Altiratinib Inhibits Tumor Growth, Invasion, Angiogenesis, and Microenvironment-Mediated Drug Resistance Via Balanced Inhibition of MET, TIE2, and VEGFR2 Bryan D

Published OnlineFirst August 18, 2015; DOI: 10.1158/1535-7163.MCT-14-1105

Small Molecule Therapeutics Molecular Cancer Therapeutics Altiratinib Inhibits Tumor Growth, Invasion, Angiogenesis, and Microenvironment-Mediated Drug Resistance via Balanced Inhibition of MET, TIE2, and VEGFR2 Bryan D. Smith1, Michael D. Kaufman1, Cynthia B. Leary1, Benjamin A. Turner1, Scott C. Wise1, Yu Mi Ahn1, R. John Booth1, Timothy M. Caldwell1, Carol L. Ensinger1, Molly M. Hood1, Wei-Ping Lu1, Tristan W. Patt1, William C. Patt1, Thomas J. Rutkoski1, Thiwanka Samarakoon1, Hanumaiah Telikepalli1, Lakshminarayana Vogeti1, Subha Vogeti1, Karen M. Yates1, Lawrence Chun2, Lance J. Stewart2, Michael Clare1, and Daniel L. Flynn1,3

Abstract

Altiratinib (DCC-2701) was designed based on the rationale of wild-type and mutated forms, in vitro and in vivo. Through its engineering a single therapeutic agent able to address multiple balanced inhibitory potency versus MET, TIE2, and VEGFR2, hallmarks of cancer (1). Specifically, altiratinib inhibits not only altiratinib provides an agent that inhibits three major evasive mechanisms of tumor initiation and progression, but also drug (re)vascularization and resistance pathways (HGF, ANG, and resistance mechanisms in the tumor and microenvironment VEGF) and blocks tumor invasion and metastasis. Altiratinib through balanced inhibition of MET, TIE2 (TEK), and VEGFR2 exhibits properties amenable to oral administration and exhibits (KDR) kinases. This profile was achieved by optimizing binding substantial blood–brain barrier penetration, an attribute of into the switch control pocket of all three kinases, inducing type II significance for eventual treatment of brain cancers and brain inactive conformations. Altiratinib durably inhibits MET, both metastases. Mol Cancer Ther; 14(9); 1–12. 2015 AACR.

Introduction predominately produced in the stroma by fibroblasts, but can be produced by tumor cells as well (5). HGF binds to MET- Cancer is recognized as a complex process involving not expressing cancer cells in a paracrine or autocrine fashion to only tumor cell transformation, but also the surrounding cause receptor activation via dimerization and trans-autopho- microenvironment. Referred to as the hallmarks of cancer, this sphorylation (6, 7). The microenvironment can mediate alter- holistic approach to understanding cancer identifies cross-talk native activation of tumor MET receptors by fibronectin, mechanisms between tumor cells and cells of the microenvi- sema4D, or TGFa (8–10). MET amplification has been dem- ronment as essential for tumor growth, invasion, and metas- onstrated to cause constitutive MET activation in glioblastomas tasis (1). New targeted therapeutics that block multiple (11, 12), medulloblastomas (13), NSCLC (14), gastroesopha- hallmark mechanisms of cancer are highly sought. Herein, we geal cancer (15), and colorectal cancer (16). Activating muta- disclose altiratinib as such an agent that addresses multiple tions have also been reported in papillary renal carcinoma (17), mechanisms of tumor and microenvironment-mediated tumor gastric cancer (18), and others (19–21). The HGF–MET axis has growth and progression. also been implicated in microenvironment-mediated drug The HGF–MET axis is involved in the etiology and progres- resistance. Stromal HGF secretion can cause resistance to BRAF sion of many human cancers (2–4). HGF (hepatocyte growth and EGFR inhibitors, as well as anti-VEGF therapy (22–28). factor), the ligand for the MET receptor tyrosine kinase, is Treatment of gliomas with anti-VEGF therapies leads to initial responses followed by hypoxia-induced epithelial-to-mesenchymal transition (EMT), which increases MET-mediated inva- 1Deciphera Pharmaceuticals, Lawrence, Kansas. 2Emerald Bio, Bain- siveness and resistance in glioma cells (25). Anti-VEGF induced 3 bridge Island, Washington. Deciphera Pharmaceuticals, Waltham, hypoxia also leads to HGF/MET activation and resistance in Massachusetts. pancreatic cancer (29). Note: Supplementary data for this article are available at Molecular Cancer Drug resistance has also been demonstrated wherein MET Therapeutics Online (http://mct.aacrjournals.org/). activation elicits rebound vascularization during anti-VEGF Corresponding Author: Daniel L. Flynn, Deciphera Pharmaceuticals, LLC, 1601 therapy. MET is expressed on endothelial cells, and stromal Trapelo Road, Suite 152, Waltham, MA 02451. Phone: 785-830-2115; Fax: 785- secretion of HGF can lead to MET-mediated angiogenesis in the 830-2150; E-mail: dfl[email protected] presence of anti-VEGF therapy (30, 31), known as evasive doi: 10.1158/1535-7163.MCT-14-1105 vascularization. A dramatic example of this has been demon- 2015 American Association for Cancer Research. strated in models of pancreatic neuroendocrine cancer, wherein

