evaluating tbk1 as a therapeutic target in cancers...

Signal Transduction

Evaluating TBK1 as a Therapeutic Target in Cancers withActivated IRF3

Asli Muvaffak1, Qi Pan1, Haiyan Yan1, Rafael Fernandez3, Jongwon Lim3, Brian Dolinski3, Thi T. Nguyen3,Peter Strack3, StephenWu1,RossanaChung1,WeiqunZhang1,ChrisHulton1, StevenRipley1,HeatherHirsch3,Kumiko Nagashima3, Kwok-Kin Wong1,2, Pasi A. J�anne1,2, Cynthia Seidel-Dugan3, Leigh Zawel3,Paul T. Kirschmeier1, Richard E. Middleton1, Erick J. Morris3, and Yan Wang3

AbstractTBK1 (TANK-binding kinase 1) is a noncanonical IkB protein kinase that phosphorylates and activates

downstream targets such as IRF3 and c-Rel and, mediates NF-kB activation in cancer. Previous reportsdemonstrated synthetic lethality of TBK1 with mutant KRAS in non–small cell lung cancer (NSCLC); thus,TBK1 could be a novel target for treatment ofKRAS-mutantNSCLC.Here, the effect of TBK1 onproliferation in apanel of cancer cells by both genetic and pharmacologic approaches was evaluated. In KRAS-mutant cancer cells,reduction of TBK1 activity by knockdown or treatment with TBK1 inhibitors did not correlate with reducedproliferation in a two-dimensional viability assay. Verification of target engagement via reduced phosphorylation ofS386 of IRF3 (pIRF3S386) was difficult to assess inNSCLCcells due to lowprotein expression.However, several celllines were identified with high pIRF3S386 levels after screening a large panel of cell lines, many of which also harborKRASmutations. Specifically, a large subset of KRAS-mutant pancreatic cancer cell lines was uncovered with highconstitutive pIRF3S386 levels, which correlated with high levels of phosphorylated S172 of TBK1 (pTBK1S172).Finally, TBK1 inhibitors dose-dependently inhibited pIRF3S386 in these cell lines, but this did not correlate withinhibition of cell growth. Taken together, these data demonstrate that the regulation of pathways important for cellproliferation in some NSCLC, pancreatic, and colorectal cell lines is not solely dependent on TBK1 activity.

Implications:TBK1has therapeutic potential under certain contexts and phosphorylation of its downstream targetIRF3 is a biomarker of TBK1 activity.

Visual Overview: http://mcr.aacrjournals.org/content/12/7/1055/F1.large.jpg.Mol Cancer Res; 12(7); 1055–66. �2014 AACR.

IntroductionTBK1 (TANK-binding kinase 1) is a noncanonical IkB

kinase, which shares 65% similarity to IKKe and is highlyexpressed in lung, breast, pancreatic, and colon cancers (1).Besides its key role in innate immune response, increasingevidence indicates that the activation of TBK1 and its closehomolog IKKe is associated with the development of humancancers (2–10). TBK1 is an 84 kDa, 729 amino acid–longprotein containing an N-terminal kinase domain, a ubiqui-tin-like domain and two C-terminal coiled-coil domains.TBK1 is an NF-kB–activating kinase, which is constitu-

tively expressed in many normal tissues, including immunecells, brain, lungs, gastrointestinal tract, and reproductiveorgans (11, 12). TBK1-deficent mice exhibit embryoniclethality due to widespread hepatic apoptosis (13, 14).TBK1 is regulated through activation byToll-like receptors

(TLR) or cytoplasmic RIG-1–like receptors, and it stimulatestype I IFN production via direct phosphorylation of IFNregulatory transcription factor 3 (IRF3) and 7 (IRF7; refs. 15–22). IRF3 phosphorylation at residues Ser-385 and Ser-386has previously been shown to control its dimerization (23–25)upon which IRF3 translocates to the nucleus (26), andcontrols transcriptional regulation of cell survival and prolif-eration (2, 11). In addition to IRF3, several other substrates, i.e., namely cRel, Akt, RalB, SIKE, etc., have been identifiedthat attribute toTBK1 function (7–9, 27–30).TBK1 requiresphosphorylation at Ser-172 within the kinase domain acti-vation loop to be activated. Phosphorylation at Ser-172 hasbeen shown previously to induce reorganization of the acti-vation loop allowing the active site in the kinase domain tobind to the substrate (31). TBK1 inhibitors of differentchemical structures have been developed by multiple groupsand it was shown previously that a PDK1/TBK1 inhibitor,BX795, enhanced phosphorylation of TBK1while inhibiting

Authors' Affiliations: 1Belfer Institute for Applied Cancer Science;2Department of Medical Oncology, Dana-Farber Cancer Institute; and3Merck Research Laboratories, Boston, Massachusetts

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

Corresponding Authors:Asli Muvaffak, Belfer Institute for Applied CancerScience, Dana-Farber Cancer Institute, 4 Blackfan Street, HIM Building,Room 320A. Boston, MA 02115. Phone: 857-453-0907; Fax: 617-582-9702; E-mail: [email protected], and Yan Wang, TESAROInc., 1000 Winter Street, Waltham, MA, [email protected]

doi: 10.1158/1541-7786.MCR-13-0642

�2014 American Association for Cancer Research.

MolecularCancer

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its activity, suggesting a feedback control mechanism thatstimulates phosphorylation of TBK1 (10, 32, 33).Although the role of TBK1 in cancer still remains unclear,

recent studies suggested its role in regulation of cell growthand proliferation, and oncogenic transformation (2, 4, 9).Shen andHahn (1) first demonstrated the role of both TBK1and IKKe in cell transformation. IKKe, a breast canceroncogene that is amplified in 30% of breast cancers, med-iates activation of NF-kB signaling and is required fortransformation in breast cancer (34–36). TBK1 was iden-tified through a functional genomics screen, which showedthat a subset of KRAS-mutant non–small cell lung cancer(NSCLC) cell lines was dependent on TBK1 and that TBK1was essential for KRAS mutant cancer cell lines (6). Thisgenerated an interest in small molecule inhibitors of TBK1that may provide a novel therapeutic strategy for KRAS-mutant tumors. However, subsequent studies were not ableto confirm the proposed synthetic lethal relationship ofoncogenic KRAS and TBK1 (5, 8). In these studies, knock-down of TBK1 did not consistently result in a significantalteration of growth, and the sensitivity to TBK1 inhibitionby either siRNA or small-molecule inhibitors did not cor-relate with KRAS status in lung, pancreatic, or colorectalcancer cell lines (5).Our study aimed to further investigate the role of TBK1 in

