Evaluating TBK1 As a Therapeutic Target in Cancers with Activated IRF3

Published OnlineFirst April 21, 2014; DOI: 10.1158/1541-7786.MCR-13-0642

Molecular Cancer Signal Transduction Research

Evaluating TBK1 as a Therapeutic Target in Cancers with Activated IRF3

Asli Muvaffak1, Qi Pan1, Haiyan Yan1, Rafael Fernandez3, Jongwon Lim3, Brian Dolinski3, Thi T. Nguyen3, Peter Strack3, Stephen Wu1, Rossana Chung1, Weiqun Zhang1, Chris Hulton1, Steven Ripley1, Heather Hirsch3, Kumiko Nagashima3, Kwok-Kin Wong1,2, Pasi A. Janne 1,2, Cynthia Seidel-Dugan3, Leigh Zawel3, Paul T. Kirschmeier1, Richard E. Middleton1, Erick J. Morris3, and Yan Wang3

Abstract TBK1 (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 reports demonstrated synthetic lethality of TBK1 with mutant KRAS in non–small cell lung cancer (NSCLC); thus, TBK1 could be a novel target for treatment of KRAS-mutant NSCLC. Here, the effect of TBK1 on proliferation in a panel 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 reduced proliferation in a two-dimensional viability assay. Verification of target engagement via reduced phosphorylation of S386 of IRF3 (pIRF3S386) was difficult to assess in NSCLC cells due to low protein expression. However, several cell lines were identified with high pIRF3S386 levels after screening a large panel of cell lines, many of which also harbor KRAS mutations. Specifically, a large subset of KRAS-mutant pancreatic cancer cell lines was uncovered with high constitutive 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 with inhibition of cell growth. Taken together, these data demonstrate that the regulation of pathways important for cell proliferation in some NSCLC, pancreatic, and colorectal cell lines is not solely dependent on TBK1 activity. Implications: TBK1 has therapeutic potential under certain contexts and phosphorylation of its downstream target IRF3 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.

Introduction tively expressed in many normal tissues, including immune k cells, brain, lungs, gastrointestinal tract, and reproductive TBK1 (TANK-binding kinase 1) is a noncanonical I B fi kinase, which shares 65% similarity to IKKe and is highly organs (11, 12). TBK1-de cent mice exhibit embryonic expressed in lung, breast, pancreatic, and colon cancers (1). lethality due to widespread hepatic apoptosis (13, 14). TBK1 is regulated through activation by Toll-like receptors Besides its key role in innate immune response, increasing – evidence indicates that the activation of TBK1 and its close (TLR) or cytoplasmic RIG-1 like receptors, and it stimulates e type I IFN production via direct phosphorylation of IFN homolog IKK is associated with the development of human – cancers (2–10). TBK1 is an 84 kDa, 729 amino acid–long regulatory transcription factor 3 (IRF3) and 7 (IRF7; refs. 15 22). IRF3 phosphorylation at residues Ser-385 and Ser-386 protein containing an N-terminal kinase domain, a ubiqui- – tin-like domain and two C-terminal coiled-coil domains. has previously been shown to control its dimerization (23 25) TBK1 is an NF-kB–activating kinase, which is constitu- upon which IRF3 translocates to the nucleus (26), and controls transcriptional regulation of cell survival and prolif- Authors' Affiliations: 1Belfer Institute for Applied Cancer Science; eration (2, 11). In addition to IRF3, several other substrates, i. 2Department of Medical Oncology, Dana-Farber Cancer Institute; and RalB fi 3 e., namely cRel, Akt, , SIKE, etc., have been identi ed Merck Research Laboratories, Boston, Massachusetts that attribute to TBK1 function (7–9, 27–30). TBK1 requires Note: Supplementary data for this article are available at Molecular Cancer phosphorylation at Ser-172 within the kinase domain acti- Research Online (http://mcr.aacrjournals.org/). vation loop to be activated. Phosphorylation at Ser-172 has Corresponding Authors: Asli Muvaffak, Belfer Institute for Applied Cancer been shown previously to induce reorganization of the acti- Science, Dana-Farber Cancer Institute, 4 Blackfan Street, HIM Building, Room 320A. Boston, MA 02115. Phone: 857-453-0907; Fax: 617-582- vation loop allowing the active site in the kinase domain to 9702; E-mail: [email protected], and Yan Wang, TESARO bind to the substrate (31). TBK1 inhibitors of different Inc., 1000 Winter Street, Waltham, MA, [email protected] chemical structures have been developed by multiple groups doi: 10.1158/1541-7786.MCR-13-0642 and it was shown previously that a PDK1/TBK1 inhibitor, 2014 American Association for Cancer Research. BX795, enhanced phosphorylation of TBK1 while inhibiting

www.aacrjournals.org 1055

Muvaffak et al.

