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Small Molecule Therapeutics

BI 885578, a Novel IGF1R/INSR Tyrosine KinaseInhibitor with Pharmacokinetic Properties ThatDissociate Antitumor Efficacy and Perturbation ofGlucose HomeostasisMichael P. Sanderson1, Joshua Apgar2, Pilar Garin-Chesa1, Marco H. Hofmann1,Dirk Kessler1, Jens Quant1, Alexander Savchenko1, Otmar Schaaf1, Matthias Treu1,Heather Tye3, Stephan K. Zahn1, Andreas Zoephel1, Eric Haaksma1,G€unther R. Adolf1, and Norbert Kraut1

Abstract

Inhibition of the IGF1R, INSRA, and INSRB receptor tyrosinekinases represents an attractive approach of pharmacologic inter-vention in cancer, owing to the roles of the IGF1R and INSRA inpromoting cell proliferation and survival. However, the centralrole of the INSRB isoform in glucose homeostasis suggests thatprolonged inhibition of this kinase could result in metabolictoxicity. We describe here the profile of the novel compound BI885578, a potent and selective ATP-competitive IGF1R/INSRtyrosine kinase inhibitor distinguished by rapid intestinal absorp-tion and a short in vivo half-life as a result of rapid metabolicclearance. BI 885578, administered daily per os, displayed anacceptable tolerability profile in mice at doses that significantlyreduced the growth of xenografted human GEO and CL-14colon carcinoma tumors. We found that treatment with BI

885578 is accompanied by increases in circulating glucoseand insulin levels, which in turn leads to compensatory hyper-phosphorylation of muscle INSRs and subsequent normaliza-tion of blood glucose within a few hours. In contrast, thenormalization of IGF1R and INSR phosphorylation in GEOtumors occurs at a much slower rate. In accordance with this, BI885578 led to a prolonged inhibition of cell proliferation andinduction of apoptosis in GEO tumors. We propose that theremarkable therapeutic window observed for BI 885578 isachieved by virtue of the distinctive pharmacokinetic propertiesof the compound, capitalizing on the physiologic mechanismsof glucose homeostasis and differential levels of IGF1R andINSR expression in tumors and normal tissues. Mol Cancer Ther;14(12); 2762–72. �2015 AACR.

IntroductionThe insulin like growth factor-1 receptor (IGF1R) and insulin

receptor (INSR) isoforms A and B are structurally related proteinsconsisting of an extracellular a subunit disulphide linked to atransmembrane b subunit with an intracellular tyrosine kinasedomain (1). Dimerization via disulphide bridging between the bsubunits of twoprotein leads to the formationof receptorswith ana-b-b-a arrangement. In cells coexpressing all three proteins, theformation of six different receptor dimers is enabled: IGF1R:

IGF1R, INSRA:INSRA, and INSRB:INSRB homodimers and"hybrids" thereof (e.g., IGF1R:INSRA; ref. 2).

Signal transduction from the receptors is dependent on thebinding of the ligands IGF1, IGF2, or insulin. The affinity of eachligand for the receptors has been extensively investigated (3–7).Briefly, IGF1 binds with high affinity to receptors containing anIGF1R protein. IGF2 displays a similar pattern of receptor bind-ing; however, its high affinity to the INSRA protein enablesbinding to the INSRA homodimer and INSRA:B hybrid. Con-versely, insulin only binds the INSRA and INSRB homodimersand INSRA:B hybrid. Upon ligand binding, the receptors under-go a structural rearrangement leading to activation of the kinasedomains and autophosphorylation of intracellular tyrosines(8). Adaptor proteins including Shc, IRS1, and -2 dock on thephospho tyrosines and transduce signaling via the MAPK andPI3K cascades.

The IGF1R is ubiquitously expressed in adult and embryonictissues and in multiple cancers (8). Genetic evidence in rodentsand humans suggest that the IGF1R and its ligands are essentialfor the genesis and homeostasis of multiple tissues (9, 10).Meanwhile, in diverse cancers, IGF1R signaling promotes onco-genic proliferation and survival (1, 8). The INSRAdisplays restrict-ed expression during early embryonic development but is aber-rantly re-expressed in diverse cancers (6, 11). INSRA signaling,particularly in cancer cells expressing autocrine IGF2, promotes

1Boehringer Ingelheim RCV GmbH & Co KG, Department of Pharma-cology, Dr. Boehringer-Gasse,Vienna, Austria. 2Boehringer IngelheimPharmaceuticals, Inc., Ridgefield, Connecticut. 3Evotec (UK) Ltd.,Abingdon, Oxfordshire, United Kingdom.

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

Current address for J. Apgar: Applied BioMath LLC, Winchester, Massachusetts.

Corresponding Author: Michael P. Sanderson, Department of Pharmacology,Boehringer Ingelheim RCV GmbH & Co KG, Dr. Boehringer-Gasse 5-11. A-1121,Vienna, Austria. Phone: 4318-0105-2794; Fax: 4318-0105-2366; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-15-0539

�2015 American Association for Cancer Research.

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oncogenic cellular responses in a similar manner to the IGF1R(12–15). As such, the IGF1R and INSRA both represent attractivetargets for pharmacologic intervention in cancer. Conversely,insulin-INSRB signaling plays a central role in glucose disposalin adult cell types such as hepatocytes, skeletal muscle, andadipocytes (16–19).

Three predominant therapeutic strategies of inhibiting signal-ing via the receptors and ligands (herein referred to as the "IGF-pathway") have been explored in human clinical trials; IGF1Rantibodies (20), IGF1/2 antibodies (21), and ATP-competitiveIGF1R/INSR tyrosine kinase inhibitors (TKI; ref. 22). Multiplephase II and III clinical trials of IGF1R antibodies have yieldeddisappointing efficacy outcomes (23, 24). Several preclinicalstudies have suggested this may be due to incomplete pathwayinhibition as a result of "sparing" of INSRA signaling (14, 25, 26).

IGF1R/INSR TKIs equipotently inhibit the IGF1R, INSRA, andINSRB kinases, as a consequence of complete identity of thereceptor ATP-binding pockets, and as such block the IGF pathwayto a broader extent than IGF1R antibodies (27–29). In keepingwith this, IGF1R/INSR TKIs deliver superior efficacy comparedto IGF1R antibodies in various preclinical cancer models(25, 26, 30). However, inhibition of INSRB-mediated glucosedisposal represents a tolerability liability of IGF1R/INSR TKIs.Indeed, hyperglycemia was recently reported as a commonadverse event in a phase I trial of the IGF1R/INSR TKI linsitinib(22). This toxicity hurdle could potentially limit the dosing ofIGF1R/INSR TKIs to levels too low to providemeaningful efficacy.