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initial efficacy of anti-VEGF therapy provokes HGF/MET-medi- (GraphPad). Kinetic determination of off-rate from MET was ated revascularization and resistance (32, 33). The angiopoietin performed as previously published (42). Michaelis–Menten anal- (ANG)–TIE2 signaling axis on endothelial cells and proangio- ysis using the PK/LDH assay evaluated competitive or noncom- genic macrophages also contributes to tumor vascularization petitive inhibition versus ATP. Kinome-wide profiling was per- and can mediate angiogenesis after anti-VEGF therapy. ANG/ formed at Reaction Biology. TIE2-mediated tumor revascularization has been demonstrated in breast, pancreatic, glioblastoma, and ovarian cancer Cell lines – (34 40). Moreover, combination treatment with anti-VEGF A375 (year of purchase, 2006), A549 (2008), BT-474 (2007), and anti-ANG2 therapy leads to a more durable reduction in CHO-K1 (2009), EA.hy926 (2012), HCT-116 (2006), HMVEC tumor growth in preclinical models (39). (2012), HUVEC (for TIE2 studies; 2012–2015), K562 (2006), The discovery of altiratinib (DCC-2701) was based on the M-NFS-60 (2009), MRC-5 (2013), MV-4-11 (2006), PC-3 rationale of incorporating balanced inhibition of MET, TIE2, (2008), SK-MEL-5 (2007), SK-MEL-28 (2006), SK-N-SH and VEGFR2 kinases within a single therapeutic. This design (2011), THP-1 (2006), and U-87-MG (2008) cells were concept addresses multiple hallmarks of cancer. Although some obtained from the American Type Culture Collection (ATCC). disclosed MET inhibitors have been described to inhibit these EBC-1 (2008) and MKN-45 (2009) cells were obtained from three kinases, inhibition of one kinase predominates, such that the Japan Health Science Research Resources Bank (Osaka, saturating inhibition of one kinase does not allow for achiev- Japan). KM-12 cells (2012) were obtained from the Division able or safe inhibition of all three. We report that altiratinib of Cancer Treatment and Diagnosis Tumor Repository, Nation- in vitro in vivo exhibits balanced inhibition and , and inhibits al Cancer Institute (Frederick, MD). HUVEC cells (for VEGFR2 three major microenvironment (re)vascularization and drug and MET studies; 2009–2014) were obtained from Lonza, Inc. resistance pathways (HGF, VEGF, ANG), allows for pronounced Luciferase-enabled B16/F10 cells (2012) were obtained from MET inhibition in tumors, and blocks tumor invasion and Caliper Life Sciences. All cell lines were cultured as recom- metastasis. mended by the supplier, unless otherwise indicated. Media reagents were purchased from Life Technologies or Lonza, Inc. Materials and Methods All cell lines were passaged fewer than 6 months after resus- citation. The ATCC performs STR analysis for characterization. MET crystallography Cells obtained from the NCI Repository and the Japan Health fi Puri ed, unphosphorylated MET kinase was dialyzed against Science Research Resources Bank were authenticated at DNA 20 mmol/L Tris pH 8.5, 100 mmol/L NaCl, 14 mmol/L 2-mer- Diagnostics Center (Fairfield, OH) using STR profiling. Lucif- – captoethanol for crystallography. MET kinase inhibitor com- erase-transfected B16/F10 cells were generated using cells fi plexes were prepared by preincubating puri ed enzyme samples obtained from ATCC and were not further characterized. overnight with a 5-fold molar excess of inhibitor and concentrat- ing to 9.5 mg/mL for crystallization trials. Crystals of the MET–DP 4157 complex were grown by vapor diffusion against crystalliza- Cell proliferation assays tion conditions of 1.0 mol/L diammonium hydrogen phosphate, Test compound in DMSO was dispensed into assay plates. 0.2 mol/L sodium chloride, 0.1 mol/L citrate pH 5.0, and 7.5% Cells were added to 96-well (EBC-1, M-NFS-60, and SK-MEL-28: glycerol. A complete x-ray diffraction dataset was collected at the 2,500 cells/well; MKN-45: 5,000 cells/well; MV-4-11: 10,000 Advanced Light Source beam line 5.0.2 and integrated and scaled cells/well) or 384-well plates (A375 and HCT-116: 625 cells/well; to 2.6-Å resolution. The structure was solved via Molecular BT-474, KM-12, PC-3, and U-87-MG: 1,250 cells/well). Plates fi Replacement using the PDB entry 2g15 as the search model. were incubated for 72 hours. Viable cells were quanti ed using The data collection and refinement statistics are shown in Sup- resazurin using a plate reader with excitation at 540 nm and plementary Table S1. emission at 600 nm.

Enzyme-linked immunosorbent assays MET modeling studies EBC-1 cells (15,000 cells/well) or MKN-45 cells (25,000 cells/ Models of the MET kinase inhibitors used in these studies were well) were added to 96-well plates in culture media. HUVECs initially constructed from standard molecular fragments, using (250,000 cells/well) were added to 12-well plates in EBM-2 the Tripos SYBYL modeling system. Inhibitor docking trials were media (Lonza, Inc.) containing 2% FBS. Cells were then incu- run using a set of standard macromolecular mechanics para- bated overnight. Diluted compound was added to cells and in- meters, and a modified torsion-only variant of the AMBER mac- cubated for 6 hours. Cells were stimulated with 40 ng/mL HGF romolecular force-field3, YETI4, with suitable electrostatic poten- (R&D Systems) for 10 minutes. For HUVECs, cells were incu- tial fit point charges on the inhibitor models. bated for 4 hours with compound, then stimulated with 200 ng/mL HGF for 15 minutes. Phospho-MET in cell lysates was Kinase assays detected using an ELISA (R&D Systems). Phospho-VEGFR2 Kinase activity was determined using the pyruvate kinase/ ELISAs were performed as above, except HUVECs were plated lactate dehydrogenase (PK/LDH) system (41). Kinases are in 96-well plates (25,000 cells/well). Cells were incubated over- described in Supplementary Table S2. Assay mixtures were mixed night, and compound was then added for 4 hours. Cells were with test compound. ATP was added to start the reaction imme- stimulated with 100 ng/mL VEGF (VEGFA) (R&D Systems) for diately or following preincubation. The absorbance at 340 nm 5 minutes. Phospho-VEGFR2 in cell lysates was detected using was measured at 30C. Reaction rates were compared with an ELISA (R&D Systems). For the phospho-FMS (CSF-1R) ELISA, controls and IC50 values were calculated using Prism software THP-1 cells (150,000 cells/well) were added to a 96-well plate

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containing compound. Cells were incubated for 4 hours and well. The cell/compound suspensions were then transferred to then stimulated with 25 ng/mL MCSF (CSF1) (R&D Systems) the Matrigel plate wells and incubated overnight. The next day, for 5 minutes. Phospho-FMS in cell lysates was detected using cells were stained with Calcein-AM dye (Life Technologies). an anti-FMS capture antibody (R&D Systems) and an anti- Images obtained via ﬂuorescent microscopy were analyzed for phospho-tyrosine antibody conjugated to horseradish peroxi- tube length using ImagePro Analyzer (Media Cybernetics) with dase (Life Technologies). a macro to automatically detect and measure tube formation.