its activity, suggesting a feedback control mechanism that following cell seeding, the cells were treated with the tool stimulates phosphorylation of TBK1 (10, 32, 33). inhibitor titrations for 4 days at 37C and then assayed by Although the role of TBK1 in cancer still remains unclear, using the ATP CellTiter-Glo luminescent cell viability assay recent studies suggested its role in regulation of cell growth (Promega; G7570). and proliferation, and oncogenic transformation (2, 4, 9). For Incucyte analysis (Essen BioScience, Inc.), cells were Shen and Hahn (1) first demonstrated the role of both TBK1 plated as described above in 96-well plates, and image-based and IKKe in cell transformation. IKKe, a breast cancer cell confluence data were collected every 2 hours during live oncogene that is amplified in 30% of breast cancers, med- growth. iates activation of NF-kB signaling and is required for – Plasmid constructs transformation in breast cancer (34 36). TBK1 was iden- – tified through a functional genomics screen, which showed pLKO.1 shRNAs [targeting TBK1 Coding Sequence KRAS – (CDS) regions] were purchased from the Broad Institute that a subset of -mutant non small cell lung cancer 0 – (NSCLC) cell lines was dependent on TBK1 and that TBK1 (Cambridge, MA). Of note, 3 untranslated region (UTR) KRAS targeting shRNAs and control shRNAs for GFP and KRAS was essential for mutant cancer cell lines (6). This fi generated an interest in small molecule inhibitors of TBK1 sequences were speci cally designed for this study (see that may provide a novel therapeutic strategy for KRAS- Supplementary Data). Human full-length TBK1 cDNA mutant tumors. However, subsequent studies were not able was purchased from Origene (SC111251). shRNA-resistant TBK1 was made by PCR cloning of TBK-WT without to confirm the proposed synthetic lethal relationship of 0 oncogenic KRAS and TBK1 (5, 8). In these studies, knock- the 3 UTR region using newly designed primers, and then down of TBK1 did not consistently result in a significant subcloned into the pLenti-CMV-Hygro-DEST (w117-1) vector (Addgene) using Gateway cloning. All shRNAs and alteration of growth, and the sensitivity to TBK1 inhibition fi by either siRNA or small-molecule inhibitors did not cor- cDNAs were con rmed by sequencing. KRAS relate with status in lung, pancreatic, or colorectal Lentiviral shRNA transduction and proliferation assay cancer cell lines (5). Lentiviral shRNAs were prepared via transfection of the Our study aimed to further investigate the role of TBK1 in corresponding plasmid DNAs into 293 T cells using Lipo- cancer by studying TBK1 knockdown using RNAi techno- fectamine 2000 (Invitrogen; 52887) in OptiMEM (Gibco; fi logy as well as small molecule inhibitors. We identi ed a 31985-070) media. The lentiviruses were packaged using subset of pancreatic ductal adenocarcinoma cell lines (PDAC) D S836 the p 8.9 and VSVG packaging vectors in early passage 293 that have high constitutive pIRF3 levels and showed that T cells. TBK1 inhibitors dose-dependently inhibited pIRF3S386.In S836 Both NSCLC and PDAC cancer cell lines were plated at addition, shRNA knockdown of TBK1 reduced pIRF3 150 to 250 K cells per well density in 6-well plates on day 0, in pancreatic cancer cell lines, and, thus, further supported S836 and the lentiviral transductions were performed on day 1 pIRF3 as a target engagement marker for TBK1 activity using polybrene (8 mg/mL; EMD Millipore # TR-1003-G). in these cancer cell lines. However, no robust inhibition of The cells were selected in 2 mg/mL puromycin for 3 days and growth was achieved in the two-dimensional proliferation on day 4 cells were plated in 1 mg/mL puromycin (Life assay in these PDAC cell lines as well as in the majority of Technologies; # A1113803) into 96-well assay plates. Cell NSCLC cell lines either by genetic knockdown or by using viability was assessed on day 8 after viral transductions using TBK1 inhibitors. Our data demonstrate that reduction of S836 the ATP CellTiter-Glo luminescent cell viability assay (Pro- TBK1 activity and the subsequent elimination of pIRF3 mega; G7570). The growth profiles of cell lines with and fi are not suf cient on their own to regulate cell proliferation in without shRNA knockdown were also recorded and analyzed the NSCLC and PDAC cell lines that were tested. by the Incucyte Imaging System (Essen BioScience, Inc.). Materials and Methods The level of knockdown of TBK1 protein was determined by Western blot analysis using the samples collected on day 8. Cell lines and tool inhibitor treatment conditions Cell lines (see Supplementary Data) were obtained from Protein detection various commercial vendors (ATCC, DSMZ, JCRB, and Cell lysates for Western blotting were collected using RIPA ECACC). The cell line authentication details are listed in the lysis buffer (Boston Bioproducts; BP-115) supplemented Supplementary Data. All cell lines were grown in recom- with 1 Halt Protease inhibitor (VWR International; VWR mended media by the ATCC supplemented with 10% fetal # PI78415), 5 mmol/L NaF (Sigma-Aldrich Co.) and 10 bovine serum (Atlanta Biologicals Inc.). All cultures were mmol/L b-glycerol phosphate (Sigma-Aldrich Co.). Protein fi – m maintained at 37 C in a humidi ed 5% CO2 atmosphere (10 15 g) was separated on 4% to 15% Mini-PROTEAN and tested periodically to ensure the absence of mycoplasma. TGX Precast Gels (Bio-Rad Laboratories; 456-1086), and on Generally, cells were passaged upon reaching approximately 4% to 15% Criterion TGX Precast Gel (Bio-Rad Labora- 75% confluence. tories; 5671085). The proteins were transferred onto a Tool inhibitor stocks and dilutions were made in dimethyl polyvinylidene difluoride membrane using the TransBlot sulfoxide solvent. Cell proliferation experiments were carried Turbo Transfer System (Bio-Rad Laboratories; 170–4155). out in a 96-well format (6 replicates), the cells were plated at a Protein levels from Western blots were quantified by density of 2,000 to 5,000 cells per well. At 24 hours densitometry using the ImageJ software and this was

1056 Mol Cancer Res; 12(7) July 2014 Molecular Cancer Research

Phosphorylated IRF3 Is a Biomarker of TBK1 Activity

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

KRAS shRNAs tested Growth inhibition (%; Cell Tissue mutation (which had TBK1 normalized to shGFP; line origin status pIRF3S386 IRF3 pTBK1S172 TBK1 KD 90% by WB) average SD) H23 Lung G12C ()NA()(þþ) shT15 shT15: 1.26 1.90 shT17 shT17: 82.74 1.41 sh3182 sh3182: 51.74 1.96 sh3183 sh3183: 35.02 3.10 sh3185 sh3185: 48.84 0.95 sh3186 sh3186: 46.53 0.77 549 Lung G12S (þ)NA(þþ)(þþ) shT15 shT15: 3.17 1.33 shT17 shT17: 77.34 0.64 sh3182 sh3182: 36.94 2.23 sh3183 sh3183: 17.44 1.95 sh3185 sh3185: 26.63 0.49 sh3186 sh3186: 34.88 1.99 H28 Lung WT ()NA()(þþ) shT15 shT15: 10.06 6.35 shT17 shT17: 14.30 2.62 Panc02.13 PDAC Q61R (þþþ)(þ)(þþþ)(þþ) shT15 shT15: 12.66 2.39 sh3182 sh3182: 16.26 1.77 sh3185 sh3185: 37.84 3.83 sh3186 sh3186: 39.24 4.03 Panc05.04 PDAC G12D (þþþ)(þ)(þþ)(þþ) shT15 shT15: 6.99 4.09 sh3182 sh3182: 26.02 5.07 sh3185 sh3185: 1.49 5.19 sh3186 sh3186: 1.44 3.44 CFPAC-1 PDAC G12V ()()()(þþ) shT15 shT15: 35.85 9.41 sh3182 sh3182: 21.19 7.01 SUIT2 PDAC G12D ()(þ)()(þþ) shT15 shT15: 35.52 9.44 sh3182 sh3182: 42.06 9.21 PL45 PDAC G12D (þþþ)(þ)(þþþ)(þþ) shT15 shT15: 0.39 6.65 sh3182 sh3182: 19.10 6.45 LOVO Colon G13D (þþ)(þþ)()(þþ) shT15 shT15: 6.00 4.99 shT17 shT17: 47.00 1.44 sh3182 sh3182: 48.00 2.99 WiDr Colon WT (þ)(þþ)()(þþ) shT15 shT15: 13.68 2.39 sh3182 sh3182: 31.00 2.61

