pharmacological and structural characterizations of ... · the crystal structure of the egfr t790m...

Small Molecule Therapeutics

Pharmacological and Structural Characterizationsof Naquotinib, a Novel Third-Generation EGFRTyrosine Kinase Inhibitor, in EGFR-MutatedNon–Small Cell Lung CancerToshiyuki Hirano1, Hiroyuki Yasuda1, Junko Hamamoto1, Shigenari Nukaga1,Keita Masuzawa1, Ichiro Kawada1, Katsuhiko Naoki2, Tatsuya Niimi3, Shinya Mimasu3,Hideki Sakagami4, Kenzo Soejima1, and Tomoko Betsuyaku1

Abstract

Multiple epidermal growth factor receptor (EGFR) tyrosinekinase inhibitors (EGFR-TKI) have been developed to effec-tively inhibit EGFR-derived signals in non–small cell lungcancer (NSCLC). In this study, we assessed the efficacy ofEGFR-TKIs, including a novel third-generation inhibitornaquotinib (ASP8273), in clinically relevant EGFR mutations,including L858R, exon 19 deletion, L858RþT790M, exon 19deletionþT790M with or without a C797S mutation, andseveral exon 20 insertion mutations. Using structural analyses,we also elucidated the mechanism of activation and sensitiv-ity/resistance to EGFR-TKIs in EGFR exon 20 insertion muta-tions. The efficacy of naquotinib in cells with L858R, exon 19deletion and exon 19 deletionþT790M was comparable withthat of osimertinib. Interestingly, naquotinib was more potent

than osimertinib for L858RþT790M. Additionally, naquotiniband osimertinib had comparable efficacy and a wide thera-peutic window for cells with EGFR exon 20 insertions. Struc-tural modeling partly elucidated the mechanism of activationand sensitivity/resistance to EGFR-TKIs in two EGFR exon 20insertion mutants, A767_V769dupASV and Y764_V765insHH.In summary, we have characterized the efficacy of EGFR-TKIsfor NSCLC using in vitro and structural analyses and suggestedthe mechanism of activation and resistance to EGFR-TKIs ofEGFR exon 20 insertion mutations. Our findings should guidethe selection of appropriate EGFR-TKIs for the treatment ofNSCLC with EGFR mutations and help clarify the biologyof EGFR exon 20 insertion mutations. Mol Cancer Ther; 17(4);740–50. �2018 AACR.

IntroductionLung cancers with mutation in the epidermal growth factor

receptor (EGFR) account for a significant subgroup of non–smallcell lung cancers (NSCLC; refs. 1, 2). Somatic mutations of thetyrosine kinase domain of EGFR, such as in-frame deletionsaround the LREA motif in exon 19 (exon 19 deletions) and theL858R point mutation in exon 21, are classic mutations, account-ing for approximately 80% to 90% of EGFRmutations in NSCLC(3, 4). These mutations destabilize the inactive conformation ofEGFR and induce its constitutive activation (5–7). Activated EGFR

transduces downstream signals, including the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK)and phosphatidylinositol-3-kinase/protein kinase B (AKT) path-ways (8, 9). Studies demonstrating the efficacy of first-generationEGFR tyrosine kinase inhibitors (EGFR-TKI) such as gefitinib anderlotinib against NSCLC harboring the aforementioned muta-tions had significantly changed the treatment strategy for NSCLC(10–15). In addition to first-generation drugs, the efficacy ofsecond-generation EGFR-TKIs, such as afatinib, in patients withNSCLC bearing these mutations has been demonstrated (16).However, NSCLC was shown to acquire resistance to first- andsecond-generation EGFR-TKIs in approximately 1 year. EGFRT790M mutation is found in approximately 50% of NSCLCresistant to first- and second-generation EGFR-TKIs (17, 18).To overcome the EGFR T790M-mediated resistance, third-generation EGFR-TKIs have been developed (19–22). Third-generation EGFR-TKIs bind irreversibly to the ATP binding pocketof EGFRby covalently binding toCys797, thereby blocking EGFR-mediated signals. Osimertinib is an FDA-approved third-generation EGFR-TKI that has been shown to significantlyimprove the prognosis of patients with NSCLC bearing the EGFRT790M mutation in multiple clinical trials (23, 24). Naquotinib,previously known as ASP8273, is a novel third-generation EGFR-TKI (25), and a preclinical study has reported its efficacy for celllines harboring EGFRmutations with or without a T790M muta-tion (26). However, naquotinib has not been fully characterized

