of treatment.

To confirm in vivo mechanism of action of entrectinib in thismodel, biomarker analysis by Western Blot of tumor lysates wasperformed on tumors excised at different time points after a singleoral administration of entrectinib. This analysis confirmed strongdose-dependentmodulation of ALKphosphorylation aswell as ofthe downstream signaling component AKT consistent with theefficacy observed (Fig. 6D). Additional efficacy studies wereperformed in NCI-H2228 tumor-bearing animals to exploredose/scheduling of the drug: Similarly, high efficacy was main-tained at the doses of 15 and 30 mg/kg per os twice daily for 10

Figure 5.In vivo activity of entrectinib in anNPM-ALK transgenic model. Magneticresonance images of the thymicmass ina representative animal before andfollowing 2 days of treatment withentrectinib at the dose of 60 mg/kgtwice daily.






consecutive days (data not shown). Interestingly, also in thismodel strong efficacy was observed after repeated cycles ofadministration with the 4 days on/3 days off scheduling currentlyused in the clinical development of entrectinib (SupplementaryFig. S5).

As mentioned above, entrectinib resulted to be well tolerated,with no signs of overt toxicity at any of the doses tested inefficacy studies. Interestingly, toxicologic studies revealed thatentrectinib efficiently crosses the BBB (i.e., with drug levelswithin the brain comparable with or exceeding plasma levels)in all three preclinical species tested (data not shown). Becausemetastasis to the brain represents a major problem in theclinical care of NSCLC and is the cause of relapse in manypatients treated with the ALK inhibitor crizotinib (24), thisfinding prompted us to evaluate the efficacy of entrectinib ina murine xenograft model of brain metastasis, in which humanNCI-H2228 cells were injected intracranially in nude mice, withanimals subsequently monitored by MRI until tumor massescould be clearly detected in the brain. Animals were then treatedwith entrectinib per os twice daily for 10 consecutive days.Because in mouse the drug distributes to brain at approximately50% of concurrent plasma concentrations (Supplementary TableS2), a dosing of 120 mg/kg/d was initially selected for this studyand crizotinibwasusedas comparator. Treatmentwith entrectinibresulted in a favorable disease control rate compared with vehicletreated animals (Fig. 6E and Supplementary Fig. S6A and S6B),that translated into a significant increase in overall survival(Fig. 6F). In addition a lower dose of entrectinib correspondingto 60mg/kgwas also testedwith partial but still significant activityachieved (Supplementary Fig. S6C). As expected due to its well-known low brain penetration, crizotinib was poorly active in thismodel (Supplementary Fig. S6D).

Clinical experience with crizotinib as well as with second-generation ALK inhibitors reveals that a frequent cause of relapsein patients after initial clinical efficacy is the acquired resistanceassociated with the introduction of single point mutations thatimpair the binding of the inhibitor to the active site of ALK kinase.Interestingly, clinical data demonstrated that patients whorelapsed due to appearance of such mutations might still benefitfrom treatment with an ALK inhibitor with a different bindingmode (22,26,37).

With the aim of testing the activity of entrectinib on differentcrizotinib or ceritinib-resistant mutated forms of ALK, Ba/F3 cellswere made ALK-dependent upon the expression of activatedEML4–ALK wt or harboring the most relevant mutationsdescribed to date. Entrectinib showed good antiproliferativeactivity on ALK-driven Ba/F3 cells (Table 3), which, despite aslight loss of potency with respect to wt ALK for the latter one, ismaintained in the presence of C1156Y and L1196M mutations.More than 10-fold lower activity was found when Ba/F3 cellsdriven by G1269A-mutated form of ALKwere tested. As expected,on the basis of predicted binding mode of entrectinib, very lowactivity was found on G1202R mutant, a feature that is incommonwithmost of the second-generation ALK inhibitors suchas ceritinib and alectinib. This finding is particularly relevant asthe G1202R mutation in ALK is predicted to correspond to theG2032R mutation in ROS1 kinase, the most important acquiredresistance mechanism that has been described to date in ROS1-positive NSCLC, and so based on these data wewould not predictsignificant activity of entrectinib in such clinical cases. Neverthe-less, G2032 is, as of today, the only acquired resistance mutant

reported for ROS1-positive patients treatedwith crizotinib andwecannot exclude, based on previous experience on ALK that addi-tional, entrectinib-sensitive ROS1 mutations will be identified.

DiscussionIn the present study, we disclose for the first time the

structure of entrectinib and describe the preclinical character-ization of this novel, orally available, small-molecule inhibitor,which is currently being investigated in two clinical trials inadult patients with TRK-positive tumors (29–31). Its preclinicalactivity on ROS1- and ALK-positive tumor models additionallysupports the enrollment of ROS1- and ALK-positive patients inthe clinical trial. In the initial characterization of the molecule,the high biochemical potency observed against TRKA, ROS1,and ALK was reflected by remarkable activity and selectivity incellular models. When profiled on a panel of more than 200human tumor cell lines, entrectinib selectively inhibited pro-liferation of tumor cells dependent upon endogenous expres-sion of these kinases with sub 100 nmol/L activity, withnegligible activity against unrelated cell lines. Subsequent stud-ies were conducted to confirm mechanism of action of entrec-tinib in TRK-, ROS1- and ALK-dependent models, and toexplore in vivo antitumor activity.