Whereas signal transduction inhibitors used in cancer therapyare generally dosed to provide maximal target inhibition duringthe entire dosing interval, we hypothesized that an IGF1R/INSRTKI that only transiently inhibits its targets could provide animproved therapeutic window. This hypothesis assumes thatupon inhibitor dissociation, INSRB-mediated glucose homeosta-sis, a highly adaptable and tightly regulated process (31), wouldrecover at a faster rate than IGF1R- and INSRA-mediated tumorproliferation and survival. As such, we optimized IGF1R/INSRTKIs to display, in addition to high target potency and selectivity,short target residence times and in vivo half-life. This led to thediscovery of the novel compound BI 885578. We demonstratethat BI 885578 delivers significant efficacy in mouse xenografttumor models at doses associated with only transient and there-fore well-tolerated inhibition of glucosemetabolism and proposeamechanisticmodel explaining the observed therapeutic windowbased on compound characteristics, receptor expression, andfeedback regulation of signal transduction.

Materials and MethodsCompounds

BI 885578 and afatinib were synthesized at Boehringer Ingel-heim. PHA665752 was purchased from Sigma Aldrich.

In vitro biochemical and cellular assaysThe DELFIA (Perkin Elmer) kinase assay using IGF1R-GST has

been described previously (32). For DELFIA INSR kinase assays,an identical assay setup was performed using INSR-GST protein.All other kinase assays were performed at Life Technologies usingthe SelectScreen platform.

IGF1R null mouse embryonic fibroblasts (MEF) stably expres-sing human IGF1R or INSRB were a kind gift from R. Vigneri(University of Catania, Italy, obtained April 26, 2008) and were

cultured in DMEM with 10% FCS, 5% sodium pyruvate, 5%nonessential amino acids, 5% glutamine, and 0.3 mg/mL puro-mycin. Protocols for assessment of IGF1R and INSRB phosphor-ylation in theseMEFs were described previously (33). The humanGEOcell linewas generously providedbyS. Pepe (Universita degliStudi di Napoli, Italy, obtained April 15, 2008) and was culturedin McCoy 5a medium with 10% FCS and 20 mmol/L HEPES.These cell lineswere not authenticated by STR, owing to the lack ofpublished reference signatures. The following cell lines werecultured according to the manufacturer's instruction and authen-ticated by STR analysis at Boehringer Ingelheim on the datesindicated. HCT 116 (obtained June 17, 2011; STR April 17,2012) and Calu-6 (obtained January 17, 2000; STR June 24,2011) were from the ATCC. TC71 (obtained April 4, 2008; STRMay 4, 2011), andCL-14 (obtained Jaunary 2, 2013; STRApril 20,2013) were from the DSMZ.

For proliferation assays, cells were cultured in BI 885578 for 72hours then incubated with AlamarBlue (Serotec Ltd.) for 7 hours.Fluorescence (extinction wavelength 544 nm, emission 590 nm)was measured and IC50 values generated using GraphPad Prism.

For assessment of protein phosphorylation, GEO cells incu-bated with compound or DMSO (control), washed with ice-coldPBS, and lysed for 10 minutes in ice-cold Lysis Buffer containingprotease andphosphatase inhibitors (Cell Signaling Technology).Homogenates were cleared by centrifugation (30minutes, 14,000rpm, 4�C). SDS-PAGE and Western blotting was performed asdescribed previously (33). The antibodies used are described inthe Supplementary Materials and Methods section. Lysates wereanalyzed using ProteomeProfiler Antibody Arrays (R&D Systems)according to the manufacturer's instructions.

Surface plasmon resonanceSurface plasmon resonance (SPR) experiments were performed

using the Sprint platform (Beactica AB). GST-IGF1R and GST-INSR proteins were immobilized by anti-GST capture usingAmine coupling buffer (10 mmol/L PBS, 0.05% Tween 20) andcapture buffer (10mmol/L TBS, 20mmol/LMgCl2, 0.05% Tween20). After capture, the surface was cross-linked with N-hydroxy-succinimide (NHS) and 1-Ethyl-3-(3-dimethylaminopropyl) car-bodiimide (EDC). Interaction experiments were performed at25�C in running buffer (10 mmol/L TBS, 20 mmol/L MgCl2,0.05% Tween 20, 5% DMSO). BI 885578 was injected for60 seconds over the immobilized proteins. Sensorgrams wereanalyzed with the BiacoreT200 evaluation software 1.0. A 1:1interactionmodelwasfitted globally to sensorgrams inmulticycleexperiments.

X-ray crystallographyThe INSR kinase domain (V1005-K1310, with mutations

C1008S, D1159N) was used for crystallization using the hang-ing-drop method. One microliter of INSR solution (13 mg/mLINSR in 50 mmol/L Tris HCl pH 7.5, 170 mmol/L NaCl) wasincubated with BI 885578 for 1 hour at 4�C with 1 mL reservoirsolution (100 mmol/L Tris pH 7.6, 22% PEG8000, 50 mmol/LNaCl, 5 mmol/L DTT). Crystals were flash-frozen in liquid nitro-gen after immersion in20%ethylene glycol.Datawere collected atthe SLS beam line X06SA (Swiss Light Source, Scherrer Institute)using a PILATUS 6M detector with a crystal that diffracted to 2.26Å. Data processing and analysis was performed using the auto-PROC toolbox (34). The structure was solved by molecularreplacement using a published INSR structure (PDB ID 1IRK;

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ref. 35). Model building and refinement was performed usingCCP4 (36), COOT (37), and autoBUSTER (38). The X-ray crys-tallography collection and processing data and refinement statis-tics are presented in Supplementary Tables S1 and S2, respectively.

In vivo tumor modelsThe protocol for the GEO xenograft model was described

previously (33). CL-14 xenograft tumors (80–110 mm3) wereestablished from CL-14 cells (5 � 106 in matrigel) by subcuta-neous injection into the right flanks of female BomTac:NMRI-Foxn1nu mice. After six weeks, mice were randomized (7 pergroup) for treatment. Tumor volumes were determined threetimes per week with a digital caliper and body weight wasmeasured daily. BI 885578 was formulated in 0.5% Natrosol(hydroxyethylcellulose natrosol 250HX, VWR International), 3%Tween80, and1mol/L citric acid. For 10mLof solution, amixtureconsisting of 9.8 mL of 0.5% natrosol and 20 mL of 3% Tween 80was prepared and citric acid then used to adjust to pH 3. All doseswere calculated relative to the mouse body weight on the treat-ment day. BI 885578 and the vehicle control (0.5% Natrosol)were administered orally (per os) with a volume of 10mL/kg bodyweight.

One-sided decreasing Mann–Whitney tests were used to com-pare tumor volumes (efficacy). Two-sided decreasing Mann–Whitney tests were used to compare body weights (tolerability).The P values for the tumor volume assessment were adjustedfor multiple comparisons according to Bonferroni–Holm (39),whereas P values for body weight remained unadjusted. For allanalyses, P values under 0.05 represented a statistically significanteffect.