1Division of Pulmonary Medicine, Department of Medicine, Keio University,School of Medicine, Tokyo, Japan. 2Kitasato University School of Medicine,Department of Respiratory Medicine, Kanagawa, Japan. 3Medicinal ChemistryResearch Labs, Drug Discovery Research, Astellas Pharma Inc., Tokyo, Japan.4Research Program Management Office, Drug Discovery Research, AstellasPharma Inc., Tokyo, Japan.

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

Corresponding Author: Hiroyuki Yasuda, Division of Pulmonary Medicine,Department of Medicine, Keio University, School of Medicine, 35 Shinanomachi,Shinjuku-ku, Tokyo, 160-8582, Japan. Phone: 81-3-3353-1211; Fax: 81-3-3353-2502; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-17-1033

�2018 American Association for Cancer Research.

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and directly compared with other EGFR-TKIs including osimerti-nib. In this study, we compared the efficacy of naquotinib withthat of other EGFR-TKIs in several clinically relevant NSCLC celllines harboring EGFR mutations.

In addition, we performed structural evaluations of severalEGFR exon 20 insertion mutants. It is noteworthy to mentionthat exon 20 insertions are the third major subgroup of EGFRmutations, accounting for approximately 4% to 10% of all EGFRmutations (27, 28).EGFR exon20 insertions havebeen associatedwith resistance to first- and second-generation EGFR-TKIs. Oneexception is the A763_Y764insFQEA mutation, which we previ-ously reported as a sensitizing mutation to first-generationEGFR-TKIs (29). We have also reported the potential efficacy ofosimertinib against tumors with exon 20 insertions in a previousstudy (30). However, findings that clarify the mechanism ofactivation and resistance of NSCLC with exon 20 insertions tofirst- and second-generation EGFR-TKIs are limited, and thereforethe mechanism was addressed in this study. Our findings shouldguide the selection of appropriate EGFR-TKIs for the treatment ofNSCLCwith EGFRmutations andhelp clarify the biology of EGFRexon 20 insertion mutations.

Materials and MethodsCell lines

Five human NSCLC cell lines were used: PC-9 [EGFR exon 19deletion (delE746-A750)]; H3255 (EGFR L858R); PC-9ER [EGFRexon19deletion (delE746-A750)þ T790M]; BID007 [EGFR exon20 insertion (A763_Y764insFQEA)]; and H1975 (EGFR L858RþT790M). PC-9, H3255, and BID007 cells were a kind gift fromDr. Susumu Kobayashi (Beth Israel Deaconess Medical Center,Boston,MA). H1975 cells were purchased from the ATCC in April2017. PC-9ER cells developed resistance to erlotinib after chronicexposure to the drug and the acquisition of a T790M mutation.Cell authentication for PC-9 and H3255 was performed by theauthors in June 2015 using genetic profiling of polymorphic shorttandem repeat (STR) loci (Takara).

Ba/F3 stable cell linesIL3-dependent murine pro–B cell line Ba/F3 stably expressing

the wild-type or mutated EGFR was created as previouslydescribed (29). Ba/F3 cells harboring EGFR mutations weremaintained in RPMI-1640 growth medium supplemented with10% fetal bovine serum and 1% penicillin/streptomycin at 37�Cin a humidified 5% CO2 incubator. Ba/F3 cells expressingwild-type EGFR were maintained in RPMI-1640 growth mediumsupplemented with 10% fetal bovine serum at 37�C in a humid-ified5%CO2 incubator in thepresence of epidermal growth factor(EGF; 10 ng/mL). The EGFR mutations examined in thisstudy include the delL747_P753insS (exon 19 del), L858R,delL747_P753insSþT790M(exon19delþT790M),L858RþT790M,delL747_P753insSþT790MþC797S(exon19delþT790MþC797S),L858RþT790MþC797S, A763_Y764insFQEA, Y764_V765insHH,A767_V769dupASV, and D770_N771insNPG.

ReagentsErlotinib and afatinib were purchased from LC Laboratories.