A549 cell migration assays HGF-stimulated melanoma cell assays A549 cells (40,000 cells/well) were added to a 96-well Oris SK-MEL-5 and SK-MEL-28 cells were added to 12-well plates at Collagen-Coated plate containing cell seeding stoppers (Platypus 450,000 and 250,000 cells/well, respectively, and incubated Technologies). After overnight incubation, stoppers were overnight. Compounds were added and plates incubated for removed, exposing an area for cell migration in the center of each 4 hours. Cells were then stimulated with 50 ng/mL HGF for well. Diluted compound in DMEM/0.5% FBS was added. After a 1 hour. Antibodies were obtained from Cell Signaling Technol- 4-hour incubation, 40 ng/mL HGF was added, and cells were ogy. HGF-stimulated proliferation assays were performed as listed incubated for 48 hours. Calcein-AM (Life Technologies) was in the cell proliferation assay methods, except that SK-MEL-28 added to the plate for 20 minutes to fluorescently labeled cells. cells were added to assay plates in complete media, media con- An Oris plate mask that exposes only the area for cell migration taining 50 ng/mL HGF, or media mixed 1:1 with MRC-5 fibro- was attached to the plate and fluorescence was detected using a blast-conditioned media. plate reader.

fi Western blot assays Mouse xenograft ef cacy models Cells (B16/F10 and CHO K1: 100,000 cells/well; U-87: All procedures were performed in compliance with all the 125,000 cells/well; A549, KM-12, EA.hy926, SK-N-SH, and laws, regulations, and guidelines of the NIH and with the HUVEC: 250,000 cells/well) were added to 24-well plates and approval of the Animal Care and Use Committee of Molecular incubated overnight. SK-N-SH cells [differentiated with Imaging, Inc. (Ann Arbor, MI), an AAALAC accredited facility. 10 mmol/L all-trans retinoic acid (ATRA)] were added to 12- For the A375 xenograft study, female nude mice were inocu- well plates at 500,000 cells/well. K562 (1,000,000 cells/well in lated subcutaneously with 5 million cells. When tumor burdens serum-free media) were added to plates directly prior to com- reached 121 mg on average on day 8, mice were randomly pound addition. CHO cells were transfected with 0.5 mgof assigned into groups of 10 and were treated with 0.4% hydro- plasmid (TIE2 cloned into the pcDNA3.2/V5-DEST expression xypropylmethylcellulose (HPMC) vehicle or test compound for vector) using Lipofectamine LTX for 18 hours. For A549, 28 days. Tumor sections were stained with CD31 antibody at EA.hy926, HUVECs, and ATRA-differentiated SK-N-SH cells, Premier Laboratory LLC. Images were obtained and analyzed serum-free media was added. RPMI-1640/10% FBS/1% NEAA with a macro for microvessel area using ImagePro Analyzer 7.0 fi was added to TIE2-transfected CHO cells. Diluted compound (Media Cybernetics, Inc.). The rst MKN-45 xenograft study was added and cells were incubated for 4 hours. A549 cells were was performed as above, except treatments began on day 7, stimulated with 40 ng/mL HGF for 15 minutes. HUVECs and when the mean tumor burden was 195 mg, and continued for EA.hy926 cells were stimulated with 800 ng/mL ANG-1 14 days. In a second MKN-45 xenograft study, treatments began (ANGPT1) (R&D Systems) multimerized with an anti-polyhis- on day 7, when the mean tumor burden was 151 mg and tidine antibody (R&D Systems) for 15 minutes. K562 and SK- continued for 21 days. For the B16/F10 study, female C57BL/6 N-SH cells were stimulated with 100 ng/mL NGF (R&D Sys- mice were implanted subcutaneously in the right lower back tems) for 10 minutes. ATRA-differentiated SK-N-SH cells were with one million luciferase-enabled B16/F10 cells. Treatments stimulated with 200 ng/mL BDNF (R&D Systems) for 5 min- began 1 hour prior to cell implant, and continued for 21 days. Ex vivo utes. Cells were lysed and equal protein amounts were sepa- bioluminescence imaging was performed on the lungs of ratedbySDS-PAGEandtransferredtoPVDF.Blotswereprobed all animals on day 21. Animals were injected intraperitoneally and bands were detected using ECL Plus (Pierce). Band (i.p.) with 150 mg/kg D-Luciferin and then euthanized. The volumes were quantified using the ImageQuant software (GE lungs of each animal were placed in a black plate containing m Healthcare). Antibodies against phospho-MET Tyr1234/1235, 300 g/mL D-Luciferin. Images were analyzed using Living total MET, phospho-TIE2 Tyr992, phosphoTRKA/B Tyr674/ Image software (Xenogen). 675, and rabbit and mouse IgG (HRP-conjugated) were obtained from Cell Signaling Technology. Antibodies against MKN-45 xenograft mouse pharmacokinetic/pharmacodynamic total TIE2, TRKA (NTRK1), and TRKB (NTRK2) were obtained model from Santa Cruz Biotechnology, Inc. Female nude mice were inoculated subcutaneously as above. On days 9 to 10, when tumor volumes reached 326 mg on Tube formation assays average, mice were randomly assigned to groups and dosed Growth factor reduced Matrigel gel solution (BD Bio- once orally with 0.4% HMPC, (n ¼ 3); altiratinib at 30 mg/kg sciences) was added to a tissue-culture treated plate. In another (n ¼ 21); or altiratinib at 10 mg/kg (n ¼ 21). At specified time 96-well plate, test compound was spotted into each well, fol- points, whole blood and tumors were collected. Pharmacoki- lowed by the addition of HMVECs (15,000 cells/well in serum- netic analysis was performed at Xenometrics, LLC. Tumor free EBM-2 media; in the presence or absence of 200 ng/mL samples were processed as described in the Western blot assay ANG2 (ANGPT2), 40 ng/mL HGF, or 100 ng/mL VEGF) to each methods.