cancer by studying TBK1 knockdown using RNAi techno-logy as well as small molecule inhibitors. We identified asubset of pancreatic ductal adenocarcinoma cell lines (PDAC)that have high constitutive pIRF3S836 levels and showed thatTBK1 inhibitors dose-dependently inhibited pIRF3S386. Inaddition, shRNA knockdown of TBK1 reduced pIRF3S836

in pancreatic cancer cell lines, and, thus, further supportedpIRF3S836 as a target engagement marker for TBK1 activityin these cancer cell lines. However, no robust inhibition ofgrowth was achieved in the two-dimensional proliferationassay in these PDAC cell lines as well as in the majority ofNSCLC cell lines either by genetic knockdown or by usingTBK1 inhibitors. Our data demonstrate that reduction ofTBK1 activity and the subsequent elimination of pIRF3S836

are not sufficient on their own to regulate cell proliferation inthe NSCLC and PDAC cell lines that were tested.

Materials and MethodsCell lines and tool inhibitor treatment conditionsCell lines (see Supplementary Data) were obtained from

various commercial vendors (ATCC, DSMZ, JCRB, andECACC). The cell line authentication details are listed in theSupplementary Data. All cell lines were grown in recom-mended media by the ATCC supplemented with 10% fetalbovine serum (Atlanta Biologicals Inc.). All cultures weremaintained at 37�C in a humidified 5% CO2 atmosphereand tested periodically to ensure the absence of mycoplasma.Generally, cells were passaged upon reaching approximately75% confluence.Tool inhibitor stocks and dilutions weremade in dimethyl

sulfoxide solvent. Cell proliferation experiments were carriedout in a 96-well format (6 replicates), the cells were plated at adensity of 2,000 to 5,000 cells per well. At 24 hours

following cell seeding, the cells were treated with the toolinhibitor titrations for 4 days at 37�C and then assayed byusing the ATP CellTiter-Glo luminescent cell viability assay(Promega; G7570).For Incucyte analysis (Essen BioScience, Inc.), cells were

plated as described above in 96-well plates, and image-basedcell confluence data were collected every 2 hours during livegrowth.

Plasmid constructspLKO.1–shRNAs [targeting TBK1 Coding Sequence

(CDS) regions] were purchased from the Broad Institute(Cambridge, MA). Of note, 30 untranslated region (UTR)–targeting shRNAs and control shRNAs for GFP and KRASsequences were specifically designed for this study (seeSupplementary Data). Human full-length TBK1 cDNAwas purchased from Origene (SC111251). shRNA-resistantTBK1 was made by PCR cloning of TBK-WT withoutthe 30UTR region using newly designed primers, and thensubcloned into the pLenti-CMV-Hygro-DEST (w117-1)vector (Addgene) using Gateway cloning. All shRNAs andcDNAs were confirmed by sequencing.

Lentiviral shRNA transduction and proliferation assayLentiviral shRNAs were prepared via transfection of the

corresponding plasmid DNAs into 293 T cells using Lipo-fectamine 2000 (Invitrogen; 52887) in OptiMEM (Gibco;31985-070) media. The lentiviruses were packaged usingthe pD8.9 and VSVG packaging vectors in early passage 293T cells.Both NSCLC and PDAC cancer cell lines were plated at

150 to 250 K cells per well density in 6-well plates on day 0,and the lentiviral transductions were performed on day 1using polybrene (8 mg/mL; EMDMillipore # TR-1003-G).The cells were selected in 2 mg/mL puromycin for 3 days andon day 4 cells were plated in 1 mg/mL puromycin (LifeTechnologies; # A1113803) into 96-well assay plates. Cellviability was assessed on day 8 after viral transductions usingthe ATP CellTiter-Glo luminescent cell viability assay (Pro-mega; G7570). The growth profiles of cell lines with andwithout shRNA knockdownwere also recorded and analyzedby the Incucyte Imaging System (Essen BioScience, Inc.).The level of knockdown of TBK1 protein was determined byWestern blot analysis using the samples collected on day 8.

Protein detectionCell lysates forWestern blottingwere collected usingRIPA

lysis buffer (Boston Bioproducts; BP-115) supplementedwith 1�Halt Protease inhibitor (VWR International; VWR# PI78415), 5 mmol/L NaF (Sigma-Aldrich Co.) and 10mmol/L b-glycerol phosphate (Sigma-Aldrich Co.). Protein(10–15 mg) was separated on 4% to 15%Mini-PROTEANTGXPrecastGels (Bio-RadLaboratories; 456-1086), and on4% to 15% Criterion TGX Precast Gel (Bio-Rad Labora-tories; 5671085). The proteins were transferred onto apolyvinylidene difluoride membrane using the TransBlotTurbo Transfer System (Bio-Rad Laboratories; 170–4155).Protein levels from Western blots were quantified by

densitometry using the ImageJ software and this was

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normalized to the loading control for each sample. The bandsignal intensities were from the raw chemiluminescentimages on films. For IC50 calculations, these data were fitwith a standard three-parameter dose–response nonlinearregression fit using GraphPad Prism software.

AntibodiesWestern blots were probed with antibodies against TBK1

(Cell Signaling Technology; D1B4, CST 3504), phospho-

TBK1 S172 (Abgent; AP3627a), IRF3 (Cell Signaling Tech-nology;D83B9,CST4302), phospho-IRF3S386 (Epitomics;Clone ID EPR2346, 2562-1), KRAS (Santa Cruz Biotech-nology Inc., F234, sc-30) GAPDH (Cell Signaling Technol-ogy; horseradish peroxidase-conjugate, 14C10, CST 3683).