Osimertinib (AZD9291) was purchased from Selleck Chemicals.Naquotinib was provided by Astellas Pharma. These compoundswere dissolved in dimethyl sulfoxide (DMSO). Total EGFR anti-body (#4267S), total AKT antibody (#9272), phospho-AKT

(S473) antibody (D9E; #4060), total p44/42 MAPK antibody(#9102S), and phospho-p44/42 MAPK (T202/204) antibody(#9101S) were purchased from Cell Signaling Technology. Phos-pho-EGFR (Y1068) antibody (44788G) was purchased fromInvitrogen/Life Technologies, and actin antibody was purchasedfrom Sigma-Aldrich.

Kinase inhibitory profilingThe inhibitory effect of naquotinib at the concentration of 1

and 10 nmol/L against 102 human tyrosine kinases and 28human serine/threonine kinases was investigated by MobilityShift Assay at Carna Biosciences, Inc. Naquotinib was incubatedwith each kinase for 30 minutes at room temperature. Afterincubation, ATP and substrate mixture was added to startenzymatic reaction. The reaction mixture was applied to LabChip3000 (PerkinElmer), and the product and substrate peptide peakswere separated and quantitated.

Cell proliferation assayThe lung cancer cells or Ba/F3 cellswereplated in a 96-well plate

with or without EGFR-TKI. Control cells were treated with anequivalent concentration of DMSO. At 72 hours after treatment,cell proliferation assay was performed by adding 20 mL CellTiter96 Aqueous One Solution Reagent (Promega) to each well. Theabsorbance at 490 nm was read using a multiple reader. Data areexpressed as the percentage of growth relative to that of thecontrols. All experiments were performed at least three times.

Immunoblotting analysisCellswere treatedwith EGFR-TKI at the indicated concentration

for 4 hours. Cells were lysed in Cell Lysis Buffer (Cell SignalingTechnology). Equal amounts of protein (20 mg) were loaded perlane on sodium dodecyl sulfate-polyacrylamide gels, and sepa-rated proteins were transferred to polyvinylidene fluoride mem-branes. After blockingwith 5% skimmilk (BectonDickinson), themembranes were incubated overnight with primary antibodies at4�C and thenwith secondary antibodies for 30minutes. To detectthe proteins, the membranes were incubated with agitation inLumiGLO reagent and peroxide (Cell Signaling Technology) andthen exposed to an X-ray film.

Apoptosis assayBa/F3 cells harboring EGFR exon 19delþT790M or EGFR

L858RþT790M were maintained in 6-well plates. The cells weretreated with different EGFR-TKIs at the indicated concentrationsfor 48 hours. Control cells were treated with an equivalentconcentration of DMSO. We analyzed cell apoptosis using theAnnexin V Apoptosis Detection Kit APC (eBioscience) accordingto the manufacturer's protocol. The proportion of apoptotic cellswas evaluated by flow cytometry using the Gallios flow cytometersystem (Beckman Coulter).

Y764_V765insHH and A767_V769dupASV model structuresThe crystal structure of EGFR T790M in a complex with naquo-

tinib was used as the template model. Briefly, the EGFR kinasedomain harboring the T790Mmutation (residues 695–1022)wasexpressed in insect cells and purified accordingly. The purifiedEGFR T790Mprotein was crystallized using the sitting drop vapordiffusion method. The PDB accession number for the EGFRT790M crystal structure in a complex with naquotinib is 5Y9T.The Y764_V765insHH and A767_V769dupASV sequences were

Characterization of Naquotinib in EGFR-Mutated Lung Cancer

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alignedusing the Structure PredictionWizard.HHorASV residueswere inserted to conserve the Glu762 residue, which is indispens-able for the kinase activity, resulting in a C-terminus shift.

The crystal structure of the EGFR T790M complex was super-imposed with the structural model of Y764_V765insHH orA767_V769dupASV with three PDB coordinates (PDBID: 4ZAU,4G5P, and 4HJO) using the Molecular Operating Environment(MOE; Chemical Computing Group), and images were generatedusing the MOE tool.

Statistical analysisStatistical analysis was performed using the GraphPad Prism

software, version 4.0 (GraphPad Software). The 50% inhibitoryconcentration (IC50) values were calculated using the GraphPadPrism software.