Pharmacodynamic biomarker analysesFor analysis of pharmacodynamic biomarkers, GEO tumor-

bearing mice were treated with BI 885578 and blood, tumor, andmuscle samples isolated (3 animals per time point). Blood waswithdrawn retroorbitally under isoflurane anesthesia and theglucose concentration in each sample measured with the Accu-Chek compact plus device (Roche). Blood was centrifuged for5 minutes (10,000 rpm, 4�C) to isolate plasma for ELISA mea-surement of IGF1 (#MG100, R&D Systems) and insulin (#90080,Crystal Chem) according to the manufacturer's instructions.

Mice were sacrificed prior to explanting tumors and muscle(right hind limb). Tumor andmuscle samples were homogenizedin ice-cold Cell Lysis Buffer using Eppendorf micropestles (Sig-ma). Homogenates were incubated on ice for 60 minutes andcleared as described above. Bio-Plex kits (Bio-Rad) were usedaccording to the manufacturer's instructions to measure phos-phorylated IGF1R (Tyrosine 1131) and INSR (Tyrosine 1146).The statistical comparison of data was performed using unpairedparametric tests.

Histomorphologic and immunohistochemical analysesGEO tumor-bearing mice were treated with BI 885578 as

described above. Two hours prior to tumor excision, mice wereadministered with 10 mL/kg of a bromodeoxyuridine (BrdUrd)solution (Invitrogen). GEO tumors were explanted as above andembedded in paraffin (FFPE). Immunohistochemistry (IHC) wasperformed on 4 mm sections, which had been pretreated withcitrate buffer (pH 6.0). The EnVisionþ System-HRP LabelledPolymer (No. 4003, DakoCytomation) method was used fol-lowed by diaminobenzidine (DAB) and hematoxylin counter-

staining. Details of the antibodies used are in the SupplementaryMaterials and Methods section.

HPLC-MS/MS quantitation of BI 885578BI 885578 in plasma was quantified by high performance

liquid chromatography/tandem mass spectrometry (HPLC)-MS/MS inESIþmodeusing an analyticalHPLC (AgilentHP1200)with a Waters XBridge BEH C18 column (2.5 mm, 2.1 � 50 mm)coupled to an ABSCIEX API5000 triple quadrupole mass spec-trometer. Samples were extracted with ethyl acetate/0.1 mol/LNaOH, evaporated under N2, and then redissolved in 25%methanol/75% water/0.01% formic acid. Ion transitions forMRM, declustering potential, and collision energy settings wereoptimized using DiscoveryQuant 2.1 and peak areas determinedusing Analyst 1.5.1 (ABSCIEX). Pharmacokinetic parameters werecalculated by noncompartmental analysis using the BoehringerIngelheim software ATLAS.

Physicochemical and DMPK assaysCaco-2 parameters were determined as described previously

(40). Aqueous solubilities were determined from 1 mg/mL solidcompound dispensed into aqueous McIlvaine buffer (pH 4.5or 6.8), or dissolved in acetonitrile/water (1:1) as reference.Dissolved concentrations were determined with an Agilent1200 HPLC/DAD-UV system.

Plasma protein binding (PPB) was determined by equilibriumdialysis. Plasma was spiked with BI 885578 and dialyzed againstSoerensen buffer (pH 7.4) for 3 hours at 37�C. PPBwas calculatedby quantifying BI 885578 concentrations in plasma and buffer byHPLC/MS-MS.

In vivo hepatic clearance (CL) was predicted by in vitro incuba-tions of BI 885578with cryopreserved hepatocytes (Celsis IVT) for30minutes followed by quantitation of BI 885578 by LC/MS/MSand using the well-stirred model.

ResultsChemical structure and target binding mode

BI 885578 is a chiral compound bearing a methyl-group at thestereogenic center located in the cycloaliphatic bridge linking thepyrimidine and pyrazole rings (Fig. 1A). The capped piperazinemoiety exposed to the solvent serves as a solubilizing group withmoderate basicity. The crystal structure of INSR with BI 885578shows a typical bilobal architecture of protein kinases (Fig. 1B).BI 885578 binds noncovalently to the ATP-binding pocket in theINSR catalytic site and forms two hydrogen bonds to the back-bone of Met1079 at the kinase hinge region (Fig. 1B and C).

Surface plasmon resonance (SPR) assays demonstrated that BI885578has ahigh affinity for the IGF1Rand INSRkinase domains(Kd¼ 9 and 12 nmol/L, respectively; Table 1). BI 885578 showedrapid association and dissociation kinetics for the IGF1R andINSR indicative of short residence times, in the range of minutes.

Molecular and cellular potency and selectivityThe potency and selectivity of BI 885578 were investigated

using recombinant in vitro kinase assays. BI 885578 potentlyinhibited the kinase activity of the IGF1R and INSR with an IC50

value of 1 nmol/L for both kinases (Table 1). When tested on199 other kinases, BI 885578 was found to be highly selective,with only 25 enzymes displaying IC50 values below 1,000nmol/L (Supplementary Table S3). A selectivity window of

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above 30-fold was demonstrated between IGF1R/INSR and thenext most potently inhibited kinases (IC50 ¼ 33 nmol/L forCLK2 and FER).

The effect of BI 885578 on ligand-stimulated IGF1R and INSRautophosphorylation incellswas assessed. IGF1RnullmouseMEFsstably expressing human IGF1R or INSRB (6) were stimulatedwithIGF1 or insulin, respectively, in the presence of BI 885578. IGF1Rand INSRB autophosphorylation was potently inhibited by BI885578 (IC50 ¼ 5 nmol/L for both targets; Table 1). BI 885578also potently inhibited the proliferation of the human cancer celllines TC71 (bone sarcoma; IC50 ¼ 4 nmol/L) and GEO (coloncarcinoma; IC50 ¼ 9 nmol/L), which have previously been shownto be sensitive to the IGF pathway inhibitors BMS-754807, linsi-tinib, BI 836845, and/or figitumumab (28, 33, 41, 42). BI 885578was comparatively inactive (IC50 > 2000 nmol/L) in proliferationassays using the IGF pathway inhibitor insensitive cell lines HCT116 (colon carcinoma) and Calu-6 (non–small cell lung carcino-ma), indicative of a lack of off-target cytotoxicity.

The ability of BI 885578 to inhibit signal transduction down-stream of the IGF1R/INSR in GEO cells was examined byWesternblotting. Treatment with BI 885578 at concentrations above20 nmol/L led to a marked reduction in the levels of phosphor-ylated IGF1R/INSR. The antibody used for this Western blotanalysis does not distinguish between the phospho forms of theIGF1R, INSRA, and INSRB. BI 885578 also potently reduced thephosphorylation of the downstream proteins Akt, Erk1/2, and S6(Fig. 2A). Levels of total IGF1R, Akt, Erk1/2, and S6 protein werenot affected.