Assays used for initial validation of tool inhibitorsTheTBK1 tool inhibitors were initially screened using the

full-length TBK1 protein in a biochemical assay (Ulight

Table 1. List of cancer cell lines tested with multiple shRNAs targeting TBK1 and those that resulted in�90% knockdown are listed

Western blot protein expressiona

Cellline

Tissueorigin

KRASmutationstatus pIRF3S386 IRF3 pTBK1S172 TBK1

shRNAs tested(which had TBK1KD � 90% by WB)

Growth inhibition (%;normalized to shGFP;average � SD)

H23 Lung G12C (�) NA (�) (þþ) shT15 shT15: �1.26 � 1.90shT17 shT17: 82.74 � 1.41sh3182 sh3182: 51.74 � 1.96sh3183 sh3183: �35.02 � 3.10sh3185 sh3185: 48.84 � 0.95sh3186 sh3186: 46.53 � 0.77

549 Lung G12S (þ) NA (þþ) (þþ) shT15 shT15: �3.17 � 1.33shT17 shT17: 77.34 � 0.64sh3182 sh3182: 36.94 � 2.23sh3183 sh3183: 17.44 � 1.95sh3185 sh3185: 26.63 � 0.49sh3186 sh3186: 34.88 � 1.99

H28 Lung WT (�) NA (�) (þþ) shT15 shT15: �10.06 � 6.35shT17 shT17: 14.30 � 2.62

Panc02.13 PDAC Q61R (þþþ) (þ) (þþþ) (þþ) shT15 shT15: 12.66 � 2.39sh3182 sh3182: 16.26 � 1.77sh3185 sh3185: 37.84 � 3.83sh3186 sh3186: 39.24 � 4.03

Panc05.04 PDAC G12D (þþþ) (þ) (þþ) (þþ) shT15 shT15: �6.99 � 4.09sh3182 sh3182: �26.02 � 5.07sh3185 sh3185: 1.49 � 5.19sh3186 sh3186: 1.44 � 3.44

CFPAC-1 PDAC G12V (�) (�) (�) (þþ) shT15 shT15: �35.85 � 9.41sh3182 sh3182: �21.19 � 7.01

SUIT2 PDAC G12D (�) (þ) (�) (þþ) shT15 shT15: 35.52 � 9.44sh3182 sh3182: 42.06 � 9.21

PL45 PDAC G12D (þþþ) (þ) (þþþ) (þþ) shT15 shT15: �0.39 � 6.65sh3182 sh3182: �19.10 � 6.45

LOVO Colon G13D (þþ) (þþ) (�) (þþ) shT15 shT15: �6.00 � 4.99shT17 shT17: 47.00 � 1.44sh3182 sh3182: 48.00 � 2.99

WiDr Colon WT (þ) (þþ) (�) (þþ) shT15 shT15: 13.68 � 2.39sh3182 sh3182: 31.00 � 2.61

NOTE: The corresponding effects in the two-dimensional cell proliferation assay obtained from ATP CellTiter-Glo (CTG) luminescentcell viability assay usingNSCLC, colorectal, andPDACcancer cell lines is reported as cell growth inhibition as thepercentageof shGFPcontrol.aBasal protein levels at 10% fetal bovine serum.

Phosphorylated IRF3 Is a Biomarker of TBK1 Activity

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kinase assay; LANCEUltra ULight, Perkin Elmer) in whichtwo lead series were identified. Six tool inhibitors (Table 3)were selected and these underwent profiling studies in a listof KRAS–wild-type (wt) and -mutant NSCLC cell lines (asdescribed above and in Table 3). In addition, these toolinhibitors were further characterized for their ability toinhibit IRF3 nuclear translocation using a high-contentimmunofluorescence imaging assay. This assay quantifiedthe amount of IRF3 protein which translocated into thenucleus of the HeLa cell line, a surrogate cell line for toolinhibitor screening; The HeLa cells were stimulated withPoly I:C (Invivogen; tlrl-pic) following treatment withTBK1 tool inhibitors; (Table 3). The assay uses an anti-IRF3 mAb (Epitomics; cat.# 2241-1), and a secondary Ablabeled with a red fluorophore (goat anti-rabbit IgG(HþL), DyLight 549 Conjugated; Thermo Scientific catno. 35558). Immunofluorescence image data were ana-lyzed using a Columbus script (Opera/Acapella/Columbusplatform for high-content screening—PerkinElmer) thatquantified mean intensity of IRF3 signal in the nucleusafter image segmentation.

Kinase panel screening of tool inhibitorsThe kinase inhibitory profiles of inhibitors were eval-

uated by using 101, 266, and 305 kinase panels atInvitrogen.

RAS pathway signature analysisGene transcription signature of the RAS pathway was

defined as described by Loboda and colleagues (see Supple-mentary Data; ref. 37).

ResultsTBK1 dependency did not correlate with the KRASmutation statusTBK1 has been proposed as a potential therapeutic target

for KRAS-dependent cancer (6). We evaluated the role ofTBK1 inKRAS-mutantNSCLC cell lines further using bothgenetic and pharmacologic approaches. Six shRNA con-structs distributed throughout both the coding and the30UTR regions were observed to reduce TBK1 protein by�90% and reduced viability up to approximately 90%whenmeasured by total cellular ATP levels (Fig. 1). All six shRNAstested gave a robust knockdown of TBK1 protein in twoKRAS-mutant NSCLC cell lines, H23 and A549 (Fig. 1B,top). However, the degree of protein knockdown did notcorrelate with decreased viability (Fig. 1B, bottom). Specif-ically, shTBK1-T15 (30UTR), which showed a very stronglevel of TBK1 knockdown, had no impact on cell growth inboth of these NSCLC cell lines as well as in 7 cell lines testedfrom PDAC and colorectal cancer cell line panels, includingboth KRAS-mutant (G12A, G12C, G12D, G12S, andQ61H) and KRAS-wt genotypic profiles (Table 1). Amongthe six shRNAs tested, only shTBK1-T15 (30UTR) andshTBK1-3182 (CDS) repeatedly gave�90%knockdown inmultiple cell lines. On the other hand, shTBK1-T17(30UTR) with a partial knockdown of TBK1 protein hadpotent antiproliferative activity in both H23 and A549 celllines. The lack of growth inhibition with shTBK1-3182 andshTBK1-T15 in most of the tested PDAC and colorectalcancer cell lines suggested that in these cell lines specifically,antiproliferative effects were not dependent on TBK1.