ResultsEfficacy of naquotinib and other EGFR-TKIs for human lungcancer–derived cell lines

To evaluate the efficacy of EGFR-TKIs including naquotinib forhuman lung cancer–derived cell lines harboring EGFRmutations,the cells were subjected to MTS cell proliferation assays in thepresence or absence of EGFR-TKIs. We observed a clear sensitivityand resistance pattern; in lung cancer cell lines harboring theclassical EGFRmutations, i.e., the exon 19 deletion (exon 19del)and L858R mutations, all EGFR-TKIs evaluated were able todramatically inhibit cell proliferation (Fig. 1A). The calculatedIC50 values are shown in Supplementary Table S1. For lung cancercell lines harboring the EGFR T790M mutations, i.e., the PC-9ER(exon 19delþT790M) and H1975 (L858RþT790M) cells, osi-mertinib and naquotinib also dramatically inhibited cell prolif-eration. We subsequently performed immunoblot analyses toconfirm that the inhibition of cell proliferation was mediatedthrough the inhibition of EGFR and its downstream signaling. Ingeneral, the immunoblotting results showed inhibition patternsthat were consistent with the results of MTS cell proliferationassays described above. For cell lines harboring the classical EGFRmutations (PC-9 and H3255), all EGFR-TKIs dramatically inhib-ited the phosphorylation of EGFR, AKT, and ERK. In PC-9ER andH1975 cells, all EGFR-TKIs except erlotinib efficiently inhibitedthe phosphorylation of EGFR, AKT, and ERK (Fig. 1B). Thesefindings clarified the sensitivity and resistance profile for differentEGFR-TKIs including naquotinib in human lung cancer cells.

Direct comparison of various EGFR-TKIs in cells with clinicallyrelevant EGFR mutations

To directly compare the mutation-selective efficacy ofEGFR-TKIs including naquotinib, we generated Ba/F3 cells stablyexpressing different EGFR mutations. These include the exon19del, L858R, exon 19delþT790M, L858RþT790M, exon19delþT790MþC797S, and L858RþT790MþC797S mutations.We performed MTS cell proliferation assays for these cells in thepresence or absence of EGFR-TKIs. As in the human lung cancercells, a clear sensitivity and resistance pattern was also observed inthese Ba/F3 cells (Fig. 2). The calculated IC50 values are shownin Table 1. For Ba/F3 cells expressing the classic EGFRmutations,we observed a clear inhibition of cell proliferation with allEGFR-TKIs evaluated. For Ba/F3 cells harboring a T790M muta-tion, exon 19delþT790M, or L858RþT790M, all EGFR-TKIsexcept erlotinib inhibited cell proliferation. For Ba/F3 cells

with a C797S mutation (exon 19delþT790MþC797S orL858RþT790MþC797S; ref. 31), none of the EGFR-TKIsinhibited the cell proliferation. Of note, for cells with theL858RþT790M mutation, the IC50 value of naquotinib wasslightly lower than that of osimertinib. The calculated IC50 valueswere 74 and 9 nmol/L for osimertinib and naquotinib,respectively. In addition, apoptosis wasmore profound in naquo-tinib-treated cells than in osimertinib-treated cells (Supplemen-tary Fig. S1).

In wild-type cells, afatinib demonstrated the strongest inhibi-tion of cell growth. To determine the therapeutic window of theEGFR-TKIs, we used a previously established model in which wecalculated the IC50 ratios of Ba/F3 cells transduced with mutantand wild-type EGFR. For cells bearing the classical EGFRmutations, all EGFR-TKIs showed a wide therapeutic windowwith a selectivity index (SI) value of approximately�2, indicatingthat the IC50 values for the mutants were approximately 100-foldlower than those of the wild-type (Supplementary Fig. S2).For cells with exon 19delþT790M or L858RþT790M,osimertinib and naquotinib, but not afatinib, demonstrated lowSI values. For cells with exon 19delþT790MþC797S orL858RþT790MþC797S, none of the EGFR-TKIs showed low SIvalues. These data are consistent with the findings from humanlung cancer–derived cell lines described above.