NOTE: The corresponding effects in the two-dimensional cell proliferation assay obtained from ATP CellTiter-Glo (CTG) luminescent cell viability assay using NSCLC, colorectal, and PDAC cancer cell lines is reported as cell growth inhibition as the percentage of shGFP control. aBasal protein levels at 10% fetal bovine serum.

normalized to the loading control for each sample. The band TBK1 S172 (Abgent; AP3627a), IRF3 (Cell Signaling Tech- signal intensities were from the raw chemiluminescent nology; D83B9,CST 4302), phospho-IRF3 S386(Epitomics; fi fi images on lms. For IC50 calculations, these data were t Clone ID EPR2346, 2562-1), KRAS (Santa Cruz Biotech- with a standard three-parameter dose–response nonlinear nology Inc., F234, sc-30) GAPDH (Cell Signaling Technol- regression fit using GraphPad Prism software. ogy; horseradish peroxidase-conjugate, 14C10, CST 3683).

Antibodies Assays used for initial validation of tool inhibitors Western blots were probed with antibodies against TBK1 The TBK1 tool inhibitors were initially screened using the (Cell Signaling Technology; D1B4, CST 3504), phospho- full-length TBK1 protein in a biochemical assay (Ulight

www.aacrjournals.org Mol Cancer Res; 12(7) July 2014 1057

Muvaffak et al.

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 G12V 2 CRL-2553 ATCC Panc02.03 G12D 3 P09 Belfer HuPt4 G12V 4 CRL-1687 ATCC Bxpc-3 WT 5 HTB-134 ATCC Hs766T Q61H 6 CRL-1469 ATCC Panc-1 G12D 7 CRL-2558 ATCC PL-45 G12D 8 CRL-1918 ATCC Cfpac-1 G12V 9 P15 Belfer Dang G12V 10 CRL-1997 ATCC Hpaf-II G12D 11 CRL-1682 ATCC Aspc-1 G12D 12 JCRB0182 JCRB KP-4 G12D 13 JCRB0177.1 JCRB KP-1NL G12D 14 P21 Belfer Patu 8902 G12V 15 JCRB1094 JCRB Suit-2 Not determined 16 CRL-2554 ATCC Panc02.13 Q61R 17 CRL-2557 ATCC Panc05.04 G12D 18 CRL-2380 ATCC Mpanc96 G12V 19 JCRB0178.1 JCRB KP-3L Not determined 20 JCRB0178.0 JCRB KP-3 G12V

kinase assay; LANCE Ultra ULight, Perkin Elmer) in which Results fi two lead series were identi ed. Six tool inhibitors (Table 3) TBK1 dependency did not correlate with the KRAS were selected and these underwent profiling studies in a list KRAS– mutation status of wild-type (wt) and -mutant NSCLC cell lines (as TBK1 has been proposed as a potential therapeutic target described above and in Table 3). In addition, these tool for KRAS-dependent cancer (6). We evaluated the role of inhibitors were further characterized for their ability to TBK1 in KRAS-mutant NSCLC cell lines further using both inhibit IRF3 nuclear translocation using a high-content fl fi genetic and pharmacologic approaches. Six shRNA con- immuno uorescence imaging assay. This assay quanti ed structs distributed throughout both the coding and the the amount of IRF3 protein which translocated into the 30UTR regions were observed to reduce TBK1 protein by nucleus of the HeLa cell line, a surrogate cell line for tool 90% and reduced viability up to approximately 90% when inhibitor screening; The HeLa cells were stimulated with measured by total cellular ATP levels (Fig. 1). All six shRNAs Poly I:C (Invivogen; tlrl-pic) following treatment with tested gave a robust knockdown of TBK1 protein in two TBK1 tool inhibitors; (Table 3). The assay uses an anti- KRAS-mutant NSCLC cell lines, H23 and A549 (Fig. 1B, IRF3 mAb (Epitomics; cat.# 2241-1), and a secondary Ab fl top). However, the degree of protein knockdown did not labeled with a red uorophore (goat anti-rabbit IgG correlate with decreased viability (Fig. 1B, bottom). Specif- (HþL), DyLight 549 Conjugated; Thermo Scientificcat 0 fl ically, shTBK1-T15 (3 UTR), which showed a very strong no.35558).Immunouorescence image data were ana- level of TBK1 knockdown, had no impact on cell growth in lyzed using a Columbus script (Opera/Acapella/Columbus both of these NSCLC cell lines as well as in 7 cell lines tested platform for high-content screening—PerkinElmer) that fi from PDAC and colorectal cancer cell line panels, including quanti ed mean intensity of IRF3 signal in the nucleus both KRAS-mutant (G12A, G12C, G12D, G12S, and after image segmentation. Q61H) and KRAS-wt genotypic profiles (Table 1). Among the six shRNAs tested, only shTBK1-T15 (30UTR) and Kinase panel screening of tool inhibitors Thekinaseinhibitoryprofilesofinhibitorswereeval- shTBK1-3182 (CDS) repeatedly gave 90% knockdown in multiple cell lines. On the other hand, shTBK1-T17 uated by using 101, 266, and 305 kinase panels at 0 Invitrogen. (3 UTR) with a partial knockdown of TBK1 protein had potent antiproliferative activity in both H23 and A549 cell RAS pathway signature analysis lines. The lack of growth inhibition with shTBK1-3182 and Gene transcription signature of the RAS pathway was shTBK1-T15 in most of the tested PDAC and colorectal defined as described by Loboda and colleagues (see Supple- cancer cell lines suggested that in these cell lines specifically, mentary Data; ref. 37). antiproliferative effects were not dependent on TBK1.