Table 2. List of PDACs used for endogenous protein expression profiling for pTBK1S172, TBK1, IRF3,pIRF3S386, and GAPDH for Western blotting (as presented in Fig. 2A)

Cell line ID Source Pancreatic cell lines Kras status

1 CRL-1420 ATCC MiaPaca-2 G12V2 CRL-2553 ATCC Panc02.03 G12D3 P09 Belfer HuPt4 G12V4 CRL-1687 ATCC Bxpc-3 WT5 HTB-134 ATCC Hs766T Q61H6 CRL-1469 ATCC Panc-1 G12D7 CRL-2558 ATCC PL-45 G12D8 CRL-1918 ATCC Cfpac-1 G12V9 P15 Belfer Dang G12V10 CRL-1997 ATCC Hpaf-II G12D11 CRL-1682 ATCC Aspc-1 G12D12 JCRB0182 JCRB KP-4 G12D13 JCRB0177.1 JCRB KP-1NL G12D14 P21 Belfer Patu 8902 G12V15 JCRB1094 JCRB Suit-2 Not determined16 CRL-2554 ATCC Panc02.13 Q61R17 CRL-2557 ATCC Panc05.04 G12D18 CRL-2380 ATCC Mpanc96 G12V19 JCRB0178.1 JCRB KP-3L Not determined20 JCRB0178.0 JCRB KP-3 G12V

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pIRF3S386 is a target engagement marker for TBK1 incancer cell linesTo identify a target engagement biomarker for TBK1

activity, we screened six different cancer cell line panels (atotal of 79 cancer cell lines; see Supplementary Data),consisting of 16 NSCLC cell lines, 20 PDAC, 10 colorectalcancer, 16 ovarian cancer, six lymphoma, and 11 glioblas-toma multiforma (GBM), for expression of the TBK1pathway proteins (i.e., IRF3, pIRF3S386, cRel, and RalB)by Western blot analysis (data not shown). From thisprofiling analysis, PDAC cell lines had the highest basalpIRF3S386 levels that correlated well with high basalpTBK1S172 levels (Fig. 2A and Table 2). However, manyof the NSCLC cell lines had very low or undetectablepIRF3S386 levels even following poly I:C stimulation (datanot shown). This limited our interpretation of the shRNAknockdown experiments in NSCLC cell lines due to theabsence of a viable target engagement marker for the assess-ment of the TBK1 activity.Thus, we used the two KRAS-mutant PDAC cell lines,

Panc 02.13 and Panc 05.04, which had high basal pIRF3S386

levels, to validate pIRF3S386 as a target engagement markerfor TBK1 activity.We performed TBK1 knockdown experi-ments using four different shRNAs (shT15, sh3182,sh3185, and sh3186) in these two PDAC cell lines. UponshRNAknockdown ofTBK1 in Panc 02.13 and Panc 05.04,the level of TBK1 protein was significantly reduced and thiscorrelated with a concomitant reduction in pIRF3S386 levels,indicating that pIRF3S386 could be used as a target engage-ment marker for TBK1 activity in cells (Fig. 2B). However,the knockdown of TBK1 did not result in a prominentinhibition of cell proliferation in these twoKRAS-dependentPDAC cell lines that had high pIRF3S386 levels (Fig. 2C).

Evaluation of TBK1 tool inhibitors in cancer cell lineswith high endogenous pIRF3S386

Complimentary data were obtained by evaluating theimpact of a set of potent ATP-competitive TBK1 inhibitors(Table 3). A total of 6 potent TBK1 inhibitors, which werefrom three different structural series (32, 38, 39), have beensynthesized. These inhibitors were characterized by bothbiochemical as well as cell-based assays (Table 3). The IC50

Table 3. TBK1 tool inhibitors: The biochemical and biologic activities and chemical selectivity profiles

Compound #1 Compound #2 Compound #3 Compound #4 Compound #5 Compound #6

Ulight @ 5 mmol/LATP IC50 (nmol/L)

0.6 0.7 0.6 0.6 4.8 3.6

Ulight @ 250 mmol/LATP IC50 (nmol/L)

2.6 2.3 4.3 2.5 32 14.5

IKKe IC50 (nmol/L) 3.9 2.2 2.3 3.2 220 66plRF3 IC50 (Panc05.04nmol/L)

1.8 ND ND ND 140 39

IRF3 Translocation IC50

(nmol/L)95 270 130 76 100 220

CTG H23 IC50 (nmol/L) 3,100 2,000 1,700 2,600 >20,000 7,000CTG H28 IC50 (nmol/L) 2,400 910 710 880 8,400 3,000CTG A549 IC50 (nmol/L) 1,900 3,200 1,400 350 11,000 4,400CTG H441 IC50 (nmol/L) 3,300 5,900 1,300 7,000 11,000 ND

Invitrogen (>100�, %) 98 (305) 95 (266) 97 (101) 98 (266) 90 (101) 91 (266)

NOTE: Top row section, First and second rows represent the IC50 values of TBK1 inhibitors obtained fromUlight kinase assay at 5 and250 mmol/L ATP concentration for inhibition of TBK1 biochemical function; and the third row represents the IC50 values of inhibitors inthe same type of assay for inhibition of IKKe biochemical function (at 10 mmol/L ATP). Middle row section, pIRF3 IC50 values calculatedfrom Western blotting quantification of pIRF3S386 levels, IRF3 IC50 values obtained from IRF3 nuclear translocation assay, and thecellular IC50 values obtained from ATP CellTiter-Glo (CTG) luminescent cell viability assay in H23, H28, A549 and H441 cell lines, arerepresented. Bottom row section, Invitrogen kinase panel screening summary representing the percentage of kinases testedwith eachTBK1 inhibitor that had an IC50 > 100� of the IC50 for TBK1 biochemical function in the same screening assay. [(The synthesis of thesesix tool compounds were reported previously, i.e., Compounds #1–4 (32), compound #5 (38), and compound #6 (39)].Abbreviation: ND, not determined.






values of these inhibitors ranged between 2.3 to 32 nmol/Lin the biochemical assay and they were active in the cellularIRF3 translocation assay (Table 3). The compounds wereused to treat Panc02.13 cells for 4 hours and displayednear full inhibition of pIRF3S386 signal by Western blotting(Fig. 3A). Interestingly, a compensatory increase inpTBK1S172 levels was observed, suggesting a feedback reac-tivation response of the TBK1 pathway. All three inhibitorsmodulated the phosphorylation of pTBK1S172 to varyingdegrees, but not the total TBK1 levels, which remainedunchanged (Fig. 3A).Multiple PDAC cell lines showed potent inhibition of

pIRF3S386 by TBK1 inhibitors (Fig. 3B) and this furthersupported pIRF3S386 as a valid target engagement markerfor measurement of TBK1 cellular activity. Similar datawere obtained in multiple colorectal cancer cell lines (datanot shown) by these TBK1 inhibitors that substantiatedpIRF3-S386 as the target engagement marker for TBK1activity. For several cell lines, we quantified this reductionin pIRF3S386 and then fit the data to a three-parameterhyperbolic–binding function to determine the apparentpotency on each cell line based on inhibition of pIRF3S386