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Orthotopic U87-MG xenograft efficacy model tumor fragments from MMTV-PyMT donor mice. Treatments Female nude mice were implanted intracranially with one began on day 31 when the mean tumor burden in the experiment million luciferase-enabled U87-MG cells. Treatments began on was 843 milligrams. Mice (n ¼ 10/group) were dosed for 3 weeks, day 31. Brain BLI signal was determined on day 45 using an IVIS then primary tumor and lungs were collected and fixed. Lung 50 optical imaging system (Xenogen). Animals were injected i.p. sections were stained with H&E at Premier Laboratory. Lung with 150 mg/kg D-Luciferin and imaged 10 minutes after metastases (10 cells) were counted manually via microscopy. injection. Data were analyzed via one-way analysis of variance (ANOVA), with post hoc analysis by the method of Holm–Sidak. For analysis þ of TIE2 macrophages, tumor sections were stained with TIE2 Orthotopic U87-MG xenograft survival model þ Female nude mice were implanted as above. Treatments began and CD31 antibodies at Premier Laboratory. The density of TIE2 on day 12. Mice (n ¼ 10/group) were dosed until the end of study. cells at the stroma/tumor boundary in each tumor was scored Three additional mice were treated as above for pharmacody- using 0, no staining; 1, low; 2, medium; and 3, high. namic analysis after 5 weeks. Results Flow cytometry of TIE2/MET-expressing monocytes Drug design and structural biology Peripheral blood samples were fixed using Lyse/Fix buffer (BD Altiratinib is a type II "switch control pocket" kinase inhibitor Biosciences). BD Fc Block was added and cells were stained (Fig. 1A). A kinase activation loop ("switch") fluxes between on with CD11b (ITGAM)-PECy7 and Gr1 (LY6C/G)-APC antibodies and off conformations through binding into or displacement (BD Pharmingen), and TIE2-PE and MET-FITC antibodies from an embedded complementary switch pocket (43). The (eBioscience) or isotype controls. Data were collected on an Accuri conformational flux controlling the "kinase on" conformation is C6 cytometer. Monocytes were gated using side and forward intercepted by a switch control pocket inhibitor effectively out- þ scatter. CD11b /Gr1 cells were gated, and TIE2-positive and competing the activation loop for binding this deep allosteric MET-positive cells were quantified. switch pocket. Supplementary Fig. S1A illustrates these concepts with the altiratinib analogue DP-4157, demarking the MET kinase PyMT syngeneic breast cancer model embedded switch (green ribbon), its cognate switch control Female FVB/NJ mice were implanted in the fourth mammary pocket (dashed green oval), and the enzymatic ATP and substrate fat pad with one million cells that had been dissociated from pockets (dashed white ovals). Occupancy of the switch pocket by

Figure 1. A, chemical structure of altiratinib. B, docked structure of altiratinib into MET kinase. C, view of the docked structure highlighting deep penetration of altiratinib's para- ﬂuorophenyl ring into the switch pocket (yellow residues). Hydrogen bonds are highlighted by dashed lines between altiratinib and switch pocket residues K1110, E1127, and D1222. Altiratinib additionally forms two hydrogen bonds with the kinase hinge region (M1160).

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DP-4157 forces the switch into the ATP and substrate pockets, AMG-208, tivantinib) MET inhibitors (Supplementary Fig. S2). conformationally blocking MET kinase activity. Switch control Altiratinib exhibited an off-rate of 0.0067 per minute, residency pocket inhibitors represent an evolution of classical type II kinase T1/2 of 103 minutes, and a Kd ¼ 0.4 nmol/L for MET (Supple- inhibitors (43). mentary Fig. S3), consistent with its binding mode deep into the MET was cocrystallized with DP-4157 (Supplementary Fig. S1 switch pocket. Michaelis–Menten analysis of altiratinib exhibited and Supplementary Table S1). A docked structure of altiratinib kinetic readouts indicative of tight-binding type II kinase inhibi- into MET is shown in Fig. 1B and C. DP-4157 induces a type II tion: increasing concentrations of altiratinib led to decreases in inactive MET conformation (Supplementary Fig. S1A). Com- Vmax even at 5,000 mmol/L ATP, demonstrating that inhibition is pared with other structures of type II inhibitors bound to MET, not surmountable by ATP (Supplementary Fig. S4). Altiratinib there is more complete electron density for all but one residue inhibited TIE2 kinase with an IC50 of 8.0 nmol/L and VEGFR2 in the switch ligand (green ribbon), confirming that DP-4157 kinase with an IC50 of 9.2 nmol/L (Table 1). Altiratinib was (and by analogy altiratinib) exerts a profound effect on MET to profiled in an internal panel of 14 additional kinases (Table 1). adopt a type II inactive conformation. Tyrosines Y1234/Y1235 Altiratinib also potently inhibited TRKA, TRKB, and TRKC (highlighted in yellow), residues that control switch conforma- (NTRK3) kinases with IC50 values of 0.85 nmol/L, 4.6 nmol/L, tion, are sequestered by p-stacking interactions of aromatic and 0.83 nmol/L, respectively. Altiratinib was >10-fold selective residues (Y1234, F1223, and the inhibitor difluorinated phenyl for MET versus FMS and KIT, and >50-fold selective for MET ring) and by hydrogen bonds between the hydroxyl moiety of versus ABL1, FYN, HER1 (EGFR), p38a (MAPK14), PDGFRa, Y1235 and aspartic acid D1228 and between the hydroxyl of PDGFRb, RET, and SRC. Altiratinib was subsequently profiled Y1234 and aspartic acid D1164 (Fig. 1B and Supplementary against 295 human kinases (Reaction Biology) and demonstrated Fig. S1A). These interactions effectively occlude key volumes of >50-fold selectivity against 273 kinases (Supplementary Table S3; both ATP and substrate pockets. Supplementary Fig. S1B high- Supplementary Fig. S5). Kinases inhibited within 50-fold of lights key hydrogen bonds formed between inhibitor and the MET include the MET family kinases SKY (TYRO3), AXL, and switch pocket region (residues D1222, K1110, and E1127) and MER (MERTK), as well as RAF kinases, a subset of ephrins, BRK the hinge region of MET kinase (residue M1160). Finally, (PTK6), DDR2, and HIPK4. This kinome-wide profiling confirm- Supplementary Fig. S1C illustrates the para-F phenyl ring of ed the potency of altiratinib versus TRK kinases. Certain kinases the inhibitor binding deeply into the hydrophobic switch pocket were further probed in cellular assays. Whereas MET and TRK were region adjacent to the a-C helix, precluding the MET switch from potently inhibited by altiratinib in cellular assays, RAF kinases occupying this region. were only weakly inhibited.