The specificity of BI 885578 for the IGF1R and INSR was nextinvestigated in a cellular context. GEO cells were treated for 24hours with 500 nmol/L BI 885578, which is 100-fold higherthan the cellular IC50 for reduction of IGF1R and INSR phos-phorylation (5 nmol/L, Table 1). The levels of 49 phosphoreceptor tyrosine kinases (pRTK) were examined in GEOextracts using pRTK arrays (Fig. 2B). Phospho signals for theEGFR, ErbB2, c-Met, IGF1R, and INSR were detected in controlGEO extracts, and BI 885578 treatment only reduced theintensity of the phospho-IGF1R and –INSR signals. This assayformat cannot distinguish between the phospho-INSRA and–INSRB, owing to the complete identity of the intracellulardomains of these proteins. The EGFR/ErbB2 inhibitor afatinib(43) and the c-Met inhibitor PHA665752 (44) reduced thephospho signals for their targets, without affecting the phos-pho-IGF1R or –INSR signal intensity.

A

B

C

Molecular weight: 527.66Empirical formula: C29H37N9O

ON

NN N

N

N

N

N

N

Figure 1.Chemical structure of BI 885578 (A), the X-ray structure of BI 885578 boundto the active site of the INSR kinase domain crystal (B), and ligand interactionplot between BI 885578 and the binding site of the INSR kinase (C).

Table 1. In vitro potency and target-binding properties of BI 885578

In vitro potency

AssayIC50

(nmol/L)

IGF1R kinase 1 � 0.2INSR kinase 1 � 0.7IGF1R cellular autophosphorylation 5 � 3INSRB cellular autophosphorylation 5 � 3TC71 proliferationa 4 � 3GEO proliferationa 9 � 3HCT 116 proliferationb >2,000Calu-6 proliferationb >2,000

Target bindingKon

(106 M�1 � s�1)Koff

(10�3 s�1)Kd

(nmol/L)IGF1R 0.7 � 0.1 6 � 0.3 9 � 1INSR 1.4 � 0.2 17 � 2 12 � 0.1

NOTE: For each assay, mean values � standard deviation are presented, whichwere determined from three or more experiments.aIGF pathway–dependent cell line.bIGF pathway–independent cell line.



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Pharmacokinetic propertiesAs mentioned above, we hypothesized that an IGF1R/INSR

inhibitor with a short systemic half-life may achieve an improvedtherapeutic window. We therefore aimed to generate a compoundwith a PK profile characterized by fast absorption, an early tmax andsubsequent rapid elimination. BI 885578 displays aqueous solu-

bility,highpermeability and lowCaco-2efflux ratio translating intorapid in vivo absorption following per os dosing in mice, rats, anddogs with tmax values under 2 hours and terminal elimination half-lives in the range of 2 to 6 hours (Table 2). BI 885578 undergoesextensive metabolism in vitro when incubated with hepatocytes,which enables thedesired short initial and terminal in vivohalf lives.

Phospho-IGF1R(Y1135/1136)/

A B

INSR (Y1150/1151) (95 kDa)

Total IGF1R (95 kDa)

Control BI 885578 Afa�nib PHA665752

pINSR

pIGF1R

pEGFR

pErbB2

p-c-Met

A123456789

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123456789

101112131415161718192021222324

123456789

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123456789

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BCDEFABCDEFABCDEFABCDEF

Phospho-Akt (T308) (60 kDa)

Phospho-Erk1/2 (T202/Y204) (42/44 kDa)

Phospho-S6 (S235/S236) (32 kDa)

Total S6 (32 kDa)

b-Ac�n (45 kDa)

BI 885578 (nmol/L)

050

010

0 20 4 0.8

Total Erk1/2 (42/44 kDa)

Total Akt (60 kDa)

Figure 2.IGF1R/INSR pathway modulation by BI 885578. A, GEO cells were treated for 30 minutes with the indicated concentrations of BI 885578. The levels of thephospho- and total forms of the indicated proteins were examined by Western blotting. b-Actin levels were also examined to demonstrate equivalentloading of cellular protein for each sample. B, GEO cells were treated for 24 hours with 500 nmol/L of either BI 885578, afatinib, or PHA665752 or an equivalentvolume of DMSO (control). Lysates were analyzed using pRTK arrays. Signals at coordinates A1/2, A23/24, and F1/2 represent reference spots to control forequivalent levels of cellular proteins. B1/2, pEGFR; B2/3, pErbB2; B17/18, pINSR; B19/20, pIGF1R; C3/4, p-c-Met.

Table 2. Key physicochemical and DMPK properties of BI 885578

Solubility (mg/mL)pH 4.5 >0.98pH 6.8 0.052

In vitro ADME and in vivo plasma PK properties across speciesHuman Mouse Rat Dog

Papp (a-b) Caco-2 cells at 10 mmol/L (10�6 cm/s) 65 NA NA NAEfflux ratio Caco-2 cells at 10 mmol/L 0.9 NA NA NAIn vitro plasma protein binding (% unbound) 7.6 6.0 7.0 21In vitro metabolic CL hepatocytes (% QH) 40 70 47 88i.v.—CL (% QH) ND 121 66 132i.v.—Vss (L/kg) ND 3.2 3.0 3.7i.v.—MRTdisp (h) ND 0.5 1.1 1.6p.o.—dose (mg/kg) ND 40 5 9p.o.—AUC(0-inf) (nmol/L�h) ND 809 500 541p.o.—bioavailability (%) ND 6.9 22 7.5p.o.—Cmax (nmol/L) ND 679 98 134p.o.—tmax (h) ND �0.2 �0.2 1.7p.o.—t1/2,z (h) ND 2.3 6.2 3.3

NOTE: Solubility data were derived from single determinations. Caco-2, plasma protein binding and hepatocytes CL data represent mean values from two separateexperiments. In vivo data are mean values from 3 animals.Abbreviations: NA, not applicable; ND, not determined.

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In vivo tolerability and efficacyThe in vivo efficacy and tolerability of BI 885578 was examined

in mice bearing xenografted GEO tumors. The GEO cell line isderived from a human colon carcinoma and carries a mutation inKRAS (G12A) in addition to displaying high expression of IGF2(45). Oral doses of 20 and 40 mg/kg applied daily (q.d.) and 80mg/kg applied every other day (q2d) were tested over 22 days. Asignificant reduction in tumor growth compared with controlswas observed in all treatment groups with median tumor growthinhibition (TGI) values of 64% (P¼ 0.0009), 85% (P¼ 0.0009),and 79% (P ¼ 0.0012), respectively (Fig. 3A).