(Fig. 3B). The IC50 values of these three TBK1 toolinhibitors were between 10 and 850 nmol/L, and this

was consistent with the expected potency based on anIRF3 nuclear translocation assay (Table 3). These dataindicated that the inhibitors were indeed getting into thecells and inhibiting the activity of TBK1.These inhibitors were then used to evaluate their impact on

proliferation in a panel of NSCLC, PDAC, colorectal cancer,ovarian cancer, GBM, and lymphoma cell lines (Fig. 4A;Supplementary Data). A typical dose–response curve isshown for a TBK1 inhibitor, compound #1, and the controlMEK inhibitor (PD-0325901) using Panc 02.13 cells (Fig.4A). The IC50 of compound #1 in the cell proliferation assay(IC50: �5 mmol/L, Fig. 4A) was significantly higher than itsIC50 in the cell-based target engagement assay, (IC50pIRF3S386 ¼ 35 nmol/L; Fig. 3B). Among the panel of18 cancer cell lines screened using these TBK1 inhibitors, asubmicromolar IC50 was rarely observed (Fig. 4B; Supple-mentary Data). Therefore, we concluded that the inhibitionof the TBK1 pathway, as measured by pIRF3S386, was notsufficient to inhibit the growth of these cancer cell lines.Using the cell line screening data for compound #1 from a

panel of 21 KRAS-wt and -mutant NSCLC cell lines (seeSupplementary Data), a RAS gene expression analysis wascarried out to assess whether the sensitivity of these NSCLCcell lines to the TBK1 inhibitor (compound #1) correlated

Figure 1. KRAS-mutant NSCLC celllines showed variable sensitivityanddependency to the knockdownof TBK1. The effect of knockdownof TBK1was studied usingmultipleshRNAs targeting both CDS and30UTR sequences of TBK1 in H23and A549 cell lines. A, thedistribution of the six shRNAstargeting TBK1 CDS and 30UTRregions is shown. B, the relativeexpression of TBK1, KRAS, andGAPDH by Western blotting isshown following lentiviraltransduction of shRNAs targetingTBK1 and KRAS at day 8posttransduction. Below eachWestern blot analysis set is a graphrepresenting the change in cellgrowth following shRNAknockdown of TBK1 and KRAS atday 8 detected using the ATPCellTiter-Glo luminescent cellviability assay.

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with the RAS signatures. This approach could potentiallyuncover a TBK1 KRAS dependency that requires >95%inhibition of its activity, which was not achieved in ourknockdown experiments. This could also explain the largeshift in the IC50 values of inhibitors that were obtained inthe cell growth assays. It was previously shown by Lobodaand colleagues (37) that the quantification of RAS-depen-dent gene expression would provide a better measure ofRAS activity in cancer cells than mutations alone. For thispurpose, the RAS pathway signatures for this NSCLC cellline panel were calculated using the methods described byLoboda and colleagues (37). The RAS score analysis usedexpression profiling data from the Broad Cancer Cell LineEncyclopedia (CCLE) database for these cell lines (seeSupplementary Data). The IC50 values of compound #1

and PD0325901 for the 21 NSCLC cell lines are listed inthe Supplementary Data. Our analysis indicated no cor-relation between the cell potency of the compound #1,and the RAS scores in this panel of 21 NSCLC cell lines(Fig. 4C). These data provided additional evidence thatthese RAS-driven NSCLC cell lines did not depend onTBK1 alone. In contrast, there was a good correlationbetween the potency of the positive control MEK inhib-itor (PD0325901) and the RAS scores of the NSCLC celllines that were tested.

Molecular rescue with shRNA-resistant TBK1 alsorestores pIRF3S386 levelsSince we validated pIRF3S386 as a biomarker for TBK1

activity, we set out to determine whether we could rescue the

Figure 2. pIRF3S386 is elevated inpancreatic cancer cell lines and is abiomarker of TBK1 activity. A, theendogenous protein expressionprofiling data for pTBK1S172, TBK1,IRF3, pIRF3S386, and GAPDH byWestern blotting in 20 PDAC celllines (listed in Table 2). B, the levelof TBK1, pIRF3S386, IRF3, andGAPDH expression followingknockdown of TBK1 by theindicated shRNAs in Panc 02.13and Panc 05.04. C, the change incell growth following shRNAknockdown of TBK1 at day 8 asdescribed in Fig. 1.






effects of TBK1 shRNA inhibition with shRNA-resistantTBK1 (Fig. 5). The H23 NSCLC cell line was chosenbecause this was the only cell line that had robust growthinhibition upon TBK1 knockdown with multiple shRNAs(Fig. 1B). As shown earlier, the shTBK1-T17 (targeting the30UTR region) effectively knocked down TBK1 in the H23cell line (�90%), which resulted in inhibition of cellproliferation (ffi80%). The KRAS knockdown by shRNAwas used as a positive control (Fig. 1B). In an engineeredH23 cell line, which stably overexpressed TBK1-WT cDNA(missing the 30UTR region), the knockdown of endogenousTBK1 using shTBK1-T17 still inhibited the proliferation ofthe H23 cell line (Fig. 5A and 5B, bottom) even thoughshRNA-resistant TBK1 levels were not reduced (byWesternblotting), as expected (Fig. 5A and 5B, top). This suggestedthat the antiproliferative effect, whichwas originally detectedin the H23 cell line after knockdown of TBK1, was not due

to on-target effects of TBK1 knockdown.Most importantly,cells expressing shRNA-resistantTBK1-WTcDNA retainedfull pIRF3S386 in the presence of shTBK1-T17, demon-strating rescue of both the TBK1 protein and its pathwayactivity, which further supports the value of pIRF3S386 as avalidated biomarker for TBK1.

DiscussionAdvances in targeted therapies for cancer have accelerated

in the past 5 years because they often have improved out-comes for selected patient populations. Identifying specificbiomarkers for patient selection and for effective pathwayinhibition is a key for the development of targeted thera-peutics. With this in mind, we evaluated the role of TBK1, anoncanonical IKK, which has been reported to be animportant kinase in cancer (4). TBK1 inhibition in a panel

Figure 3. TBK1 inhibitors reducedendogenous p-IRF3S386 in Panc02.13 cells. A, pTBK1S172,pIRF3S386, TBK1, IRF3, and b-actinexpression following treatmentwith the indicated concentrationsof the TBK1 inhibitors, compound#1, compound #5, and compound#6 at 4 hours are shown. Top set,TBK1S172and pIRF3S386; bottomset, corresponding total protein. B,TBK1 inhibitor dose–responsecurves on inhibition of pIRF3S386 inPanc 02.13, Panc 05.04, and KP-3L. The corresponding IC50 valuesfor each inhibitor are shown.