To confirm that the inhibition of Ba/F3 cell proliferation wasmediated by the inhibition of EGFR and its downstreamsignaling, we performed an immunoblot analysis. The inhibi-tion pattern observed with the immunoblot analysis was gen-erally consistent with the results of the MTS cell proliferationassay (Supplementary Fig. S3). For Ba/F3 cells with the classicEGFR mutations, all EGFR-TKIs were able to significantlyinhibit the phosphorylation of EGFR, AKT, and ERK. For Ba/F3cells bearing the exon 19delþT790M or L858RþT790Mmutation, all EGFR-TKIs except erlotinib efficiently inhibitedthe phosphorylation of EGFR, AKT, and ERK. In contrast,for Ba/F3 cells with exon 19delþT790MþC797S orL858RþT790MþC797S mutation, none of the EGFR-TKIsinhibited the phosphorylation of EGFR, AKT, and ERK. ForBa/F3cells with wild-type EGFR, afatinib demonstrated thestrongest inhibition of EGFR, AKT, and ERK phosphorylations.These findings showed fundamental differences in the muta-tion-selective efficacy of EGFR-TKIs.

Efficacy of EGFR-TKIs for EGFR exon 20 insertion mutationsEGFR exon 20 insertions have been reported in tumors resistant

to first- and second-generation EGFR-TKIs. An exception to this isthe A763_Y764insFQEAmutation, which we previously reportedas a mutation that retains sensitivity to first- and second-gener-ation EGFR-TKIs. We have previously reported the therapeuticwindow and potential efficacy of osimertinib for resistant cellswith EGFR exon 20 insertion mutations (29). To evaluate theefficacy of EGFR-TKIs including naquotinib, we performedMTS cell proliferation assays for Ba/F3 cells harboring differentEGFR exon 20 insertion mutations. The EGFR exon 20 insertionsevaluated in this study are A763_Y764insFQEA (FQEA),Y764_V765insHH (HH), A767_V769dupASV (ASV), andD770_N771insNPG (NPG). For Ba/F3 cells bearing theA763_Y764insFQEAmutation, all EGFR-TKIs were able to inhibitthe cell proliferation. For Ba/F3 cells resistant to first- and second-generation EGFR-TKIs bearing other exon 20 insertionmutations,afatinib, osimertinib, and naquotinib were able to inhibit the cell

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Figure 1.

Sensitivity of lung cancer cell lines to EGFR-TKIs. A, MTS assays of PC-9, H3255, PC-9ER, and H1975 cells exposed to different EGFR-TKIs. Data represent themean � standard deviation. B, PC-9, H3255, PC-9ER, and H1975 cells were treated with different EGFR-TKIs at the indicated concentrations for 4 hours prior toimmunoblotting for phosphorylated (p) and nonphosphorylated forms of EGFR, AKT, and ERK. Actin was used as a loading control.






proliferation (Fig. 3A). The calculated IC50 values are shown inSupplementary Table S2.

For cells with the A763_Y764insFQEAmutation, all EGFR-TKIsshowed a wide therapeutic window with an SI value of approx-imately �2, indicating that the IC50 values for the mutants wereapproximately 100-fold lower than those for the wild-type(Supplementary Fig. S4). For cells harboring other EGFR exon20 insertion mutations, osimertinib and naquotinib showed awide therapeutic window, but not for Y764_V765insHH. In animmunoblot analysis, all EGFR-TKIs evaluated showed limitedefficacy for Ba/F3 cells with a Y764_V765insHH mutation(Supplementary Fig. S5). A similar sensitivity pattern was alsoobserved in a human lung cancer–derived cell line, BID007,which also harbors the EGFR A763_Y764insFQEA mutation(Fig. 3B; Supplementary Table S1). Immunoblotting for theBID007 cells also showed a similar inhibition pattern of EGFRand its downstream signals, which was consistent with the find-ings of the MTS cell proliferation assay (Fig. 3C). These resultsindicate a comparable efficacy for naquotinib and osimertinib inseveral EGFR exon 20 insertion mutants.