1058 Mol Cancer Res; 12(7) July 2014 Molecular Cancer Research

Phosphorylated IRF3 Is a Biomarker of TBK1 Activity

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/L 0.6 0.7 0.6 0.6 4.8 3.6

ATP IC50 (nmol/L) Ulight @ 250 mmol/L 2.6 2.3 4.3 2.5 32 14.5

ATP IC50 (nmol/L)

IKKe IC50 (nmol/L) 3.9 2.2 2.3 3.2 220 66

plRF3 IC50 (Panc05.04 1.8 ND ND ND 140 39 nmol/L)

IRF3 Translocation IC50 95 270 130 76 100 220 (nmol/L)

CTG H23 IC50 (nmol/L) 3,100 2,000 1,700 2,600 >20,000 7,000

CTG H28 IC50 (nmol/L) 2,400 910 710 880 8,400 3,000

CTG A549 IC50 (nmol/L) 1,900 3,200 1,400 350 11,000 4,400

CTG 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 from Ulight kinase assay at 5 and

250 mmol/L ATP concentration for inhibition of TBK1 biochemical function; and the third row represents the IC50 values of inhibitors in

the same type of assay for inhibition of IKKe biochemical function (at 10 mmol/L ATP). Middle row section, pIRF3 IC50 values calculated S386 from Western blotting quantiﬁcation of pIRF3 levels, IRF3 IC50 values obtained from IRF3 nuclear translocation assay, and the

cellular IC50 values obtained from ATP CellTiter-Glo (CTG) luminescent cell viability assay in H23, H28, A549 and H441 cell lines, are represented. Bottom row section, Invitrogen kinase panel screening summary representing the percentage of kinases tested with each

TBK1 inhibitor that had an IC50 > 100 of the IC50 for TBK1 biochemical function in the same screening assay. [(The synthesis of these six tool compounds were reported previously, i.e., Compounds #1–4 (32), compound #5 (38), and compound #6 (39)]. Abbreviation: ND, not determined. pIRF3S386 is a target engagement marker for TBK1 in levels, to validate pIRF3S386 as a target engagement marker cancer cell lines for TBK1 activity. We performed TBK1 knockdown experi- To identify a target engagement biomarker for TBK1 ments using four different shRNAs (shT15, sh3182, activity, we screened six different cancer cell line panels (a sh3185, and sh3186) in these two PDAC cell lines. Upon total of 79 cancer cell lines; see Supplementary Data), shRNA knockdown of TBK1 in Panc 02.13 and Panc 05.04, consisting of 16 NSCLC cell lines, 20 PDAC, 10 colorectal the level of TBK1 protein was signiﬁcantly reduced and this cancer, 16 ovarian cancer, six lymphoma, and 11 glioblas- correlated with a concomitant reduction in pIRF3S386 levels, toma multiforma (GBM), for expression of the TBK1 indicating that pIRF3S386 could be used as a target engage- pathway proteins (i.e., IRF3, pIRF3S386, cRel, and RalB) ment marker for TBK1 activity in cells (Fig. 2B). However, by Western blot analysis (data not shown). From this the knockdown of TBK1 did not result in a prominent proﬁling analysis, PDAC cell lines had the highest basal inhibition of cell proliferation in these two KRAS-dependent pIRF3S386 levels that correlated well with high basal PDAC cell lines that had high pIRF3S386 levels (Fig. 2C). pTBK1S172 levels (Fig. 2A and Table 2). However, many of the NSCLC cell lines had very low or undetectable Evaluation of TBK1 tool inhibitors in cancer cell lines pIRF3S386 levels even following poly I:C stimulation (data with high endogenous pIRF3S386 not shown). This limited our interpretation of the shRNA Complimentary data were obtained by evaluating the knockdown experiments in NSCLC cell lines due to the impact of a set of potent ATP-competitive TBK1 inhibitors absence of a viable target engagement marker for the assess- (Table 3). A total of 6 potent TBK1 inhibitors, which were ment of the TBK1 activity. from three different structural series (32, 38, 39), have been Thus, we used the two KRAS-mutant PDAC cell lines, synthesized. These inhibitors were characterized by both S386 Panc 02.13 and Panc 05.04, which had high basal pIRF3 biochemical as well as cell-based assays (Table 3). The IC50

www.aacrjournals.org Mol Cancer Res; 12(7) July 2014 1059

Muvaffak et al.

Figure 1. KRAS-mutant NSCLC cell lines showed variable sensitivity and dependency to the knockdown of TBK1. The effect of knockdown of TBK1 was studied using multiple shRNAs targeting both CDS and 30UTR sequences of TBK1 in H23 and A549 cell lines. A, the distribution of the six shRNAs targeting TBK1 CDS and 30UTR regions is shown. B, the relative expression of TBK1, KRAS, and GAPDH by Western blotting is shown following lentiviral transduction of shRNAs targeting TBK1 and KRAS at day 8 posttransduction. Below each Western blot analysis set is a graph representing the change in cell growth following shRNA knockdown of TBK1 and KRAS at day 8 detected using the ATP CellTiter-Glo luminescent cell viability assay.

values of these inhibitors ranged between 2.3 to 32 nmol/L was consistent with the expected potency based on an in the biochemical assay and they were active in the cellular IRF3 nuclear translocation assay (Table 3). These data IRF3 translocation assay (Table 3). The compounds were indicated that the inhibitors were indeed getting into the used to treat Panc02.13 cells for 4 hours and displayed cells and inhibiting the activity of TBK1. near full inhibition of pIRF3S386 signal by Western blotting These inhibitors were then used to evaluate their impact on (Fig. 3A). Interestingly, a compensatory increase in proliferation in a panel of NSCLC, PDAC, colorectal cancer, pTBK1S172 levels was observed, suggesting a feedback reac- ovarian cancer, GBM, and lymphoma cell lines (Fig. 4A; tivation response of the TBK1 pathway. All three inhibitors Supplementary Data). A typical dose–response curve is modulated the phosphorylation of pTBK1S172 to varying shown for a TBK1 inhibitor, compound #1, and the control degrees, but not the total TBK1 levels, which remained MEK inhibitor (PD-0325901) using Panc 02.13 cells (Fig. unchanged (Fig. 3A). 4A). The IC50 of compound #1 in the cell proliferation assay m fi Multiple PDAC cell lines showed potent inhibition of (IC50: 5 mol/L, Fig. 4A) was signi cantly higher than its S386 pIRF3 by TBK1 inhibitors (Fig. 3B) and this further IC50 in the cell-based target engagement assay, (IC50 supported pIRF3S386 as a valid target engagement marker pIRF3S386 ¼ 35 nmol/L; Fig. 3B). Among the panel of for measurement of TBK1 cellular activity. Similar data 18 cancer cell lines screened using these TBK1 inhibitors, a were obtained in multiple colorectal cancer cell lines (data submicromolar IC50 was rarely observed (Fig. 4B; Supple- not shown) by these TBK1 inhibitors that substantiated mentary Data). Therefore, we concluded that the inhibition pIRF3-S386 as the target engagement marker for TBK1 of the TBK1 pathway, as measured by pIRF3S386, was not activity. For several cell lines, we quantified this reduction sufficient to inhibit the growth of these cancer cell lines. in pIRF3S386 and then fit the data to a three-parameter Using the cell line screening data for compound #1 from a hyperbolic–binding function to determine the apparent panel of 21 KRAS-wt and -mutant NSCLC cell lines (see potency on each cell line based on inhibition of pIRF3S386 Supplementary Data), a RAS gene expression analysis was (Fig.3B).TheIC50 values of these three TBK1 tool carried out to assess whether the sensitivity of these NSCLC inhibitors were between 10 and 850 nmol/L, and this cell lines to the TBK1 inhibitor (compound #1) correlated