Muvaffak et al.





of 79 cancer cell lines (35 KRAS-mutant and 18 KRAS-wt,confirmed by CCLE) was achieved using small-moleculeinhibitors as well as shRNAs to knockdownTBK1; however,we did not observe consistent growth reduction in vitro. Toevaluate the corresponding pathway effects in cells, wevalidated pIRF3S386, which is a direct substrate of TBK1,as a target engagement biomarker for TBK1 activity. Wealso demonstrated good correlation between activatedTBK1 (pTBK1S172) and pIRF3S386 in multiple cancercell lines. Although TBK1 activity was effectively reducedby TBK1 inhibitors and by some short hairpins targetingTBK1, there was no clear evidence of TBK1 beingessential in regulation of cell growth and proliferation inthese cancer cell lines.The role of TBK1 in activation of the NF-kB pathway in

immune cells via phosphorylation of IkB and IRF3/IRF7has been extensively documented (6–9); however, themechanisms that activate proteins and pathways down-stream of TBK1 in cancer still remain incompletely under-stood. Some studies have placed TBK1 downstream of RASsignaling, increasing the potential for TBK1 therapeutics incancer (6, 8). Upon activation of RAS signaling, RalB, a

target of TBK1, was reported to be downregulated and thiswas shown to control the expression of other downstreamtargets, which were important in regulation of apoptosis andcell survival (i.e., Bcl-XL and Akt). This further suggestedthat TBK1 was related to proteins downstream of RAS,which were involved in regulation of cancer cell survival(3, 9). Another study showed that shRNA knockdown ofTBK1 inhibited phosphorylation of AKT at Ser-473 and,thus, mediated its prosurvival role in U2OS and HBEC celllines (8). In our preliminary studies using Western blotanalysis to assess the changes in pAKTS473 and pAKTT308

levels in cancer cell lines, no changes were observed eitherfollowing shRNA knockdown of TBK1 or treating the celllines withTBK1 inhibitors. Recently,Marion and colleagues(27) also identified SIKE as a TBK1 substrate, and have alsoshown thatTBK1 inhibition resulted in inhibition of TBK1-mediated IRF3 phosphorylation. Furthermore, a systematicRNAi screening study (6) revealed that TBK1 was specif-ically required for survival of lung cancer cells harboringoncogenic KRAS mutations and also provided additionalevidence, suggesting that Bcl-XL was important for TBK1-sensitive lung cancer survival.

Figure 4. Multiple TBK1 kinaseinhibitors showed only modestactivity in three different cancer cellline panels. A, example growthinhibition curves from Panc 02.13cells after treatingwith the titrationsof the indicatedTBK1 inhibitors andthe MEK inhibitor PD-0325901 atday 4 are shown. The viability wasassessed using the same methodasdescribed in Fig. 1.B, IC50 valuesfrom the selected PDAC, colorectalcancer, and NSCLC cancer celllines using TBK1 inhibitorcompound #1. C, correlationanalysis of the RAS score versussensitivity to the TBK1 inhibitorcompound #1 in a panel of 26NSCLC cell lines is shown.






Our results did not demonstrate a dependency of TBK1 inKRAS-mutant NSCLC cell lines, consistent with some otherrecent reports, even though some shRNAs and inhibitors canreduce cell proliferation (5, 8). In two prior TBK1 shRNAknockdown studies, there was no overlap in NSCLC celllines tested, and the reported growth inhibition used differ-ent shRNAs (6, 8). Furthermore, in prior reports, the rescueof the growth inhibition phenotype using shRNA-resistantTBK1 was not performed. Here, we used a larger over-lapping set of shRNAs, including two novel 30UTR-target-ing shRNAs. We also included two NSCLC cell linespreviously shown to be sensitive to TBK1 knockdown(A549 and H23) as well as many other KRAS-mutant cancercell lines. Collectively, these knockdown studies argue that

the role of TBK1 in cell proliferation and growth mechan-isms still remain to be elucidated. Identifying these deter-minants and codependencies, especially in the context ofRAS mutations, could still have significant implications fortargeted therapy of KRAS-mutant cancers (40).Using small-molecule kinase inhibitors of TBK1 is a

complementary approach to a genetic knockdown strategyfor clarifying the role of TBK1 in cancer. Recently, small-molecule inhibitors of TBK1 tested in lung, pancreas, andcolon cancer cell lines revealed a small subset of cell linessensitive to TBK1 inhibition but this sensitivity did notcorrelate with KRAS dependence (5, 8). Here, we usedTBK1 inhibitors of three different structural classes andfound only weak growth reduction and this activity didnot correlate with reduction of pIRF3S386 levels. Inaddition, the activity observed in a large panel of celllines did not correlate with the RAS signatures of those celllines (37). Therefore, TBK1 inhibition alone is notsufficient to drive a robust growth inhibition phenotypein these cell lines. It is possible that by combininginhibitors of other KRAS-activated pathways would be amore effective treatment paradigm (40).TBK1 integrates a variety of upstream signals via stimu-

lation of TLRs and directly modulates the function ofnumerous downstream targets. Indeed, it is unclear whatTBK1 substrates are important for cancer survival, and wehave provided evidence that pIRF3S386 is a direct substrate ofTBK1 for some NSCLC, PDAC, and colorectal cancer celllines. Previous studies have demonstrated the potential forTBK1 to autophosphorylate when overexpressed in 293 Tcells (3). However, it is unclear what other endogenouskinases can activate TBK1, though it is known that ubiqui-tination regulated by its binding partners TRAM, TRIF,TRAF3, and RalB, controls its activation. In the presence ofTBK1 inhibitors, TBK1 remains phosphorylated and in fact,TBK1 phosphorylation seems to increase, suggesting afeedback mechanism that stimulates the endogenous acti-vating kinase (10). It also is likely that depending on whatupstream signals are activating TBK1 (i.e., TLR4, TGFa,etc.) different downstream targets might be involved inregulating the pathway. Recent advances in the availabilityand specificity of TBK1 inhibitors coupled with validatedbiomarkers such as pIRF3S386 should provide improvedtools to help address these issues and hopefully providebetter insights into our understating of the complex networkof TBK1 regulatory pathways in cancer. Continued researchon these emerging pathways will hopefully yield key insightsinto TBK1 biology and establish important directions intotreating diseases targeted through TBK1.

Disclosure of Potential Conflicts of InterestQ. Pan is a senior principal scientist at Boehringer Ingelheim. P. Strack is a director

at Merck & Co. E.J. Morris is a Scientist at Merck. No potential conflicts of interestwere disclosed by the other authors.