With regard to these distinct oncogenic events, it is fair to saythat until recently, TRKA was not considered by the pharmaceu-tical industry as a high priority therapeutic target in the cancerfield, because the best characterized oncogenic rearrangements ofits gene,NTRK1, were primarily associated with a subset of PTC, atumor type that has a good prognosis, with thyroidectomy gen-erally being a curative surgical procedure. No TRKA inhibitorswere explored in clinical trials with the possible exception oflestaurtinib (CEP-701), a staurosporine-derivative multikinaseinhibitor that was investigated in clinical settings with reportedTRKA overexpression, without evidence of efficacy (7,38). How-ever, recent studies that have identified recurring oncogenicNTRK1 rearrangements in subsets of NSCLC and colorectal car-cinoma patients (11,12), as well as other studies suggesting apotential role of activated TRKA in other tumor types, includingglioblastoma and Spitz melanoma (14–16,39), have createdmuch renewed interest in the identification and clinical investi-gation of effective TRKA inhibitors (13). In a rapidly evolvingscenario, these findings indicate that TRKA inhibition may rep-resent an innovative opportunity for the therapy of selectedpatients whose tumors harbor NTRK1 gene rearrangements andthis has supported the enrollment of TRKA-positive patients inongoing clinical trials of entrectinib. Here, we report that

Table 3. Antiproliferative activity of entrectinib on Ba/F3 cells expressingdifferent EML4–ALK mutants

Cell lines IC50 (mmol/L)

Ba/F3 þ IL3 2.650Ba/F3-EML4-ALK-wt - 0.028

þ IL3 1.490Ba/F3-EML4-ALK-C1156Y - 0.029

þ IL3 1.070Ba/F3-EML4-ALK-L1196M - 0.067

þ IL3 1.380Ba/F3-EML4-ALK-G1202R - 0.897

þ IL3 2.780Ba/F3-EML4-ALK-G1269A - 0.390

þ IL3 1.150

Ardini et al.





Figure 6.Activity of entrectinib againstNCI-H2228NSCLC tumors. A,NCI-H2228cellswere treatedwith entrectinib at different concentrations for 2 or96hours. Levelsof proteinsand phospho-proteins were detected by immunoblot analysis using specific antibodies. B, NCI-H2228 cells were treated with 50 nmol/L entrectinib for 24 and96 hours and cell-cycle distribution was evaluated by PI staining and cytofluorimetric analysis (top). Induction of apoptosis was confirmed byWestern Blot analysis ofPARP cleavage in treated cells (bottom). C, nu/nu mice bearing established NCI-H2228 xenografts were administered entrectinib per os twice daily at 60 mg/kgor 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(n¼ 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 administeredorally 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 wereanalyzed by Western Blot analysis with the indicated antibodies. E, mice bearing intracranial tumors were treated with vehicle or with entrectinib orally at thedose of 120mg/kg twice daily for 10 consecutive days. Representativemagnetic resonance images of vehicle or entrectinib-treatedmice are shown. To gain insight intoviability 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).






entrectinib is extremely effective against the human TRKA-depen-dent colorectal carcinoma cell line KM12, with efficacy studiesshowing that oral administration yields potent tumor growthinhibition at well-tolerated doses. Ex vivo target modulationanalysis showed strong correlation between in vivo antitumoractivity and target inhibition, as assessed by the phosphorylationstatus of TRKA, as well as of the downstream signaling transducerPLCg1, thus providing strong evidence of on-targetmechanism ofaction. These data support the rationale for clinical developmentof entrectinib in TRKA-dependent indications. As entrectinib wasfound to be also a potent inhibitor of the other members of theTRK family, the drug is currently being explored in TRKB- andTRKC-dependent clinical settings.

While being an extremely potent TRK inhibitor, we found thatentrectinib also possesses excellent biochemical activity againstROS1 kinase, which translates into potent in vitro and in vivoefficacy in a ROS1-driven tumor model. Although detailed dataon remarkable clinical benefit observed in ROS1-positive patientpopulation enrolled in an expansion cohort of the phase I clinicaltrial with crizotinib have been recently reported (28), we believethat our report of the high in vitro and in vivo activity of entrectinibagainst ROS1-driven tumor models is an additional significantfinding. In particular, cellular proliferation and mechanism ofaction data indicate that entrectinib is in the range of 10- to 100-fold more potent against ROS1 than crizotinib, and we thuspropose that it holds potential for exciting clinical activity inspecific subsets of NSCLC, as well as additional settings such ascholangiocarcinoma and inflammatory myofibroblastic tumors,which harbor genetic aberrancies in ROS1 (40).