Inhibition of recombinant kinases Inhibition of tumor cell kinases Altiratinib inhibited MET kinase activity with an IC50 value of EBC-1 (NSCLC) and MKN-45 (gastric cancer) human cancer 2.7 nmol/L. Activating oncogenic MET mutations in the switch cell lines genomically amplify and overexpress MET with subse- region (residues 1228, 1230, and 1250; ref. 17) were also potently quent ligand-independent MET activation. Altiratinib inhibited inhibited by altiratinib (Table 1). This proﬁle was superior to other MET phosphorylation with IC50 values of 0.85 nmol/L and type II (cabozantinib, E-7050) and type I (crizotinib, PF-4217903, 2.2 nmol/L, respectively (Table 2-A). In the U-87 glioblastoma cell line, MET and HGF are both expressed. Altiratinib blocked autocrine activation of MET phosphorylation in these cells (IC50 Table 1. Kinase inhibition proﬁle of altiratinib of 6.2 nmol/L, Table 2-A; ref. 44). Kinase IC50, nmol/L Altiratinib was evaluated for inhibition of NGF-stimulated MET 2.7 0.7 TRKA phosphorylation in K562 and SK-N-SH cancer cells, and MET D1228H 3.6 0.5 for inhibition of constitutive TRKA phosphorylation in KM-12 MET D1228N 1.3 0.4 MET Y1230C 1.2 1.1 cancer cells, which have an oncogenic TPM3-TRKA fusion. Altir- MET Y1230D 0.37 0.28 atinib exhibited IC50s of 0.69 nmol/L in K562 cells, 1.2 nmol/L MET Y1230H 1.5 0.4 in SK-N-SH cells, and 1.4 nmol/L in KM-12 cells (Table 2). In MET M1250T 6.0 0.3 SK-N-SH cells differentiated by treatment with ATRA, altiratinib TIE2 8.0 1.7 inhibited TRKB phosphorylation with an IC50 of 0.24 nmol/L. VEGFR2 9.2 3.3 TRKA 0.85 0.22 TRKB 4.6 0.4 Inhibition of tumor microenvironment cell kinases TRKC 0.83 0.39 MET, TIE2, and VEGFR2 are highly expressed on stromal cells FLT-3 9.3 FMS 32 of the tumor microenvironment, especially on endothelial KIT 68 cells (45). Altiratinib inhibited HGF-stimulated MET phosphor- ABL1 215 ylation in HUVECs, exhibiting an IC50 of 2.3 nmol/L (Table 2-B). PDGFRa 340 In ANG1-stimulated HUVECs and EA.hy926 cells, altiratinib p38a 392 exhibited IC50 values of 1.0 nmol/L and 2.6 nmol/L, respectively, SRC 424 for inhibition of TIE2 phosphorylation. In VEGF-stimulated RET 1,400 PDGFRb 1,600 HUVECs, altiratinib inhibited VEGFR2 phosphorylation with an HER1 >3,300 IC50 of 4.7 nmol/L. Altiratinib exhibited a balanced potency for FYN >5,000 inhibiting these multiple angiogenic signaling pathways com-

NOTE: IC50 values were determined as described in experimental procedures vs. pared with other multitargeted MET inhibitors E-7050, cabozan- puriﬁed kinase domains. tinib, and MGCD-265 (Supplementary Fig. S6).

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Table 2. Altiratinib inhibition in cellular assays

Kinase Cell line Phosphorylation IC50, nmol/L Proliferation IC50, nmol/L Other IC50, nmol/L A. Tumor cell MET EBC-1 0.85 0.36 1.9 0.4 MET MKN-45 2.2 1.2 5.1 0.5 MET U87 6.2 n ¼ 1 5,750 1,070a MET A549 1,360 240a 13 6d TRKA K562 0.69 n ¼ 2 TRKA SK-N-SH 1.2 0.7 TRKA KM-12 1.4 n ¼ 2 3.8 1.8 TRKB SK-N-SHb 0.24 0.14 FLT3 MV-4-11 12.1 1.4 B. Microenvironment MET HUVEC 2.3 1.1 MET HMVEC 11 10c TIE2 HUVEC 1.0 0.8 TIE2 EA.hy926 2.6 n ¼ 2 TIE2 HMVEC 7.1 5.9e TIE2 CHO 2.4 n ¼ 2 VEGFR2 HUVEC 4.7 0.9 VEGFR2 HMVEC 58 42f FMS THP-1 79 30 FMS M-NFS-60 770 220 aDriver unknown or multiple drivers. bSK-N-SH cells were differentiated with all-trans retinoic acid prior to assay to induce TRKB expression. cHGF-stimulated capillary tube formation. dHGF-stimulated motility assay. eANG2-stimulated capillary tube formation. fVEGF-stimulated capillary tube formation.