Authors' ContributionsConception and design: A. Muvaffak, Q. Pan, R. Fernandez, J. Lim, H. Hirsch,C. Seidel-Dugan, R.E. Middleton, Y. WangDevelopment of methodology: A. Muvaffak, Q. Pan, R. Fernandez, B. Dolinski,H. Hirsch, K.-K. Wong, C. Seidel-Dugan, R.E. Middleton, Y. Wang

Figure 5. Rescue experiment using an engineered H23-TBK1-WToverexpression cell line and the 30UTR-targeting shRNA-T17. A,confirmation of the efficient knockdown of TBK1 using shT17 in the H23cell line (byWestern blotting, top), and the growth inhibition phenotype ina two-dimensional cell viability assay (bottom). B, an engineered cell linestably expressing TBK1-WT cDNA (H23-TBK1-WT) was used to attemptthe rescue of the shRNA-T17 antiproliferative effect. Top, the change inexpression levels of TBK1,pTBK1S172, andpIRFS386 and IRF3byWesternblotting, and bottom is showing the change in growth profiles uponshRNA knockdown with the indicated shRNAs. shRNA lentiviraltransductionsweredone inparallel in bothH23native and theH23-TBK1-WT overexpression cell lines, and the cell growth profiles weremonitoredusing the viability assay at day 8 as described in Fig. 1. This experimentwas repeated two times.

Muvaffak et al.





Acquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): A. Muvaffak, R. Fernandez, B. Dolinski, T.T. Nguyen, S. Wu,R. Chung, W. Zhang, C. Hulton, S. Ripley, K. Nagashima, K.-K. WongAnalysis and interpretation of data (e.g., statistical analysis, biostatistics, compu-tational analysis): A. Muvaffak, R. Fernandez, B. Dolinski, T.T. Nguyen, P. Strack,R. Chung, W. Zhang, C. Hulton, H. Hirsch, K. Nagashima, C. Seidel-Dugan,R.E. MiddletonWriting, review, and/or revision of the manuscript: A. Muvaffak, J. Lim,B. Dolinski, T.T. Nguyen, H. Hirsch, P.A. J�anne, C. Seidel-Dugan, L. Zawel,P.T. Kirschmeier, R.E. Middleton, E.J. Morris, Y. WangAdministrative, technical, or material support (i.e., reporting or organizing data,constructing databases): H. Yan, R. Fernandez, P. Strack, S. Wu, R. Chung,W. Zhang, C. Hulton, K. NagashimaStudy supervision: A. Muvaffak, Q. Pan, R. Fernandez, J. Lim, P.A. J�anne,P.T. Kirschmeier, R.E. Middleton, E.J. Morris, Y. Wang

AcknowledgmentsThe authors thank Drs. Jessie English and Steve Fawell as the senior man-

agement from Belfer Institute for Applied Cancer Science, Dana-Farber CancerInstitute and Merck Inc. for their continuous support and guidance all throughoutthis project. The authors thank Minou Modabber, Director DFCI Medical ArtsCore, for her excellent work on preparing the figures and the tables and herextensive teamwork on the details.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be herebymarked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

Received December 9, 2013; revised March 12, 2014; accepted April 4, 2014;published OnlineFirst April 21, 2014.

References1. Shen RR, Hahn WC. Emerging roles for the noncanonical IKKs in

cancer. Oncogene 2011;30:631–41.2. Kim JY,Welsh EA, Oguz U, Fang B, Bai Y, Kinose F, et al. Dissection of

TBK1 signaling via phosphoproteomics in lung cancer cells. Proc NatlAcad Sci U S A 2013;110:12414–9.

3. Tu D, Zhu Z, Zhou AY, Yun CH, Lee KE, Toms AV, et al. Structure andubiquitination-dependent activation of TANK-binding kinase 1. CellRep 2013;3:747–58.

4. Kim JY, Beg AA, Haura EB. Noncanonical IKKs, IKKe and TBK1, asnovel therapeutic targets in the treatment of non–small cell lungcancer. Expert Opin Ther Targets 2013;17:1109–12.

5. Vangamudi B, Ayres AE, Burke JP, Waterson AG, Rossanese OW,Fesik SW. Evaluation of TBK1 as a novel cancer target in the KRASpathway [abstract]. In: Proceedings of the 103rd AnnualMeeting of theAmerican Association for Cancer Research; 2012 Mar 31–Apr 4;Chicago, IL: AACR; 2012. Abstract nr 1813.

6. Barbie DA, Tamayo P, Boehm JS, Kim SY, Moody SE, Dunn IF, et al.Systematic RNA interference reveals that oncogenic Kras-driven can-cers require TBK1. Nature 2009;462:108–12.

7. Xie X, Zhang D, Zhao B, Lu MK, You M, Condorelli G, et al.IkappaB kinase epsilon and TANK-binding kinase 1 activate AKT bydirect phosphorylation. Proc Natl Acad Sci U S A 2011;108:6474–9.

8. Ou YH, Torres M, Ram R, Formstecher E, Roland C, Cheng T, et al.TBK1 directly engages Akt/PKB survival signaling to support onco-genic transformation. Mol Cell 2011;41:458–470.

9. Chien Y, Kim S, Bumeister R, Loo YM, Kwon SW, Johnson CL, et al.RalB GTPase-mediated activation of the IkappaB family kinase TBK1couples innate immune signaling to tumor cell survival. Cell2006;127:157–70.

10. Clark K, Plater L, Peggie M, Cohen P. Use of the pharmacologicalinhibitor BX795 to study the regulation and physiological roles ofTBK1 and IkappaB kinase epsilon: a distinct upstream kinasemediates Ser-172 phosphorylation and activation. J Biol Chem2009;284:14136–46.

11. Larabi A, Devos JM, Ng SL, Nanao MH, Round A, Maniatis T, et al.Crystal structure andmechanism of activation of TANK-binding kinase1. Cell Rep 2013;3:734–46.

12. The Human Protein Atlas project (funded by the Knut & Alice Wallen-berg foundation), http://www.proteinatlas.org/search/tbk1.

13. Bonnard M, Mirtsos C, Suzuki S, Graham K, Huang J, Ng M, et al.Deficiency of T2K leads to apoptotic liver degeneration and impairedNf-kappaB–dependent gene transcription. EMBO J 2000;19:4976—85.

14. Li Q, Van Antwerp D, Mercurio F, Lee KF, Verma IM. Severe liverdegeneration in mice lacking the IkappaB kinase 2 gene. Science1999;284:321–5.