1060 Mol Cancer Res; 12(7) July 2014 Molecular Cancer Research

Phosphorylated IRF3 Is a Biomarker of TBK1 Activity

Figure 2. pIRF3S386 is elevated in pancreatic cancer cell lines and is a biomarker of TBK1 activity. A, the endogenous protein expression proﬁling data for pTBK1S172, TBK1, IRF3, pIRF3S386, and GAPDH by Western blotting in 20 PDAC cell lines (listed in Table 2). B, the level of TBK1, pIRF3S386, IRF3, and GAPDH expression following knockdown of TBK1 by the indicated shRNAs in Panc 02.13 and Panc 05.04. C, the change in cell growth following shRNA knockdown of TBK1 at day 8 as described in Fig. 1.

with the RAS signatures. This approach could potentially and PD0325901 for the 21 NSCLC cell lines are listed in uncover a TBK1 KRAS dependency that requires >95% the Supplementary Data. Our analysis indicated no cor- inhibition of its activity, which was not achieved in our relation between the cell potency of the compound #1, knockdown experiments. This could also explain the large and the RAS scores in this panel of 21 NSCLC cell lines shift in the IC50 values of inhibitors that were obtained in (Fig. 4C). These data provided additional evidence that the cell growth assays. It was previously shown by Loboda these RAS-driven NSCLC cell lines did not depend on and colleagues (37) that the quantiﬁcation of RAS-depen- TBK1 alone. In contrast, there was a good correlation dent gene expression would provide a better measure of between the potency of the positive control MEK inhib- RAS activity in cancer cells than mutations alone. For this itor (PD0325901) and the RAS scores of the NSCLC cell purpose, the RAS pathway signatures for this NSCLC cell lines that were tested. line panel were calculated using the methods described by Loboda and colleagues (37). The RAS score analysis used Molecular rescue with shRNA-resistant TBK1 also expression proﬁling data from the Broad Cancer Cell Line restores pIRF3S386 levels Encyclopedia (CCLE) database for these cell lines (see Since we validated pIRF3S386 as a biomarker for TBK1 Supplementary Data). The IC50 values of compound #1 activity, we set out to determine whether we could rescue the

www.aacrjournals.org Mol Cancer Res; 12(7) July 2014 1061

Muvaffak et al.

Figure 3. TBK1 inhibitors reduced endogenous p-IRF3S386 in Panc 02.13 cells. A, pTBK1S172, pIRF3S386, TBK1, IRF3, and b-actin expression following treatment with the indicated concentrations of the TBK1 inhibitors, compound #1, compound #5, and compound #6 at 4 hours are shown. Top set, TBK1S172and pIRF3S386; bottom set, corresponding total protein. B, TBK1 inhibitor dose–response curves on inhibition of pIRF3S386 in Panc 02.13, Panc 05.04, and KP- 3L. The corresponding IC50 values for each inhibitor are shown.

effects of TBK1 shRNA inhibition with shRNA-resistant to on-target effects of TBK1 knockdown. Most importantly, TBK1 (Fig. 5). The H23 NSCLC cell line was chosen cells expressing shRNA-resistant TBK1-WT cDNA retained because this was the only cell line that had robust growth full pIRF3S386 in the presence of shTBK1-T17, demon- inhibition upon TBK1 knockdown with multiple shRNAs strating rescue of both the TBK1 protein and its pathway (Fig. 1B). As shown earlier, the shTBK1-T17 (targeting the activity, which further supports the value of pIRF3S386 as a 30UTR region) effectively knocked down TBK1 in the H23 validated biomarker for TBK1. cell line (90%), which resulted in inhibition of cell proliferation (ﬃ80%). The KRAS knockdown by shRNA was used as a positive control (Fig. 1B). In an engineered Discussion H23 cell line, which stably overexpressed TBK1-WT cDNA Advances in targeted therapies for cancer have accelerated (missing the 30UTR region), the knockdown of endogenous in the past 5 years because they often have improved out- TBK1 using shTBK1-T17 still inhibited the proliferation of comes for selected patient populations. Identifying speciﬁc the H23 cell line (Fig. 5A and 5B, bottom) even though biomarkers for patient selection and for effective pathway shRNA-resistant TBK1 levels were not reduced (by Western inhibition is a key for the development of targeted thera- blotting), as expected (Fig. 5A and 5B, top). This suggested peutics. With this in mind, we evaluated the role of TBK1, a that the antiproliferative effect, which was originally detected noncanonical IKK, which has been reported to be an in the H23 cell line after knockdown of TBK1, was not due important kinase in cancer (4). TBK1 inhibition in a panel

1062 Mol Cancer Res; 12(7) July 2014 Molecular Cancer Research

Phosphorylated IRF3 Is a Biomarker of TBK1 Activity

Figure 4. Multiple TBK1 kinase inhibitors showed only modest activity in three different cancer cell line panels. A, example growth inhibition curves from Panc 02.13 cells after treating with the titrations of the indicated TBK1 inhibitors and the MEK inhibitor PD-0325901 at day 4 are shown. The viability was assessed using the same method as described in Fig. 1. B, IC50 values from the selected PDAC, colorectal cancer, and NSCLC cancer cell lines using TBK1 inhibitor compound #1. C, correlation analysis of the RAS score versus sensitivity to the TBK1 inhibitor compound #1 in a panel of 26 NSCLC cell lines is shown.