Prolonged off-rates in cellular studies melanoma cells (Fig. 2C, lane 3). However, upon HGF stim- Altiratinib exhibited a prolonged off-rate from TIE2 in CHO ulation, mimicking stromal microenvironment resistance (22, cells. After cells were incubated with 1 mmol/L altiratinib for 2 23), ERK remained phosphorylated despite the presence of hours and then washed out by exchanging media, altiratinib dabrafenib (Fig. 2C, lane 7). Altiratinib inhibited activation inhibited TIE2 phosphorylation for 24 hours (Fig. 2A). In KM- of MET and AKT (AKT1) (Fig. 2C, lane 6), and the addition of 12 cells incubated with 0.5 mmol/L altiratinib for 2 hours prior to altiratinibtodabrafenibrestoredcompleteinhibitionofERK wash out, TRKA phosphorylation was inhibited by >80% for 24 phosphorylation (Fig. 2C, lane 8). Dabrafenib potently inhib- hours (Fig. 2B). ited proliferation of the SK-MEL 28 mutant BRAF cell line (representative data in Supplementary Fig. S8A). When HGF fi Functional inhibition in cell assays or broblast-conditioned media containing HGF was added, Altiratinib potently inhibited cellular proliferation in MET- dabrafenib was rendered ineffective (Supplementary Fig. S8B amplifiedEBC-1andMKN-45cells,aswellasTPM3–TRKA and S8C). Altiratinib as a single agent did not inhibit prolif- fusion KM-12 cells (Table 2-A). Activation of MET is known to eration of SK-MEL 28 cells in the absence of stromal HGF, as increase the motility and invasiveness of cancer cells: altiratinib expected (Supplementary Fig. S8D); however, the addition of inhibited HGF-induced A549 cell migration, with an IC of altiratinib (50 nmol/L) restored sensitivity to dabrafenib in the 50 fi 13 nmol/L (Table 2-A). Altiratinib also inhibited FLT3-ITD presence of HGF or broblast-conditioned media (Supplemen- tary Fig. S8E and S8F). Similarly, trametinib (MEK inhibitor) mutant MV-4-11 cell proliferation with an IC50 of 12 nmol/L, correlating with enzyme data (Table 2-A). Altiratinib only exhibited nanomolar inhibition of SK-MEL-28 proliferation weakly inhibited other cancer cell lines, including proliferation (Supplementary Fig. S8G). In the presence of HGF, trametinib lost approximately 10- to 40-fold potency (representative data of M-NFS-60 (IC50, 770 nmol/L); A375, BT-474, HCT-116, PC- in Supplementary Fig. S8H). Altiratinib restored sensitivity to 3, SK-MEL-28, U87, and A549 cells (IC50s > 1,000 nmol/L), indicating a lack of nonspecific cytotoxicity, or potency in cell trametinib (Supplementary Fig. S8I). lines driven by mutant BRAF or Ras. Pharmacokinetic/pharmacodynamic evaluation in the MKN-45 xenograft model Functional inhibition of capillary tube formation Altiratinib was evaluated in the MET-amplified MKN-45 When stimulated with growth factors, endothelial cells can be xenograft model to determine the pharmacokinetic/pharmaco- in vitro induced to form capillaries . Altiratinib inhibited HMVEC dynamic relationship for in vivo MET target inhibition. A single capillary tube formation with upon stimulation with ANG2, HGF, oral dose of 30 mg/kg altiratinib led to >95% inhibition of and VEGF, respectively (Table 2-B, Supplementary Fig. S7). MET phosphorylation for the entire 24-hour period (Supple- mentary Table S4A). A single 10 mg/kg oral dose of altiratinib Reversal of BRAF therapy resistance mediated by stromal HGF (Fig. 2D; Supplementary Table S4B) exhibited complete inhi- Dabrafenib (BRAF inhibitor) inhibited phosphorylation of bition of MET phosphorylation through 12 hours and 73% the downstream RAF effector ERK in BRAFV600E SK-MEL-5 inhibition at 24 hours postdose [factoring in plasma protein

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Figure 2. A and B, inhibition of phosphorylated TIE2 (pTIE2) or phosphorylated TRKA (pTRKA) in CHO cells, depicting percentage inhibition of pTIE2 or pTRKA at various time points after washout of altiratinib. C, altiratinib inhibition of cellular proliferation and survival pathways in the B-RAF V600E SK-MEL-5 melanoma cell line stimulated with HGF and restoration of sensitivity to dabrafenib. D, altiratinib inhibition of MET phosphorylation after a single oral dose of 10 mg/kg in the MKN-45 xenograft pharmacodynamic model. E and F, efﬁcacy in the MKN-45 gastric cancer xenograft model. binding (98.7% bound)], the free drug concentrations required Efﬁcacy in the MKN-45 xenograft model for MET inhibition correlated with in vitro results (IC50 1.1 When altiratinib was administered at 10 mg/kg BID orally, the ng/mL ¼ 2.2 nmol/L; Table 2). mean tumor burden of treated versus control animals on day 14

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(% T/C) was 16% (P < 0.05), and T-C (median tumor growth 10 mg/kg, and altiratinib was also administered in combination delay of treated versus control animals) was 23 days (P < 0.001; with bevacizumab on the same dosing schedule. Treatment began Fig. 2E). Tumor regressions were observed in 90% of mice in the on day 12, and continued until the end of study. The median altiratinib-treated cohort at the end of dosing. A second MKN-45 survival of vehicle-treated mice was 66.5 days (black); survival of xenograft study explored lower doses of altiratinib. Mice were the bevacizumab cohort was 88 days (red, P ¼ 0.0013). In com- dosed orally for 21 days. Day 24 (% T/C) values were 31% parison, survival of altiratinib-treated mice was 112 days (blue, (P < 0.05) and 39% (P < 0.05), and median T-C values were P ¼ 0.0047), and in combination with bevacizumab, median 19 (P < 0.001) and 12 days (P ¼ 0.002), for the 10 mg/kg QD and survival further increased to 166 days (magenta, P < 0.0001 vs. 5 mg/kg BID dosing regimens, respectively (Fig. 2F). vehicle; P ¼ 0.016 vs bevacizumab single agent; Fig. 3C).

Evaluation in the U87 glioma model alone and in combination Altiratinib in combination with bevacizumab decreases with bevacizumab þ Altiratinib was evaluated alone and in combination with bev- circulating TIE2 -expressing monocytes It has been demonstrated that certain cancers, including mel- acizumab in the U87-MG xenograft glioma model. Tumor cells þ were injected intracerebroventricularly (ICV) and tumor growth anomas and gliomas, lead to an increased circulation of TIE2 was monitored in vivo by quantitation of luciferase-mediated BLI. circulating monocytes and their recruitment to distal metastatic Bevacizumab treatment (5 mg/kg i.p. every 3 days) resulted in a sites (46) or to their recruitment to anti-VEGF treated gliomas fi (40). To monitor these monocytes in the U87 glioma model, statistically insigni cant 63% decrease in BLI signal compared with þ peripheral monocytes were gated on CD11b /Gr1 surface mar- vehicle after 2 weeks of treatment. Altiratinib dosed at 10 mg/kg þ þ þ BID led to a significant 90% decrease in BLI signal (P ¼ 0.0013). kers and TIE2 and/or TIE2 /MET cells quantitated. Altiratinib Furthermore, the combination of altiratinib with bevacizumab and bevacizumab as single agents did not decrease these circu- > P ¼ lating monocyte populations. However, combination treatment resulted in a 90% decrease in BLI signal ( 0.0005; Fig. 3A). þ resulted in a 40% decrease in circulating TIE2 monocytes (P ¼ þ þ Altiratinib alone and in combination with bevacizumab 0.08) and an 80% decrease in proangiogenic TIE2 /MET mono- increases survival in the U87 glioma model cytes (P ¼ 0.005, Fig. 3B). The lowered population of both of these þ In this study, bevacizumab was dosed at 10 mg/kg intraperi- TIE2 monocyte populations was consistent with greater survival toneally twice weekly, altiratinib was dosed twice daily orally at benefit (Fig. 3C).