Structural characterization of EGFR exon 20 insertionmutations

The findings described above indicate a clear efficacy differencefor EGFR-TKIs among cells bearing different EGFR mutations,especially in cells with EGFR exon 20 insertion mutations. Pre-viously, data on the mechanism of activation and drug sensitivityof EGFR exon 20 insertion mutants based on structural analyseshad been limited. In this study, we performed a structural model-ing of EGFR bearing different exon 20 insertion mutations.Among the four EGFR exon 20 insertion mutations,D770_N771insNPG and A763_Y764insFQEA have been evalu-ated extensively in a previous study (29). In this study, we focusedon the A767_V769dupASV mutation, one of the most commonEGFR exon 20 insertion mutations, and the Y764_V765insHHmutation, the most resistant mutation examined in this study.Bothmodels assumed that the catalytically indispensable Glu762retains its position and that the inserted residues shift toward theC-terminus. We generated a model for EGFR A767_V769dupASVto structurally analyze its mechanism of activation and differencein EGFR-TKIs sensitivity. The model showed that the ASV inser-tion forms a residual loop at the end of the C-helix. This insertiondoes not directly affect EGFR-TKIs upon inhibitor superimposi-tion (Supplementary Fig. S6). Because the insertion site ofA767_V769dupASV is only one residue different from thatof D770_N771insNPG, their mechanism of activation anddifference in inhibitor sensitivity may be similar. As observed inthe crystal structure of EGFR D770_N771insNPG, the insertedresidues form a tight turn at the end of the C-helix, whichmay shift the kinase activity equilibrium to an upregulated

state (29). This model suggested that the A767_V769dupASVandD770_N771insNPGmutations have a similar conformation-al effect.

To thoroughly analyze the inhibitor binding effect of theY764_V765insHH insertion, we generated a structural model ofEGFR Y764_V765insHH. The structural model with C-terminalinsertion showed that the inserted histidine residues displaceVal765 and Met766 (Fig. 4A). By superimposing EGFR-TKIs withthis model structure, we observed that the second histidineinsertion in the Met766 position can directly affect inhibitorsthat are buried deep into the back-pocket such as erlotinib andafatinib, while other inhibitors including naquotinib and osimer-tinib should remain unaffected (Fig. 4B). The first histidineinsertion in the Val765 position would lose its hydrophobicitytoward Leu858 and thus keeping it flipped in its active confor-mation (32).

Biological validation of the EGFR Y764_V765insHH structureIn order to validate the structural models above, we performed

further in vitro analyses. First, to investigate whether the effectof the insertion (i.e., the shift toward C-terminus) alone issufficient to upregulate the kinase activity, we constructed anEGFR M766_A767insAA mutant. We did not construct aY764_V765insAA mutant because this mutation may alter thestructure of the EGFR kinase pocket, especially the Met766. Toinvestigate whether this mutation activates EGFR, we transducedit toBa/F3 cells. As expected, Ba/F3 cells transducedwithwild-typeEGFR failed to grow in the absence of EGF.However, interestingly,Ba/F3 cells transduced with EGFRM766_A767insAA grew even inthe absence of EGF at a rate comparable to cells with wild-typereceptor with EGF (Fig. 5A). These results demonstrated that theeffect of the insertion alone was sufficient to upregulate the EGFRkinase activity.

Next, to confirm the structural finding that the replacementof V765 and M766 to H765 and H766, respectively(V765HþM766H)may result in EGFR-TKI insensitivity, especial-ly to erlotinib and afatinib, we constructed EGFR bearing theL858RþV765HþM766Hmutation.We transduced thismutationto Ba/F3 cells and performed anMTS cell proliferation assay in thepresence of EGFR-TKIs. As predicted by the structural analysis, theL858RþV765HþM766H mutant showed remarkable insensitiv-ity toward erlotinib and afatinib (Fig. 5B; SupplementaryTable S3). These data supported the findings that replacementof V765 and M766 to H765 and H766 (V765HþM766H)induced insensitivity to EGFR-TKIs. In summary, we have dem-onstrated that the insertion of histidine residues in the position ofVal765 and Met766 in Y764_V765insHH upregulated the EGFRkinase activity, and that it caused a steric insensitivity to theparticular EGFR-TKIs.