15. Clark K, Takeuchi O, Akira S, Cohen P. The TRAF-associated proteinTANK facilitates cross-talk within the IkappaB kinase family duringToll-like receptor signaling. Proc Natl Acad Sci U S A 2011;108:17093–8.

16. Fitzgerald KA, McWhirter SM, Faia KL, Rowe DC, Latz E, GolenbockDT, et al. IKKepsilon and TBK1 are essential components of the IRF3signaling pathway. Nat Immunol 2003;4:491–6.

17. Sharma S, tenOever BR, Grandvaux N, Zhou GP, Lin R, Hiscott J.Triggering the interferon antiviral response through an IKK-relatedpathway. Science 2003;300:1148–51.

18. Black KE, Collins SL, Hagan RS, Hamblin MJ, Chan-Li Y, HallowellRW, Powell JD, Horton MR. Hyaluronan fragments induce IFNb via anovel TLR4-TRIF-TBK1-IRF3-dependent pathway. J Inflamm 2013;10:23.

19. Goncalves A, B€urckst€ummer T, Dixit E, Scheicher R, G�orna MW,Karayel E, et al. Functional dissection of the TBK1 molecular network.PLoS ONE 2011;6:e23971.

20. Pomerantz JL, Baltimore D. NF-kappaB activation by a signalingcomplex containing TRAF2, TANK, TBK1, a novel IKK related kinase.EMBO J 1999;18:6694–704.

21. Shimada T, Kawai T, TakedaK,MatsumotoM, Inoue J, Tatsumi Y, et al.IKK-i, a novel lipopolysaccharide-inducible kinase that is related that isrealted to IkappaB kinases. Int Immunol 1999;11:1357–62.

22. Chau TL, Gioia R, Gatot JS, Patrascu F, Carpentier I, Chapelle JP, et al.Are the IKKs and IKK-related kinases TBK1 and IKK-epsilon similarlyactivated? Trends Biochem Sci 2008;33:171–80.

23. Takahasi K, Suzuki NN, Horiuchi M, Mori M, Suhara W, Okabe Y, et al.X-ray crystal structure of IRF-3 and its functional implications. NatStruct Biol 2003;10:922–7.

24. Mori M, Yoneyama M, Ito T, Takahashi K, Inagaki F, Fujita T. Identi-fication of Ser-386 of interferon regulatory factor 3 as critical target forinducible phosphorylation that determines activation. J Biol Chem2004;279:9698–702.

25. Servant MJ, Grandvaux N, tenOever BR, Duguay D, Lin R, Hiscott J.Identification of the minimal phosphoacceptor site required for in vivoactivation of interferon regulatory factor 3 in response to virus anddouble-stranded RNA. J Biol Chem 2003;278:9441–7.

26. Ogasawara N, Sasaki M, Itoh Y, Tokudome K, Kondo Y, Ito Y, et al.Rebamipide suppresses TLR-TBK1 signaling pathway resulting inregulating IRF3/7 and IFNa/b reduction. J Clin Biochem Nutr 2011;48:154–60.

27. Marion JD, Roberts CF, Call RJ, Forbes JL, Nelson KT, Bell JE, et al.Mechanism of endogenous regulation of the type I interferon responseby suppressor of IkB kinase {epsilon} (SIKE), a novel substrate ofTANK-binding kinase 1 (TBK1). J Biol Chem 2013;288:18612–23.

28. Ishikawa H, Barber GN. STING is an endoplasmic reticulum adaptorthat facilitates innate immune signalling. Nature 2008;455:674–8.

29. Chen H, Sun H, You F, Sun W, Zhou X, Chen L, et al. Activation ofSTAT6 by STING is critical for antiviral innate immunity. Cell 2011;147:436–46.

30. Bowie A. The STING in the tail for cytosolic DNA-dependent activationof IRF3. Sci Signal 2012;5:pe9.

31. Ma X, Helgason E, Phung QT, Quan CL, Iyer RS, Lee MW, et al.Molecular basis of Tank-binding kinase 1 activation by transautopho-sphorylation. Proc Natl Acad Sci U S A 2012;109:9378–83.

32. Holcomb R, Suzuki K, Halter RJ, Sebahar PR, Mcleod DA, Shender-ovich MD, et al. Amino-Pyrimidine Compounds as Inhibitors of TBK1and/or IKK epsilon. 2011 (patent WO/2011/046970).

33. McIver EG, Bryans J, Birchall K, Chugh J, Drake T, Lewis SJ, et al.Synthesis and structure-activity relationships of a novel series of






pyrimidines as potent inhibitors of TBK1/IKKe kinases. Bioorg MedChem Lett 2012;22:7169–73.

34. Boehm JS, Zhao JJ, Yao J, Kim SY, Firestein R, Dunn IF, et al.Integrative genomic approaches idebtify IKBKE as a breast canceroncogene. Cell 2007;129:1065–79.

35. Hutti JE, Shen RR, Abbott DW, Zhou AY, Sprott KM, Asara JM, et al.Phosphorylation of the tumor suppressor CYLD by the breast canceroncogene IKKepsilon promotes cell transformation. Mol Cell2009;34:461–72.

36. Shen RR, Zhou AY, Kim E, Lim E, Habelhah H, Hahn WC. IkB kinase ephosphorylates TRAF2 to promote mammary epithelial cell transfor-mation. Mol Cell Biol 2012;32:4756–68.

37. Loboda A, NebozhynM, Klinghoffer R, Frazier J, ChastainM, ArthurW,et al. A gene expression signature of RAS pathway dependencepredicts response to PI3K and RAS pathway inhibitors and expandsthe population ofRASpathway activated tumors. BMCMedGenomics2010;3:26.

38. McIver EG, Bryans JS, Smiljanic E, Lewis SJ, Hough J, Drake T.Preparation of pyrimidine derivatives capable of inhibiting one or morekinases. 2009 (patent WO 2009122180).

39. McIver EG, Hough J. Preparation of pyrrolopyrimidines as kinaseinhibitors. 2010 (patent WO 2010100431).

40. Li J,HuangJ, JeongJH,ParkSJ,WeiR,PengJ, et al. SelectiveTBK1/IKKidual inhibitors with anticancer potency. Int J Cancer 2013;134:1972–80.


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2014;12:1055-1066. Published OnlineFirst April 21, 2014.Mol Cancer Res Asli Muvaffak, Qi Pan, Haiyan Yan, et al. IRF3Evaluating TBK1 as a Therapeutic Target in Cancers with Activated

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