of 79 cancer cell lines (35 KRAS-mutant and 18 KRAS-wt, target of TBK1, was reported to be downregulated and this conﬁrmed by CCLE) was achieved using small-molecule was shown to control the expression of other downstream inhibitors as well as shRNAs to knockdown TBK1; however, targets, which were important in regulation of apoptosis and we did not observe consistent growth reduction in vitro.To cell survival (i.e., Bcl-XL and Akt). This further suggested evaluate the corresponding pathway effects in cells, we that TBK1 was related to proteins downstream of RAS, validated pIRF3S386, which is a direct substrate of TBK1, which were involved in regulation of cancer cell survival as a target engagement biomarker for TBK1 activity. We (3, 9). Another study showed that shRNA knockdown of also demonstrated good correlation between activated TBK1 inhibited phosphorylation of AKT at Ser-473 and, TBK1 (pTBK1S172)andpIRF3S386 in multiple cancer thus, mediated its prosurvival role in U2OS and HBEC cell cell lines. Although TBK1 activity was effectively reduced lines (8). In our preliminary studies using Western blot by TBK1 inhibitors and by some short hairpins targeting analysis to assess the changes in pAKTS473 and pAKTT308 TBK1, there was no clear evidence of TBK1 being levels in cancer cell lines, no changes were observed either essential in regulation of cell growth and proliferation in following shRNA knockdown of TBK1 or treating the cell these cancer cell lines. lines with TBK1 inhibitors. Recently, Marion and colleagues The role of TBK1 in activation of the NF-kB pathway in (27) also identiﬁed SIKE as a TBK1 substrate, and have also immune cells via phosphorylation of IkB and IRF3/IRF7 shown that TBK1 inhibition resulted in inhibition of TBK1- has been extensively documented (6–9); however, the mediated IRF3 phosphorylation. Furthermore, a systematic mechanisms that activate proteins and pathways down- RNAi screening study (6) revealed that TBK1 was specif- stream of TBK1 in cancer still remain incompletely under- ically required for survival of lung cancer cells harboring stood. Some studies have placed TBK1 downstream of RAS oncogenic KRAS mutations and also provided additional signaling, increasing the potential for TBK1 therapeutics in evidence, suggesting that Bcl-XL was important for TBK1- cancer (6, 8). Upon activation of RAS signaling, RalB,a sensitive lung cancer survival.

www.aacrjournals.org Mol Cancer Res; 12(7) July 2014 1063

Muvaffak et al.

the role of TBK1 in cell proliferation and growth mechanisms still remain to be elucidated. Identifying these deter- minants and codependencies, especially in the context of RAS mutations, could still have signiﬁcant implications for targeted therapy of KRAS-mutant cancers (40). Using small-molecule kinase inhibitors of TBK1 is a complementary approach to a genetic knockdown strategy for clarifying the role of TBK1 in cancer. Recently, small- molecule inhibitors of TBK1 tested in lung, pancreas, and colon cancer cell lines revealed a small subset of cell lines sensitive to TBK1 inhibition but this sensitivity did not correlate with KRAS dependence (5, 8). Here, we used TBK1 inhibitors of three different structural classes and found only weak growth reduction and this activity did not correlate with reduction of pIRF3S386 levels. In addition, the activity observed in a large panel of cell lines did not correlate with the RAS signatures of those cell lines (37). Therefore, TBK1 inhibition alone is not sufﬁcient to drive a robust growth inhibition phenotype in these cell lines. It is possible that by combining inhibitors of other KRAS-activated pathways would be a more effective treatment paradigm (40). TBK1 integrates a variety of upstream signals via stimulation of TLRs and directly modulates the function of numerous downstream targets. Indeed, it is unclear what TBK1 substrates are important for cancer survival, and we have provided evidence that pIRF3S386 is a direct substrate of TBK1 for some NSCLC, PDAC, and colorectal cancer cell lines. Previous studies have demonstrated the potential for TBK1 to autophosphorylate when overexpressed in 293 T

Figure 5. Rescue experiment using an engineered H23-TBK1-WT cells (3). However, it is unclear what other endogenous overexpression cell line and the 30UTR-targeting shRNA-T17. A, kinases can activate TBK1, though it is known that ubiqui- confirmation of the efficient knockdown of TBK1 using shT17 in the H23 tination regulated by its binding partners TRAM, TRIF, cell line (by Western blotting, top), and the growth inhibition phenotype in TRAF3, and RalB, controls its activation. In the presence of a two-dimensional cell viability assay (bottom). B, an engineered cell line TBK1 inhibitors, TBK1 remains phosphorylated and in fact, stably expressing TBK1-WT cDNA (H23-TBK1-WT) was used to attempt the rescue of the shRNA-T17 antiproliferative effect. Top, the change in TBK1 phosphorylation seems to increase, suggesting a expression levels of TBK1, pTBK1S172, and pIRFS386 and IRF3 by Western feedback mechanism that stimulates the endogenous acti- blotting, and bottom is showing the change in growth profiles upon vating kinase (10). It also is likely that depending on what shRNA knockdown with the indicated shRNAs. shRNA lentiviral upstream signals are activating TBK1 (i.e., TLR4, TGFa, transductions were done in parallel in both H23 native and the H23-TBK1- WT overexpression cell lines, and the cell growth profiles were monitored etc.) different downstream targets might be involved in using the viability assay at day 8 as described in Fig. 1. This experiment regulating the pathway. Recent advances in the availability was repeated two times. and specificity of TBK1 inhibitors coupled with validated biomarkers such as pIRF3S386 should provide improved tools to help address these issues and hopefully provide Our results did not demonstrate a dependency of TBK1 in better insights into our understating of the complex network KRAS-mutant NSCLC cell lines, consistent with some other of TBK1 regulatory pathways in cancer. Continued research recent reports, even though some shRNAs and inhibitors can on these emerging pathways will hopefully yield key insights reduce cell proliferation (5, 8). In two prior TBK1 shRNA into TBK1 biology and establish important directions into knockdown studies, there was no overlap in NSCLC cell treating diseases targeted through TBK1. lines tested, and the reported growth inhibition used different shRNAs (6, 8). Furthermore, in prior reports, the rescue Disclosure of Potential Conflicts of Interest of the growth inhibition phenotype using shRNA-resistant Q. 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 interest TBK1 was not performed. Here, we used a larger over- were disclosed by the other authors. lapping set of shRNAs, including two novel 30UTR-targeting shRNAs. We also included two NSCLC cell lines Authors' Contributions previously shown to be sensitive to TBK1 knockdown Conception and design: A. Muvaffak, Q. Pan, R. Fernandez, J. Lim, H. Hirsch, KRAS C. Seidel-Dugan, R.E. Middleton, Y. Wang (A549 and H23) as well as many other -mutant cancer Development of methodology: A. Muvaffak, Q. Pan, R. Fernandez, B. Dolinski, cell lines. Collectively, these knockdown studies argue that H. Hirsch, K.-K. Wong, C. Seidel-Dugan, R.E. Middleton, Y. Wang