DiscussionIn this study, we have characterized the efficacy of multiple

EGFR-TKIs including a novel third-generation EGFR-TKI, naquo-tinib, for clinically relevant EGFR mutations. The inhibitoryeffects of naquotinib on various kinases are shown in Supple-mentary Table S4. Naquotinib exhibited mutation-selectiveinhibitory effects for EGFR. Other than mutated EGFR, naquoti-nib inhibited several kinases, including BTK, JAK3, and TXK. Adifference in the inhibitory profile between naquotinib andosimertinib (20) was observed, whichmay affect efficacy or safety

Table 1. IC50 values (nmol/L) of EGFR-TKIs for Ba/F3 cells harboring EGFRmutations

Erlotinib Afatinib Osimertinib Naquotinib

exon19 del 7 0.7 6 9L858R 24 0.7 16 11exon19 delþT790M 1590 176 7 10L858þT790M >10000 821 74 9exon19 delþT790MþC797S 906 1214 1180 235L858þT790MþC797S 1910 1625 1913 1994Wild-type 667 28 704 830

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Figure 2.

Sensitivity of Ba/F3 cells expressing wild-type (WT) or mutant EGFR to EGFR-TKIs. MTS assays were conducted for Ba/F3 cells expressing the indicatedEGFR genotypes. Data represent the mean � standard deviation.






Figure 3.

Effect of EGFR-TKIs on Ba/F3 or lung cancer cells harboring EGFR exon 20 insertion mutations. A, Sensitivity of Ba/F3 cells expressing EGFR exon 20insertion mutations to EGFR-TKIs. MTS assays were conducted for Ba/F3 cells expressing the indicated EGFR genotypes. Data represent the mean � standarddeviation. B, The sensitivity of BID007 cells expressing EGFR exon 20 insertion mutation, A763_Y764insFQEA, to EGFR-TKIs. MTS assays were conducted inBID007 cells. Data points represent the mean � standard deviation. C, BID007 cells were treated with the indicated concentrations of EGFR-TKIs for 4 hoursprior to immunoblotting for the phosphorylated (p) and nonphosphorylated forms of EGFR, AKT, and ERK. Actin was used as a loading control.


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of these inhibitors in lung cancer patients. In our cell-based assays,the efficacy of naquotinib and osimertinib was generally compa-rable. For most of the EGFRmutations, except for C797S-positivemutations and Y764_V765insHH, naquotinib demonstrated lowIC50 values and a wide therapeutic window. The therapeuticwindow for these EGFR-TKIs is an important determinant of theirpotential toxicity in human. To quantify this, we used an in vitromodel, which we described in a previous study (30), to determinethe ratio of IC50 values in Ba/F3 cells transduced with mutant orwild-type EGFR. For EGFR L858RþT790M, naquotinib showedlower IC50 values and a wider therapeutic window than osimer-tinib. However, whether the difference observed in the in vitrostudy is clinically relevant remains unclear. Further characteriza-tions of naquotinib through additional in vitro and in vivo studiesare required.

Structural analysis can offer insight into the mechanism ofprotein activation and relationship between protein structure anddrug sensitivity. Structural analyses for EGFR with L858R orG719S mutations have been performed in numerous studies(5, 7, 33). Both L858R and G719S mutations lock the EGFR

kinase domain in a constitutively active conformation by desta-bilizing the inactive conformation. However, for EGFRwith exon20 insertion mutations that displayed resistance to first- andsecond-generation EGFR-TKIs, the structural basis for resistanceis poorly understood. In a previous study, we have clarifiedthe mechanism of activation and sensitivity for EGFRD770_N771insNPG and A763_Y764insFQEA using crystal struc-ture and structural modeling, respectively (29). For theD770_N771insNPG mutation, the crystal structure revealed anunaltered ATP binding pocket, and the inserted residues form awedge at the end of the C-helix, a key structural component foractivation (34). For the A763_Y764insFQEAmutation, structuralmodeling revealed that the inserted residues shift the registerof the C-helix in the N-terminal direction, altering the structurein the region that is also affected by the L858R mutation. In thisstudy, we have partially elucidated the mechanism of activationand sensitivity of two EGFR exon 20 insertion mutations associ-ated with resistance to first- and second-generation EGFR-TKIs,i.e., the A767_V769dupASV and Y764_V765insHH mutations.For both mutations, the models assumed that the catalytically

Figure 4.