The 20 and 40 mg/kg q.d. treatments did not reduce mousebody weight gain (Fig. 3B), suggesting that these treatmentswere tolerated. In contrast, the 80 mg/kg q2d group displayedbody weight reductions on the treatment days. Treatment-freedays in this group were associated with a recovery of bodyweight. Following the final BI 885578 treatment in each group,blood glucose levels were monitored over 24 hours. The 20 and40mg/kg q.d. treatments induced a transient elevation of bloodglucose, with normalization observable between 2 and 6 hoursafter dosing (Fig. 3C). The 80 mg/kg q2d treatment inducedslightly more prolonged, yet not statistically significant, eleva-tion of blood glucose compared with the other dose groups.

The efficacy and tolerability of the40mg/kg (q.d., per os) dose ofBI 885578 was further examined in the CL-14 colon carcinomaxenograft model (Supplementary Fig. S1A–S1C). BI 885578 sig-nificantly inhibited the growth of CL-14 tumors with a medianTGI of 89% (P¼ 0.0003). As in theGEO xenograft, this BI 885578dose had no significant impact on body weight gain (P ¼ 0.093)and was associated with only a transient (<6 hours) elevation ofblood glucose.

In vivo pathway modulation in plasma, tumor, and muscleTo investigate mechanisms by which the 40 mg/kg dose of BI

885578 delivers efficacy and acceptable tolerability, we examinedpharmacodynamic markers of the IGF pathway in plasma andtissues. GEO tumor-bearingmice were treated once with this doseof BI 885578 and plasma, tumor, andmuscle samples were takenat multiple time points up to 24 hours after application.

Rapid absorption of BI 885578 with an early tmax andsubsequent rapid elimination was observed in plasma withconcentrations at 24 hours below the LLOQ of 2.8 nmol/L (Fig.4A). Similar to data shown above (Fig. 3C), a transient eleva-tion of plasma glucose levels was observed 15 minutes after 40mg/kg BI 885578 treatment, with normalization of glucoselevels occurring by 1 hour (Fig. 4A). Plasma insulin levels weresignificantly increased with an Emax of 34-fold above baselineobserved 1 hours after dosing but normalized between 12 and24 hours. A comparatively low variation (�2-fold of baseline)was observed for IGF1 levels and at no time point was thevariation found to be statistically significant compared withuntreated controls (Fig. 4A).

We next monitored the phosphorylation of the IGF1R andINSR in GEO tumors andmuscle. The Bio-Plex assays used do notdiscriminate between the phospho-INSRA and -INSRB. BI 885578treatment led to a significant and equivalent reduction of IGF1Rand INSR phosphorylation in GEO tumors, with an Emax ofapproximately 10% control at 1 hour after dosing (Fig. 4B). Thephosphorylation of both IGF1R and INSR remained suppressedto 10% to 25% of control until 6 hours. In this experiment,phospho-INSR levels normalized after 24 hours. Partial recovery

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Figure 3.Efficacy and tolerability of BI 885578 in the GEO xenograft model. GEOtumor-bearing mice (7 animals per group) were treated orally with vehiclecontrol (0 mg/kg) or BI 885578 at doses of 20 or 40mg/kg q.d. or 80 mg/kgq2d. A, median tumor volumes were monitored on the days indicatedby a data point. B, median changes in mouse body weight were monitoredevery day. C, following the final treatment in each group, blood glucoselevels were measured at the indicated time points. The upper limit of normal(ULN) blood glucose levels in untreated mice is indicated. � , P < 0.05 fortreated versus control group.



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of IGF1R phosphorylation in GEO tumors was observed between6 and 24 hours, but did not reach levels observed in untreatedcontrol tumors. In a separate experiment analyzing later timepoints, IGF1R and INSR phosphorylation in GEO tumors nor-malized to control levels 48 hours after dosing (SupplementaryFig. S2).

In mouse muscle, the kinetics of IGF1R phosphorylationshowed a similar profile to that of the IGF1R in GEO tumors,whereas the kinetics of INSR phosphorylation contrasted starklyto that of IGF1R and INSR phosphorylation in GEO tumors andthe IGF1R in muscle. INSR phosphorylation in muscle wassignificantly reduced to 23% of control 15 minutes after BI

885578 dosing, but then rebounded to between 200% and400% of control between 6 and 24 hours (Fig. 4B). This reboundof phospho-INSR levels inmuscle coincidedwith the induction ofplasma insulin and the normalization of blood glucose levelsdescribed above.

In vivomodulation of cell proliferation and survival markers intumors

We next examined the effect of BI 885578 onmarkers of tumorproliferation, growth, and survival. GEO tumor-bearing micewere treated once with BI 885578 (40mg/kg) and tumors excisedfor histologic and immunohistochemical analysis at multiple

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Figure 4.Effects of BI 885578 on IGF pathway pharmacodynamic biomarkers. GEO tumor-bearing mice were treated once orally with BI 885578 (40 mg/kg) andblood, tumors, and muscle sampled at 0, 0.25, 1, 2, 6, 12, and 24 hours after administration for analysis of PD biomarkers (3 animals per time point). A, levelsof circulating BI 885578, glucose, insulin, and mouse IGF1 were examined. B, the phosphorylation of the IGF1R and INSR was measured in tumor and muscleprotein extracts. � , P < 0.05 for treated versus control group.

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time points. Mice were also injected with a BrdUrd solution 2hours prior to tumor sampling to enable monitoring of nucleo-side uptake by GEO tumor cells as an indicator of proliferation.Erk1/2 and S6 are both downstream of the IGF1R and INSR andtheir phosphorylation is indicative of tumor cell proliferation andsurvival (46, 47). A reduction in the phosphorylation of Erk1/2and S6 was observed 12 to 48 hours after BI 885578 dosing (Fig.

5A; Table 3). A reduction of BrdUrd uptake at 24 hours indicatedthat GEO cell proliferation was inhibited (Fig. 5B; Table 3).Normalization of Erk1/2 and S6 phosphorylation and BrdUrduptake to levels equivalent to that in untreated tumors wasobserved at 72 hours. An increase of cleaved caspase-3 levels wasalso observed in GEO tumors at 12 and 24 hours, indicative ofapoptosis (Fig. 5B; Table 3).

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Figure 5.Effect of BI 885578 on GEO tumor cell proliferation and survival markers. GEO tumor-bearing mice were treated once orally with BI 885578 (40 mg/kg) andtumors sampled at0, 12, 24, and72hours after administration (3animals per timepoint). IHCwasused to analyze the levels of phosphorylatedErk1/2 andS6 (A) aswell asBrdUrd incorporation and cleaved caspase-3 levels (B) in each sample. Images representative of the effects observed for each time point and markers are shown.