1064 Mol Cancer Res; 12(7) July 2014 Molecular Cancer Research

Phosphorylated IRF3 Is a Biomarker of TBK1 Activity

Acquisition of data (provided animals, acquired and managed patients, provided Acknowledgments facilities, etc.): A. Muvaffak, R. Fernandez, B. Dolinski, T.T. Nguyen, S. Wu, The authors thank Drs. Jessie English and Steve Fawell as the senior man- R. Chung, W. Zhang, C. Hulton, S. Ripley, K. Nagashima, K.-K. Wong agement from Belfer Institute for Applied Cancer Science, Dana-Farber Cancer Analysis and interpretation of data (e.g., statistical analysis, biostatistics, compu- Institute and Merck Inc. for their continuous support and guidance all throughout tational analysis): A. Muvaffak, R. Fernandez, B. Dolinski, T.T. Nguyen, P. Strack, this project. The authors thank Minou Modabber, Director DFCI Medical Arts R. Chung, W. Zhang, C. Hulton, H. Hirsch, K. Nagashima, C. Seidel-Dugan, Core, for her excellent work on preparing the ﬁguresandthetablesandher R.E. Middleton extensive teamwork on the details. Writing, review, and/or revision of the manuscript: A. Muvaffak, J. Lim, B. Dolinski, T.T. Nguyen, H. Hirsch, P.A. Janne, C. Seidel-Dugan, L. Zawel, The costs of publication of this article were defrayed in part by the payment of page P.T. Kirschmeier, R.E. Middleton, E.J. Morris, Y. Wang advertisement Administrative, technical, or material support (i.e., reporting or organizing data, charges. This article must therefore be hereby marked in accordance with constructing databases): H. Yan, R. Fernandez, P. Strack, S. Wu, R. Chung, 18 U.S.C. Section 1734 solely to indicate this fact. W. Zhang, C. Hulton, K. Nagashima Study supervision: A. Muvaffak, Q. Pan, R. Fernandez, J. Lim, P.A. Janne, Received December 9, 2013; revised March 12, 2014; accepted April 4, 2014; P.T. Kirschmeier, R.E. Middleton, E.J. Morris, Y. Wang published OnlineFirst April 21, 2014.