Structural model of Y764_V765insHH.A, Alignments of EGFR wild-type,Y764_V765insHH, V765HþM766H,and M766_A767insAA. Thecatalytically indispensable Glu762is shown in the blue box. B,Superimposition of differentEGFR-TKIs on the structural model ofEGFR Y764_V765insHH. Key residuesonEGFR for kinase catalysis are shownas orange sticks. Leu858, a key residuefor EGFR activation, is shown asyellow sticks. The inserted histidineresidues are shown as magenta sticks.TKIs erlotinib, afatinib, naquotinib,and osimertinib are shown in gray,green, yellow, and cyan, respectively.Inset, chemical structures of erlotinib,afatinib, naquotinib, and osimertinib.






indispensable Glu762 retains its position and that the insertedresidues shift toward the C-terminus. This assumption was sup-ported by results of the in vitro experiments. Interestingly, a shifttoward the C-terminus by two amino acids activated the EGFR, asdemonstrated by the ligand-independent proliferation of Ba/F3cells transducedwithEGFRM766_A767insAA. Althoughdifferent

insertions may yield slight differences in the structure, insertionof several residues in this EGFR region (i.e., near the end of theC-helix) should have a similar structural effect.

In addition, we have proposed a mechanism for resistanceassociated with EGFR Y764_V765insHH mutation, one ofthe most resistant mutations. We demonstrated that in

Figure 5.

In vitro confirmation of the structural modeling of EGFR. A, Cell growth curve of EGFR wild-type (WT) and M766_A767insAA (insAA) in the presence orabsence of EGF (10 ng/mL). B, Sensitivity of Ba/F3 cells expressing mutant EGFR to EGFR-TKIs. MTS assays were conducted for Ba/F3 cells expressing theindicated EGFR genotypes. Data represent the mean � standard deviation.


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Y764_V765insHH, histidine residues inserted in the Val765 andMet766 positions upregulated the EGFR kinase activity andcaused steric insensitivity to the particular EGFR-TKIs. Thesefindings shed light on the biology of EGFR exon 20 insertionmutations.

In summary, we have characterized the efficacy of differentEGFR-TKIs including naquotinib, a novel third-generationEGFR-TKI, using in vitro and structural analyses. Resultsof this study also partially clarified the mechanism of activa-tion and resistance of EGFR exon 20 insertion mutations toEGFR-TKIs. Our findings should help to determine the appro-priate EGFR-TKIs for the treatment of NSCLC with EGFRmutations and shed light on the biology of specific subsetsof EGFR mutations.

Disclosure of Potential Conflicts of InterestK. Soejima reports receiving a commercial research grant from Taiho

and has received speakers bureau honoraria from AstraZeneca, Chugai,Ono, MSD and Eli Lilly. No potential conflicts of interest were disclosedby the others.

Authors' ContributionsConception and design: H. Yasuda, I. Kawada, K. Naoki, H. Sakagami,K. Soejima, T. BetsuyakuDevelopment of methodology: H. Yasuda, J. Hamamoto

Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): T. Hirano, J. Hamamoto, K. Masuzawa, T. Niimi,S. MimasuAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): T. Hirano, H. Yasuda, J. Hamamoto, S. Nukaga,K. Masuzawa, K. Naoki, T. Niimi,Writing, review, and/or revision of the manuscript: T. Hirano, H. Yasuda,J. Hamamoto, I. Kawada, K. Naoki, T. Niimi, S. Mimasu, H. Sakagami,T. BetsuyakuAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): T. Hirano, H. Yasuda, J. Hamamoto, K. NaokiStudy supervision: H. Yasuda, I. Kawada, K. Naoki, H. Sakagami, K. Soejima,T. Betsuyaku

AcknowledgmentsThis work was supported in part by the Grants-in-Aid for Scientific Research

on Priority Areas from the Ministry of Education, Culture, Sports, Science, andTechnology of Japan to K. Soejima (15K09229), T. Betsuyaku (15H04833), andH. Yasuda (17K09667). We thank Ms. Mikiko Shibuya for her excellenttechnical assistance. We also thank the Collaborative Research Resources atthe Keio University School of Medicine for assistance with cell sorting.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received October 21, 2017; revised December 13, 2017; accepted February 5,2018; published first February 21, 2018.

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Hirano et al.




2018;17:740-750. Published OnlineFirst February 21, 2018.Mol Cancer Ther Toshiyuki Hirano, Hiroyuki Yasuda, Junko Hamamoto, et al.

Small Cell Lung Cancer−-Mutated NonEGFRNovel Third-Generation EGFR Tyrosine Kinase Inhibitor, in

Pharmacological and Structural Characterizations of Naquotinib, a

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