A B

250 10

200 8

150 6

100 photons/sec) 4 6

50 cells of All %

(x 10 2 Mean luciferase signal 0 0 g b a e b v l ev hicle g/k b.i.d. c ini e m g zum hi B t Figure 3. V i ra + Be c Ve ti b Al A, evaluation of tumor volume after mg/k eva tini umab 5 b + 2 weeks of treatment in the orthotopic iz ltira ac inib 10 A U-87-MG glioma model. Tumor volume ev t + – + B ira CD11b /Gr1 /TIE2 lt tiratinib was quantitated by mean luciferase A l + – + + A CD11b /Gr1 /TIE2 /MET signal. B, FLOW quantitation of þ circulating TIE2 (open bars) and þ þ TIE2 /MET (black bars) monocytes C after 5 weeks of treatment. C, Kaplan–Meier survival plot in the U-87-MG ICV xenograft model orthotopic U-87-MG glioma model. Vehicle 100 90 Bevacizumab 10 mg/kg twice weekly i.p. 80 Altiratinib 10 mg/kg b.i.d. p.o. 70 Altiratinib 10 mg/kg b.i.d. p.o. 60 + bevacizumab 10 mg/kg twice weekly i.p. 50 40 30

Percent survival Percent 20 10 0 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 Time (days)

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Altiratinib alone and in combination with paclitaxel inhibits with vehicle, paclitaxel (10 mg/kg i.v. every 5 days), altiratinib þ PyMT mammary tumor growth, reduces tumoral TIE2 stromal 15 mg/kg orally twice daily, or a combination for 3 weeks. cell density, and inhibits lung metastasis Tumor growth inhibition (TGI) was determined to be 42% for Altiratinib was evaluated in the PyMT syngeneic mammary the paclitaxel cohort (P ¼ 0.013), 69% for altiratinib (P ¼ 0.002), tumor model, a model that recapitulates many features of human and 89% for the combination (P < 0.0001; Fig. 4A). Altiratinib breast cancer (47). This model has been shown to exhibit a alone and in combination with paclitaxel reduced the intratu- signiﬁcant TIE2 stromal cellular contribution to tumor growth, moral TIE2 score by 50% (P ¼ 0.043) and 62% (P ¼ 0.015), invasiveness, and metastasis (48). After implanting PyMT cells in respectively (Fig. 4B). Altiratinib reduced lung metastases by the mammary fat pad and allowing primary tumors to reach 74% (P ¼ 0.012), and the combination reduced lung metastases approximately 850 mg, cohorts were randomized and treated similarly by 64% (P ¼ 0.020; Fig. 4C).

Figure 4. A, primary tumor growth in the PyMT breast cancer model treated with vehicle, paclitaxel (10 mg/kg i.v. every 5 days), altiratinib 15 mg/kg twice daily, or combination. B, quantitation of TIE2 cellular content within the primary tumor. C, quantitation of lung metastases. D, primary tumor growth in the A375 melanoma xenograft model. E, quantiﬁcation of tumor vascularization determined with anti-CD31 antibody.

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Antiangiogenic efficacy in vivo cross-talk with the microenvironment. Altiratinib blocked stro- Altiratinib was also evaluated for efficacy in the A375 BRAF- mal HGF-mediated resistance to dabrafenib, restoring sensitivity V600E xenograft model (Fig. 4D). Although altiratinib only to drug treatment as determined by inhibition of melanoma cell weakly inhibited BRAF-V600E cell proliferation, its pan anti- proliferation and blockade of the cellular ERK kinase pathway. angiogenic properties made it a candidate for evaluation of Moreover, altiratinib blocked activation of the AKT signaling effects on tumor vascularization. Treatment began on day 8 and pathway caused by stromal HGF activation of melanoma tumor continued for 4 weeks. Altiratinib (20 mg/kg, PO, QD) pro- cell MET receptors. In a second model of stromal microenviron- duced a tumor growth delay of 15.7 days (P < 0.001), and a day ment-induced MET activation, altiratinib was evaluated in the 22 % T/C of 33% (P < 0.05). Altiratinib (10 mg/kg, PO, BID) orthotopic U87-MG glioblastoma model. Bevacizumab offers ledtotumorgrowthdelayof>12.4 days (P < 0.001) and a day temporary improvement in glioma patients but eventually results 22% T/C value of 47% (P < 0.05). Inhibition of tumor growth in tumor progression/invasion that has been linked to therapy- þ correlated with significant decreases in CD31 microvessel area induced MET activation and evasive revascularization (24–26) in tumor sections (Fig. 4E). and to anti-VEGF–mediated infiltration of protumoral TIE2- expressing monocytes (40). Altiratinib resulted in superior reduc- Distribution of altiratinib into brain tissue tion in tumor volume compared with bevacizumab, and in a The ability of altiratinib to penetrate the CNS was evaluated survival study, altiratinib combined with bevacizumab led to a in mice. Altiratinib was found to achieve a brain:plasma ratio 2-fold increase in survival compared with bevacizumab alone. of 0.23 after systemic dosing, indicating significant penetra- Significantly, 75% of altiratinib-treated mice in the combination tion of the murine blood–brain barrier (Supplementary cohort surviving to day 200 study endpoint were demonstrated Fig. S9). to be tumor-free survivors. Thus, there is rationale for clinical evaluation of altiratinib in glioma settings that are characterized by bevacizumab therapy-induced MET and/or TIE2 activation. Discussion Further support for clinical evaluation in GBM is the significant The discovery of altiratinib was based on the rationale of blood–brain barrier penetration of altiratinib. incorporating balanced inhibition of MET, TIE2, and VEGFR2 Despite the clinical success of anti-VEGF therapy, there are kinases within a single therapeutic. This design concept increasing data supporting the concept of "evasive vasculari- addresses multiple hallmarks of cancer (1), including cancer zation," wherein alternative signaling pathways are coopted by cell mechanisms of MET-mediated tumor initiation and pro- tumors for (re)vascularization. Thus, while initial treatment gression, and microenvironment mechanisms driven by MET, results in a reduction in tumor vascularization, anti-VEGF TIE2, and VEGFR2, including angiogenesis, invasion, metasta- therapy can result in revascularization via alternative mechan- sis, and inflammation. isms (31–39), including those mediated by activation of endo- Altiratinib is a potent type II switch pocket inhibitor of MET, thelial MET or TIE2 receptors. Altiratinib exhibited equipotent binding to the kinase in an inactive conformation. The exten- nanomolar inhibition of VEGFR2, MET, and TIE2 in a stan- sive interactions in the buried switch pocket of MET and the dardized HUVEC assay. This balanced inhibition of multiple cellular resiliency are consistent with the potent KD and slow angiogenic pathways was superior to other clinically active off-rate versus MET. Michaelis–Menten analysis of altiratinib multi-targeted MET/VEGFR2 inhibitors. In addition, altiratinib demonstrated an allosteric type II inhibition with respect to blocked capillary tube formation stimulated by VEGFA, HGF, ATP. Increasing concentrations of altiratinib led to decreases in or ANG2. Inhibition of microvessel density was demonstrated Vmax even at 5,000 mmol/L ATP, demonstrating that inhibition in vivo in the A375 tumor xenograft model. is not surmountable by ATP. The XRay cocrystal structure of TIE2-expressing macrophages are a highly skewed M2 mac- the analogue DP-4157 reveals significant electron density rophage phenotype that promote tumor growth, invasion, of the MET switch (activation loop) in an induced "kinase vascularization, and metastasis. In vivo depletion of TIE2 mono- off" conformation. Altiratinib was designed to effectively cytes/macrophages was demonstrated in two preclinical mod- outcompete the MET activation loop for binding to this els: (i) in the U87-MG orthotopic glioma model, altiratinib activating allosteric switch pocket. A critical test of this attribute in combination with bevacizumab led to a 80% decrease in þ is that altiratinib reverses the process of conformational activa- circulating proangiogenic TIE2 monocytes. Interestingly this tion brought about by aggressive activation loop phosphory- combination cohort also afforded the greatest survival benefit. lation or mutation. Indeed, altiratinib exhibited potent The significance of TIE2-expressing monocyte infiltration into inhibition of MET phosphorylation and proliferation in assays gliomas treated with anti-VEGF therapy has been recently characterized by MET genomic amplification, and exhibited reported (40); (ii) in the PyMT mammary tumor model, altir- subnanomolar or low nanomolar potency when evaluated in a atinib alone or in combination with paclitaxel led to a sign- þ battery of MET activating switch mutations. Altiratinib was ificant reduction in intratumoral TIE2 cells and also to a further characterized in vivo in the MET-amplified MKN-45 reductioninbothprimarytumorvolumeandlungmetastases. gastric cancer xenograft model. A single oral dose of 10 mg/ Thus, altiratinib exhibits pharmacologic effects on the levels kg afforded complete inhibition of MET activation for 12 hours, or recruitment of TIE2-expressing monocytes/macrophages, with 73% inhibition maintained through 24 hours. This dose which may have relevance in human cancers. of altiratinib led to MKN-45 tumor regressions in a multi-day In addition to inhibiting MET, TIE2, and VEGFR2, altiratinib dosing study. exhibited potent inhibition of the TRK kinase family (TRK-A, -B, In addition to its inhibition of cancer cell autonomous growth and -C). TRK-A has been shown to be expressed in pancreatic and survival driven by MET, altiratinib was evaluated in a variety ductal adenocarcinoma cells and to be involved in perineural of studies wherein tumor growth and progression were driven by invasion (49). In addition, TRK fusions have been reported in