Table 3. IHC scoring of the effect of BI 885578 on GEO tumor cell proliferation and survival markers

Control 2 h 6 h 12 h 24 h 48 h 72 h

BrdUrd þþþ þþþ þþþ þþ þ þþþ þþþCleaved casp-3 � � �/þ þ þ �/þ �pErk1/2 þþþ þþ þþ �/þ �/þ þ þþ/þþþpS6 þþ/þþþ þþ/þþþ þþ þ þ þ þþþNOTE: IHC samples from experiments described in Fig. 5A and B were scored for staining intensities based on standard histology criteria for each marker analyzed.



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DiscussionGiven the indispensable role of the INSRB in glucose homeo-

stasis, the development of IGF1R/INSR TKIs that balance efficacyand tolerability represents a significant challenge. In preclinicalstudies conducted earlier, we noted that published compoundswith relatively long half-lives in mice only achieved significantantitumor efficacy at the cost of poor tolerability, in particular iftreatment was continued for more than one or two weeks. Wehypothesized that an improved therapeutic window can beachieved with a potent and selective IGF1R/INSR TKI that under-goes rapid in vivo elimination and thus only transiently inhibits itstargets. In our compound optimization campaign (to bedescribed in a separate publication) we therefore included rapidelimination, based on susceptibility to hepatocyte-mediatedmetabolism, as a prioritized optimization parameter. Theseefforts led to the discovery of BI 885578.

BI 885578 potently and specifically inhibited the kinaseactivities of the IGF1R and INSR as well as downstream signaltransduction and this translated into potent inhibition ofthe proliferation of IGF pathway–dependent, but not IGF path-way–independent, cell lines. The activity of BI 885578 in diversetumor models and the genetic determinants of BI 885578sensitivity are currently under investigation with particular focuson IGF2 expression (48, 49; M. Sanderson, manuscript inpreparation). The physicochemical and metabolic properties ofBI 885578 enabled the desired in vivo pharmacokinetic profileupon oral dosing, characterized by rapid intestinal absorptionand fast elimination. In addition, the short residence times ofBI 885578 for the IGF1R and INSR ensured that prolongedtarget engagement, subsequent to plasma elimination, couldbe avoided.

BI 885578 induced an elevation of blood glucose in mice,indicative of inhibition of INSRB-mediated glucose homeostasis.

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Figure 6.Model for the differential kinetic effects of BI 885578 on GEO tumors and muscle. Top, under basal conditions, GEO tumors express a dominant proportion ofIGF1R homodimers and a smaller proportion of IGF1R:INSRA hybrids, which both bind circulating mouse IGF1 and autocrine human IGF2 to promote cellproliferation and survival. Muscle cells dominantly express INSRB homodimers that bind insulin to promote glucose uptake. INSRB:IGF1R hybrids, of low abundancein muscle, bind circulating IGF1. Middle, BI 885578 achieves peak concentrations soon after oral dosing and inhibits the IGF1R and INSR isoforms in all tissues,leading to an elevation of blood glucose. Glucose in turn stimulates b cells of the pancreas to immediately release their depot of insulin into the circulation.In contrast, blockade of the IGF1R does not result in increased IGF levels. Blockade of IGF1R and IGF1R:INSRA hybrids in GEO cells results in shutdown of signaltransduction, inhibiting cell proliferation and inducing apoptosis. Bottom, BI 885578 is rapidly eliminated, enabling the high circulating insulin levels topromote hyperphosphorylation of inhibitor-free INSRB homodimers in muscle. This leads to rapid glucose disposal by muscle and normalization of bloodglucose levels. With IGF1/2 levels remaining constant, re-equilibration of IGF1R homodimer and IGF1R:INSRA hybrid phosphorylation is slow and GEO cellproliferation and survival signaling never fully recover before the next BI 885578 dose is administered.

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We noted that doses associated with only a transient (<6 hourspostadministration) elevation of blood glucose levels in micewere acceptably tolerated, even during prolonged treatment.Intriguingly, these doses demonstrated efficacy in GEO and CL-14 tumor xenografts, indicating that BI 885578was able to delivera therapeutic window.

Pharmacokinetic/pharmacodynamic analyses indicated that BI885578 differentially modulated IGF pathway componentsresponsible for glucose homeostasis and tumorigenic signaling.INSR phosphorylation in muscle, a pharmacodynamic marker ofglucose homeostasis, was only briefly suppressed and subse-quently increased even beyond baseline levels, potentially as aresult of the sharp increase in circulating insulin levels. Mean-while, the phosphorylation of the IGF1R in GEO tumors andmuscle recovered only at a much slower rate. This could bepotentially explained by the finding that unlike insulin, levels ofIGF1, the predominant ligand for the IGF1R, did not undergocompensatory induction. Unlike in muscle, the INSR in GEOtumors did not appear to be sensitive to increased circulatinginsulin levels. This could potentially be explained by two separatemechanisms. First, the expression of the IGF1R by GEO cells isapproximately 15-fold higher than of the INSR, while the INSRAisoform predominates over INSRB (33). This likely results in thedominant representation at the GEO cell surface by IGF1R homo-dimers and IGF1R:INSRAhybrids, which both display low affinityfor insulin (3, 4). A consequently low proportion of INSRA orINSRB homodimers and INSRA:B hybrids, capable of high-affin-ity insulin binding, could explain the inability of GEO tumorINSRs to respond to elevated plasma insulin levels. In contrast,muscle dominantly expresses the INSRB (6), thus providingample high-affinity receptor sites for insulin binding. Second, theautocrine expression of IGF2 by GEO cells (45) could result in ahigh level of INSRA occupancy, thus reducing the availability ofligand-free receptors for insulin engagement. It would be ofinterest for future studies to examine the kinetic effects of BI885578 on INSR phosphorylation in tumor models that eitherlack autocrine IGF1/2 expression, or dominantly express theINSRA or INSRB relative to the IGF1R.

In conclusion, we propose a model based on our observationsto explain the mechanism by which the properties of BI 885578exploit the rapid adaptability of INSRB signaling in metabolictissues and slower recovery of IGF1R/INSRA signaling in GEOtumors to achieve a therapeutic window (Fig. 6). Translation ofour encouraging findings from mouse models to cancer patients

requires that the favorable pharmacokinetic characteristics of BI885578 are maintained in humans. In initial experiments usinghuman hepatocytes, we observed rapid in vitro BI 885578 metab-olism indicating that the desired short initial and terminal in vivohalf-life may be achieved in humans. Evaluation of the efficacyand tolerability of BI 885578 in cancer patients thus seemswarranted.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: M.P. Sanderson, J. Apgar, P. Garin-Chesa, M.H. Hof-mann, D. Kessler, J. Quant, O. Schaaf, M. Treu, H. Tye, S.K. Zahn, E. Haaksma,G.R. AdolfDevelopment of methodology: M.P. Sanderson, P. Garin-Chesa, M.H. Hof-mann, D. Kessler, A. Savchenko, O. Schaaf, M. Treu, H. Tye, S.K. Zahn,A. Zoephel, N. KrautAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M.P. Sanderson, P. Garin-Chesa, M.H. Hofmann,D. Kessler, A. Savchenko, O. Schaaf, M. Treu, H. Tye, S.K. Zahn, A. ZoephelAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M.P. Sanderson, J. Apgar, P. Garin-Chesa,M.H. Hofmann, D. Kessler, J. Quant, A. Savchenko, O. Schaaf, M. Treu, H. Tye,S.K. Zahn, A. Zoephel, E. Haaksma, G.R. Adolf, N. KrautWriting, review, and/or revision of the manuscript:M.P. Sanderson, J. Apgar,P. Garin-Chesa, M.H. Hofmann, D. Kessler, J. Quant, A. Savchenko, O. Schaaf,M. Treu, H. Tye, S.K. Zahn, A. Zoephel, G.R. Adolf, N. KrautAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): P. Garin-Chesa, D. Kessler, J. QuantStudy supervision:M.P. Sanderson,D. Kessler, J. Quant, S.K. Zahn, E.Haaksma,G.R. Adolf, N. Kraut