Regarding the ALK-dependent tumors examined in this study,the correlation between in vitro and in vivo antitumor activity andtarget inhibition, as assessed by the phosphorylation status ofNPM-ALK in ALCL cell lines and of EML4-ALK in the NSCLC cellline NCI-H2228, as well as detailed pharmacokinetic/pharmaco-dynamic studies provide confirmation of target-dependent activ-ity of entrectinib in these models. In ALK-driven xenograft mod-els, oral treatment with entrectinib at extremely well-tolerateddoses induced remarkable tumor regression with all treatedanimals being tumor free by the end of a 10 days treatmentperiod and again with persistent complete tumor eradicationobserved in the majority of animals.

Because thedevelopment ofbrainmetastases is amajor causeofrelapse after initial clinical benefit with crizotinib (24), we believethat a highly significant feature of entrectinib is its favorable BBBpenetration in all preclinical species tested. To explore this featureof entrectinib's profile, the drug was tested against an intracranialgrowth model of NSCLC in the mouse, showing an excellenttumor growth control rate, and suggesting that the drugmay be ofparticular clinical benefit in patients with brain metastases. Thisfinding is particularly relevant in the perspective of potentialclinical development in indications such as TRKA-positive glio-blastoma and in clinical settings where brain metastases arecommonly encountered, as occurs in fusion-driven NSCLC.

In conclusion, the studies described herein provide a scientificrationale supporting the development of entrectinib in TRK-,

ROS1-, and ALK-dependent tumor settings. Clinical trials withthe compound are currently ongoing, with promising early signsof activity (29–31). Interestingly, the active recruitment of TRKA-positive patients already in phase I trial resulted in successfultreatment of a colorectal carcinoma patient and a NSCLC patientharboring TRKA-rearranged tumors, with remarkable activityobserved after the first cycle of administration, including com-plete regression of a brain metastasis in the latter case (30,31). Inaddition, durable objective responses were observed in severalNSCLCpatientswhose tumors harboredROS1orALK fusions andin ALK-driven neuroblastoma (29). These exciting preliminaryclinical data are fully coherent with the preclinical studiesdescribed herein, and strongly support further investigation ofthe drug in the patient subsets described above.

Disclosure of Potential Conflicts of InterestD. Anderson has ownership interest (including patents) in and is a consul-

tant/advisory board member for Ignyta. G. Li has ownership interest (includingpatents) in Ignyta. No potential conflicts of interest were disclosed by the otherauthors.

Authors' ContributionsConception and design: E. Ardini, M. Menichincheri, M.B. Saccardo,D. Anderson, J. Harris, G. Li, A. Isacchi, P. Magnaghi, A. GalvaniDevelopment of methodology: C. De Ponti, D. Ballinari, G. Texido,N.Amboldi,M.B. Saccardo, P.Orsini,D.Anderson, J.Harris, E. Felder,D.DonatiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): P. Banfi, D. Ballinari, M. Ciomei, G. Texido,A. Degrassi, N. Avanzi, N. Amboldi, M.B. Saccardo, D. Casero, L. Mologni,G. Wei, G. LiAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): E. Ardini, P. Banfi, D. Ballinari, M. Ciomei,A. Degrassi, N. Avanzi, N. Amboldi, D. Anderson, G. Wei, G. Li, E. Felder,D. Donati, A. Isacchi, E. PesentiWriting, review, and/or revision of the manuscript: E. Ardini, R. Bosotti,R. Pulci, G. Texido, A. Degrassi, M.B. Saccardo, D. Anderson, G. Wei,J.-M. Vernier, G. Li, D. Donati, A. Isacchi, A. GalvaniAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): R. Bosotti, G. WeiStudy supervision: E. Ardini, A. Isacchi, E. Pesenti, P. Magnaghi, A. GalvaniOther (participated in the chemistry program that led to entrectinib):T. Bandiera

AcknowledgmentsThe authors thank the NMS Biochemical Screening and Experimental Ther-

apy Groups, as well as our colleagues in various support units for theircontribution to the experimental activities. We are indebted to Ettore Perronefor skilled synthetic chemistry support and to Valter Croci, colleague and friend,for his dedicated work. Finally, the authors thank Zachary Hornby and RobertWild for critical reading of the article.

Grant SupportThis work was financially supported by Nerviano Medical Sciences srl.The costs of publication of this articlewere defrayed inpart by the payment of

page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received September 11, 2015; revised January 20, 2016; accepted January 21,2016; published OnlineFirst March 3, 2016.

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2016;15:628-639. Published OnlineFirst March 3, 2016.Mol Cancer Ther Elena Ardini, Maria Menichincheri, Patrizia Banfi, et al. Multiple Molecularly Defined Cancer Indications

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

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