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many cancers, including thyroid, lung adenocarcinoma, gliob- Acquisition of data (provided animals, acquired and managed patients, lastoma, and fibrosarcoma (50). Altiratinib also potently inhib- provided facilities, etc.): B.D. Smith, C.B. Leary, B.A. Turner, T.M. Caldwell, ited cellular FLT-3 in the MV-4-11 AML cell line, indicating M.M. Hood, W.P. Lu, T.W. Patt, T.J. Rutkoski, L. Chun Analysis and interpretation of data (e.g., statistical analysis, biostatistics, potential utility in acute myeloid leukemia driven by FLT-3 computational analysis): B.D. Smith, M.D. Kaufman, C.B. Leary, B.A. Turner, alterations. M.M. Hood, W.P. Lu, T.W. Patt, T.J. Rutkoski, H. Telikepalli, L. Chun, M. Clare, In conclusion, altiratinib is a robust inhibitor of MET, D.L. Flynn including activating mutant forms. It exhibits robust pharma- Writing, review, and/or revision of the manuscript: B.D. Smith, M.D. cology in tumor models driven by genomic MET mutation as Kaufman, T.M. Caldwell, H. Telikepalli, L. Chun, D.L. Flynn well as in models of microenvironment activation of MET. Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): T. Samarakoon, H. Telikepalli, D.L. Flynn Altiratinib exhibits balanced inhibition of three kinase mechan- Study supervision: B.D. Smith, S.C. Wise, L.J. Stewart, D.L. Flynn isms (MET, TIE2, VEGFR2) known to be relevant in tumor Other (synthesis): Y.M. Ahn evasive vascularization, and also leads to reduction in circu- Other (chemistry support and oversight of synthesis of preclinical batches þ lating or tumoral TIE2 stromal cell populations in cancer at CMO): C.L. Ensinger models. Acknowledgments The authors thank Mike Taylor, Oliver Rosen, and James Bristol for insightful Disclosure of Potential Conflicts of Interest suggestions during the preparation of this article. No potential conflicts of interest were disclosed. Grant Support This study was funded by Deciphera Pharmaceuticals. Authors' Contributions The costs of publication of this article were defrayed in part by the payment advertisement Conception and design: B.D. Smith, M.D. Kaufman, S.C. Wise, R.J. Booth, of page charges. This article must therefore be hereby marked W.C. Patt, K.M. Yates, D.L. Flynn in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Development of methodology: B.D. Smith, M.D. Kaufman, C.B. Leary, Y.M. Ahn, R.J. Booth, T.M. Caldwell, W.P. Lu, W.C. Patt, L. Vogeti, S. Vogeti, Received January 2, 2015; revised May 28, 2015; accepted June 13, 2015; K.M. Yates, L. Chun, D.L. Flynn published OnlineFirst August 18, 2015.

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OF12 Mol Cancer Ther; 14(9) September 2015 Molecular Cancer Therapeutics

Altiratinib Inhibits Tumor Growth, Invasion, Angiogenesis, and Microenvironment-Mediated Drug Resistance via Balanced Inhibition of MET, TIE2, and VEGFR2

Bryan D. Smith, Michael D. Kaufman, Cynthia B. Leary, et al.

Mol Cancer Ther Published OnlineFirst August 18, 2015.

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