AcknowledgmentsThe authors thank the following colleagues for their excellent technical

assistance: Jack Bramham, Nora D€urkop, Thomas Fett, Stefan Fischer, AstridJeschko, Thomas Karner, Frank Maigler, Reiner Meyer, Sophie Mitzner, ThomasPecina, Carlos Ramirez-Santa Cruz, Christian Salamon, Samira Selman,Michaela Streicher, and Bernhard Wolkerstorfer.

Grant SupportThis work was financially supported by Boehringer Ingelheim.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 July 1, 2015; revised September 10, 2015; accepted September 14,2015; published OnlineFirst October 5, 2015.

References1. King H, Aleksic T, Haluska P,Macaulay VM. Can we unlock the potential of

IGF-1R inhibition in cancer therapy? Cancer Treat Rev 2014;40:1096–105.2. Belfiore A, Frasca F, Pandini G, Sciacca L, Vigneri R. Insulin receptor

isoforms and insulin receptor/insulin-like growth factor receptor hybridsin physiology and disease. Endocr Rev 2009;30:586–623.

3. Benyoucef S, Surinya KH, Hadaschik D, Siddle K. Characterization ofinsulin/IGF hybrid receptors: contributions of the insulin receptor L2 andFn1 domains and the alternatively spliced exon 11 sequence to ligandbinding and receptor activation. Biochem J 2007;403:603–13.

4. Pandini G, Frasca F, Mineo R, Sciacca L, Vigneri R, Belfiore A. Insulin/insulin-like growth factor I hybrid receptors have different biologicalcharacteristics depending on the insulin receptor isoform involved. J BiolChem 2002;277:39684–95.

5. Blanquart C, Achi J, Issad T. Characterization of IRA/IRB hybrid insulinreceptors using bioluminescence resonance energy transfer. Biochem Phar-macol 2008;76:873–83.

6. Frasca F, Pandini G, Scalia P, Sciacca L, Mineo R, Costantino A, et al.Insulin receptor isoform A, a newly recognized, high-affinity insulin-likegrowth factor II receptor in fetal and cancer cells. Mol Cell Biol 1999;19:3278–88.

7. Denley A, Bonython ER, Booker GW, Cosgrove LJ, Forbes BE, Ward CW,et al. Structural determinants for high-affinity binding of insulin-likegrowth factor II to insulin receptor (IR)-A, the exon 11 minus isoform ofthe IR. Mol Endocrinol 2004;18:2502–12.

8. Pollak M. The insulin and insulin-like growth factor receptor family inneoplasia: an update. Nat Rev Cancer 2012;12:159–69.

9. Liu JP, Baker J, Perkins AS, Robertson EJ, Efstratiadis A. Mice carrying nullmutations of the genes encoding insulin-like growth factor I (Igf-1) andtype 1 IGF receptor (Igf1r). Cell 1993;75:59–72.

10. Abuzzahab MJ, Schneider A, Goddard A, Grigorescu F, Lautier C, Keller E,et al. IGF-I receptor mutations resulting in intrauterine and postnatalgrowth retardation. N Engl J Med 2003;349:2211–22.



Dow



11. Denley A, Wallace JC, Cosgrove LJ, Forbes BE. The insulin receptor isoformexon 11- (IR-A) in cancer and other diseases: a review. Horm Metab Res2003;35:778–85.

12. Sciacca L, Mineo R, Pandini G, Murabito A, Vigneri R, Belfiore A. In IGF-Ireceptor-deficient leiomyosarcoma cells autocrine IGF-II induces cell inva-sion and protection from apoptosis via the insulin receptor isoform A.Oncogene 2002;21:8240–50.

13. Vella V, Pandini G, Sciacca L, Mineo R, Vigneri R, Pezzino V, et al. A novelautocrine loop involving IGF-II and the insulin receptor isoform-A stimu-lates growth of thyroid cancer. J Clin Endocrinol Metab 2002;87:245–54.

14. UlanetDB, LudwigDL, KahnCR,HanahanD. Insulin receptor functionallyenhances multistage tumor progression and conveys intrinsic resistance toIGF-1R targeted therapy. Proc Natl Acad Sci U S A 2010;107:10791–8.

15. Avnet S, Sciacca L, Salerno M, Gancitano G, Cassarino MF, Longhi A,et al. Insulin receptor isoform A and insulin-like growth factor II asadditional treatment targets in human osteosarcoma. Cancer Res 2009;69:2443–52.

16. Kitamura T, Kahn CR, Accili D. Insulin receptor knockout mice. Annu RevPhysiol 2003;65:313–32.

17. Lauro D, Kido Y, Castle AL, Zarnowski MJ, Hayashi H, Ebina Y, et al.Impaired glucose tolerance in mice with a targeted impairment of insulinaction in muscle and adipose tissue. Nat Genet 1998;20:294–8.

18. Moller DE, Yokota A, Caro JF, Flier JS. Tissue-specific expression of twoalternatively spliced insulin receptor mRNAs in man. Mol Endocrinol1989;3:1263–9.

19. Mosthaf L, Grako K, Dull TJ, Coussens L, Ullrich A, McClain DA. Func-tionally distinct insulin receptors generated by tissue-specific alternativesplicing. EMBO J 1990;9:2409–13.

20. Beltran PJ, Mitchell P, Chung YA, Cajulis E, Lu J, Belmontes B, et al. AMG479, a fully human anti-insulin-like growth factor receptor type I mono-clonal antibody, inhibits the growth and survival of pancreatic carcinomacells. Mol Cancer Ther 2009;8:1095–105.

21. Iguchi H, Nishina T, Nogami N, Kozuki T, Yamagiwa Y, Yagawa K. Phase Idose-escalation study evaluating safety, tolerability and pharmacokineticsof MEDI-573, a dual IGF-I/II neutralizing antibody, in Japanese patientswith advanced solid tumours. Invest New Drugs 2015;33:194–200.

22. Puzanov I, Lindsay CR, Goff L, Sosman J, Gilbert J, Berlin J, et al. A phase Istudy of continuous oral dosing ofOSI-906, a dual inhibitor of insulin-likegrowth factor-1 and insulin receptors, in patients with advanced solidtumors. Clin Cancer Res 2015;21:701–11.

23. Yee D. Insulin-like growth factor receptor inhibitors: baby or the bath-water? J Natl Cancer Inst 2012;104:975–81.

24. AllisonM. Clinical setbacks reduce IGF-1 inhibitors to cocktail mixers. NatBiotechnol 2012;30:906–7.

25. Buck E, Gokhale PC, Koujak S, Brown E, Eyzaguirre A, Tao N, et al.Compensatory insulin receptor (IR) activation on inhibition of insulin-like growth factor-1 receptor (IGF-1R): rationale for cotargeting IGF-1R andIR in cancer. Mol Cancer Ther 2010;9:2652–64.

26. Garofalo C,ManaraMC, Nicoletti G,MarinoMT, Lollini PL, AstolfiA, et al.Efficacy of and resistance to anti-IGF-1R therapies in Ewing's sarcoma isdependent on insulin receptor signaling. Oncogene 2011;30:2730–40.

27. Ullrich A, Gray A, Tam AW, Yang-Feng T, Tsubokawa M, Collins C, et al.Insulin-like growth factor I receptor primary structure: comparison withinsulin receptor suggests structural determinants that define functionalspecificity. EMBO J 1986;5:2503–12.

28. Carboni JM, Wittman M, Yang Z, Lee F, Greer A, Hurlburt W, et al. BMS-754807, a small molecule inhibitor of insulin-like growth factor-1R/IR.Mol Cancer Ther 2009;8:3341–9.

29. Buck E,MulvihillM. Smallmolecule inhibitors of the IGF-1R/IR axis for thetreatment of cancer. Expert Opin Investig Drugs 2011;20:605–21.

30. HouX, Huang F,Macedo LF, Harrington SC, Reeves KA, Greer A, et al. DualIGF-1R/InsR inhibitor BMS-754807 synergizes with hormonal agents in

treatment of estrogen-dependent breast cancer. Cancer Res 2011;71:7597–607.

31. Eberhard D, Lammert E. The pancreatic beta-cell in the islet and organcommunity. Curr Opin Genet Dev 2009;19:469–75.

32. TyeH,Guertler U,HofmannMH,MayerM, Pal S, Rast G, et al. Discovery ofnovel amino-pyrimidine inhibitors of the insulin-like growth factor 1(IGF1R) and insulin receptor (INSR) kinases; parallel optimization of cellpotency and hERG inhibition. Med Chem Commun 2015;6:1244–51.

33. Friedbichler K, Hofmann MH, Kroez M, Ostermann E, Lamche HR, KoesslC, et al. Pharmacodynamic and antineoplastic activity of BI 836845, a fullyhuman IGF ligand-neutralizing antibody, and mechanistic rationale forcombination with rapamycin. Mol Cancer Ther 2014;13:399–409.

34. VonrheinC, Flensburg C, Keller P, Sharff A, SmartO, PaciorekW, et al. Dataprocessing and analysis with the autoPROC toolbox. Acta Crystallogr DBiol Crystallogr 2011;67:293–302.

35. Hubbard SR, Wei L, Ellis L, Hendrickson WA. Crystal structure of thetyrosine kinase domain of the human insulin receptor. Nature 1994;372:746–54.

36. Dodson EJ, Winn M, Ralph A. Collaborative Computational Project,number 4: providing programs for protein crystallography. Methods Enzy-mol 1997;277:620–33.

37. Emsley P, Lohkamp B, Scott WG, Cowtan K. Features and development ofCoot. Acta Crystallogr D Biol Crystallogr 2010;66:486–501.

38. Smart OS, Womack TO, Flensburg C, Keller P, Paciorek W, Sharff A, et al.Exploiting structure similarity in refinement: automated NCS and target-structure restraints in BUSTER. Acta Crystallogr DBiol Crystallogr 2012;68:368–80.

39. Sonnenberg A. [Bonferroni-Holm sequential test procedure]. Z Gastro-enterol 1985;23:703–4.

40. Luippold AH, Arnhold T, Jorg W, Kruger B, Sussmuth RD. Application of arapid and integrated analysis system (RIAS) as a high-throughput proces-sing tool for in vitro ADME samples by liquid chromatography/tandemmass spectrometry. J Biomol Screen 2011;16:370–7.

41. Pavlicek A, Lira ME, Lee NV, Ching KA, Ye J, Cao J, et al. Molecularpredictors of sensitivity to the insulin-like growth factor 1 receptor inhib-itor Figitumumab (CP-751,871). Mol Cancer Ther 2013;12:2929–39.

42. Pitts TM, Tan AC, Kulikowski GN, Tentler JJ, Brown AM, Flanigan SA, et al.Development of an integrated genomic classifier for a novel agent incolorectal cancer: approach to individualized therapy in early develop-ment. Clin Cancer Res 2010;16:3193–204.

43. Solca F, Dahl G, Zoephel A, Bader G, Sanderson M, Klein C, et al. Targetbinding properties and cellular activity of afatinib (BIBW 2992), anirreversible ErbB family blocker. J Pharmacol Exp Ther 2012;343:342–50.

44. Ma PC, Schaefer E, Christensen JG, Salgia R. A selective small molecule c-MET Inhibitor, PHA665752, cooperates with rapamycin. Clin Cancer Res2005;11:2312–9.

45. Ji QS, Mulvihill MJ, Rosenfeld-FranklinM, Cooke A, Feng L, Mak G, et al. Anovel, potent, and selective insulin-like growth factor-I receptor kinaseinhibitor blocks insulin-like growth factor-I receptor signaling in vitro andinhibits insulin-like growth factor-I receptor dependent tumor growth invivo. Mol Cancer Ther 2007;6:2158–67.

46. NelsonV,Altman JK, Platanias LC.Next generation ofmammalian target ofrapamycin inhibitors for the treatment of cancer. Expert Opin InvestigDrugs 2013;22:715–22.

47. BurottoM,Chiou VL, Lee JM, Kohn EC. TheMAPKpathway across differentmalignancies: a new perspective. Cancer 2014;120:3446–56.

48. Zanella ER, Galimi F, Sassi F, Migliardi G, Cottino F, Leto SM, et al. IGF2 isan actionable target that identifies a distinct subpopulation of colorectalcancer patients with marginal response to anti-EGFR therapies. Sci TranslMed 2015;7:272ra12.

49. Cancer Genome Atlas Network. Comprehensive molecular characteriza-tion of human colon and rectal cancer. Nature 2012;487:330–7.


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