References 1. Shen RR, Hahn WC. Emerging roles for the noncanonical IKKs in 17. Sharma S, tenOever BR, Grandvaux N, Zhou GP, Lin R, Hiscott J. cancer. Oncogene 2011;30:631–41. Triggering the interferon antiviral response through an IKK-related 2. Kim JY, Welsh EA, Oguz U, Fang B, Bai Y, Kinose F, et al. Dissection of pathway. Science 2003;300:1148–51. TBK1 signaling via phosphoproteomics in lung cancer cells. Proc Natl 18. Black KE, Collins SL, Hagan RS, Hamblin MJ, Chan-Li Y, Hallowell Acad Sci U S A 2013;110:12414–9. RW, Powell JD, Horton MR. Hyaluronan fragments induce IFNb via a 3. Tu D, Zhu Z, Zhou AY, Yun CH, Lee KE, Toms AV, et al. Structure and novel TLR4-TRIF-TBK1-IRF3-dependent pathway. J Inflamm 2013; ubiquitination-dependent activation of TANK-binding kinase 1. Cell 10:23. Rep 2013;3:747–58. 19. Goncalves A, Burckst€ ummer€ T, Dixit E, Scheicher R, Gorna MW, 4. Kim JY, Beg AA, Haura EB. Noncanonical IKKs, IKKe and TBK1, as Karayel E, et al. Functional dissection of the TBK1 molecular network. novel therapeutic targets in the treatment of non–small cell lung PLoS ONE 2011;6:e23971. cancer. Expert Opin Ther Targets 2013;17:1109–12. 20. Pomerantz JL, Baltimore D. NF-kappaB activation by a signaling 5. Vangamudi B, Ayres AE, Burke JP, Waterson AG, Rossanese OW, complex containing TRAF2, TANK, TBK1, a novel IKK related kinase. Fesik SW. Evaluation of TBK1 as a novel cancer target in the KRAS EMBO J 1999;18:6694–704. pathway [abstract]. In: Proceedings of the 103rd Annual Meeting of the 21. Shimada T, Kawai T, Takeda K, Matsumoto M, Inoue J, Tatsumi Y, et al. American Association for Cancer Research; 2012 Mar 31–Apr 4; IKK-i, a novel lipopolysaccharide-inducible kinase that is related that is Chicago, IL: AACR; 2012. Abstract nr 1813. realted to IkappaB kinases. Int Immunol 1999;11:1357–62. 6. Barbie DA, Tamayo P, Boehm JS, Kim SY, Moody SE, Dunn IF, et al. 22. Chau TL, Gioia R, Gatot JS, Patrascu F, Carpentier I, Chapelle JP, et al. Systematic RNA interference reveals that oncogenic Kras-driven can- Are the IKKs and IKK-related kinases TBK1 and IKK-epsilon similarly cers require TBK1. Nature 2009;462:108–12. activated? Trends Biochem Sci 2008;33:171–80. 7. Xie X, Zhang D, Zhao B, Lu MK, You M, Condorelli G, et al. 23. Takahasi K, Suzuki NN, Horiuchi M, Mori M, Suhara W, Okabe Y, et al. IkappaB kinase epsilon and TANK-binding kinase 1 activate AKT by X-ray crystal structure of IRF-3 and its functional implications. Nat direct phosphorylation. Proc Natl Acad Sci U S A 2011;108:6474–9. Struct Biol 2003;10:922–7. 8. Ou YH, Torres M, Ram R, Formstecher E, Roland C, Cheng T, et al. 24. Mori M, Yoneyama M, Ito T, Takahashi K, Inagaki F, Fujita T. Identi- TBK1 directly engages Akt/PKB survival signaling to support onco- fication of Ser-386 of interferon regulatory factor 3 as critical target for genic transformation. Mol Cell 2011;41:458–470. inducible phosphorylation that determines activation. J Biol Chem 9. Chien Y, Kim S, Bumeister R, Loo YM, Kwon SW, Johnson CL, et al. 2004;279:9698–702. RalB GTPase-mediated activation of the IkappaB family kinase TBK1 25. Servant MJ, Grandvaux N, tenOever BR, Duguay D, Lin R, Hiscott J. couples innate immune signaling to tumor cell survival. Cell Identification of the minimal phosphoacceptor site required for in vivo 2006;127:157–70. activation of interferon regulatory factor 3 in response to virus and 10. Clark K, Plater L, Peggie M, Cohen P. Use of the pharmacological double-stranded RNA. J Biol Chem 2003;278:9441–7. inhibitor BX795 to study the regulation and physiological roles of 26. Ogasawara N, Sasaki M, Itoh Y, Tokudome K, Kondo Y, Ito Y, et al. TBK1 and IkappaB kinase epsilon: a distinct upstream kinase Rebamipide suppresses TLR-TBK1 signaling pathway resulting in mediates Ser-172 phosphorylation and activation. J Biol Chem regulating IRF3/7 and IFNa/b reduction. J Clin Biochem Nutr 2011; 2009;284:14136–46. 48:154–60. 11. Larabi A, Devos JM, Ng SL, Nanao MH, Round A, Maniatis T, et al. 27. Marion JD, Roberts CF, Call RJ, Forbes JL, Nelson KT, Bell JE, et al. Crystal structure and mechanism of activation of TANK-binding kinase Mechanism of endogenous regulation of the type I interferon response 1. Cell Rep 2013;3:734–46. by suppressor of IkB kinase {epsilon} (SIKE), a novel substrate of 12. The Human Protein Atlas project (funded by the Knut & Alice Wallen- TANK-binding kinase 1 (TBK1). J Biol Chem 2013;288:18612–23. berg foundation), http://www.proteinatlas.org/search/tbk1. 28. Ishikawa H, Barber GN. STING is an endoplasmic reticulum adaptor 13. Bonnard M, Mirtsos C, Suzuki S, Graham K, Huang J, Ng M, et al. that facilitates innate immune signalling. Nature 2008;455:674–8. Deficiency of T2K leads to apoptotic liver degeneration and impaired 29. Chen H, Sun H, You F, Sun W, Zhou X, Chen L, et al. Activation of Nf-kappaB–dependent gene transcription. EMBO J 2000;19: STAT6 by STING is critical for antiviral innate immunity. Cell 2011; 4976—85. 147:436–46. 14. Li Q, Van Antwerp D, Mercurio F, Lee KF, Verma IM. Severe liver 30. Bowie A. The STING in the tail for cytosolic DNA-dependent activation degeneration in mice lacking the IkappaB kinase 2 gene. Science of IRF3. Sci Signal 2012;5:pe9. 1999;284:321–5. 31. Ma X, Helgason E, Phung QT, Quan CL, Iyer RS, Lee MW, et al. 15. Clark K, Takeuchi O, Akira S, Cohen P. The TRAF-associated protein Molecular basis of Tank-binding kinase 1 activation by transautopho- TANK facilitates cross-talk within the IkappaB kinase family during sphorylation. Proc Natl Acad Sci U S A 2012;109:9378–83. Toll-like receptor signaling. Proc Natl Acad Sci U S A 2011;108: 32. Holcomb R, Suzuki K, Halter RJ, Sebahar PR, Mcleod DA, Shender- 17093–8. ovich MD, et al. Amino-Pyrimidine Compounds as Inhibitors of TBK1 16. Fitzgerald KA, McWhirter SM, Faia KL, Rowe DC, Latz E, Golenbock and/or IKK epsilon. 2011 (patent WO/2011/046970). DT, et al. IKKepsilon and TBK1 are essential components of the IRF3 33. McIver EG, Bryans J, Birchall K, Chugh J, Drake T, Lewis SJ, et al. signaling pathway. Nat Immunol 2003;4:491–6. Synthesis and structure-activity relationships of a novel series of

www.aacrjournals.org Mol Cancer Res; 12(7) July 2014 1065

Muvaffak et al.

pyrimidines as potent inhibitors of TBK1/IKKe kinases. Bioorg Med 37. Loboda A, Nebozhyn M, Klinghoffer R, Frazier J, Chastain M, Arthur W, Chem Lett 2012;22:7169–73. et al. A gene expression signature of RAS pathway dependence 34. Boehm JS, Zhao JJ, Yao J, Kim SY, Firestein R, Dunn IF, et al. predicts response to PI3K and RAS pathway inhibitors and expands Integrative genomic approaches idebtify IKBKE as a breast cancer the population of RAS pathway activated tumors. BMC Med Genomics oncogene. Cell 2007;129:1065–79. 2010;3:26. 35. Hutti JE, Shen RR, Abbott DW, Zhou AY, Sprott KM, Asara JM, et al. 38. McIver EG, Bryans JS, Smiljanic E, Lewis SJ, Hough J, Drake T. Phosphorylation of the tumor suppressor CYLD by the breast cancer Preparation of pyrimidine derivatives capable of inhibiting one or more oncogene IKKepsilon promotes cell transformation. Mol Cell kinases. 2009 (patent WO 2009122180). 2009;34:461–72. 39. McIver EG, Hough J. Preparation of pyrrolopyrimidines as kinase 36. Shen RR, Zhou AY, Kim E, Lim E, Habelhah H, Hahn WC. IkB kinase e inhibitors. 2010 (patent WO 2010100431). phosphorylates TRAF2 to promote mammary epithelial cell transfor- 40. Li J, Huang J, Jeong JH, Park SJ, Wei R, Peng J, et al. Selective TBK1/IKKi mation. Mol Cell Biol 2012;32:4756–68. dual inhibitors with anticancer potency. Int J Cancer 2013;134:1972–80.

1066 Mol Cancer Res; 12(7) July 2014 Molecular Cancer Research

Evaluating TBK1 as a Therapeutic Target in Cancers with Activated IRF3

Asli Muvaffak, Qi Pan, Haiyan Yan, et al.

Mol Cancer Res 2014;12:1055-1066. Published OnlineFirst April 21, 2014.

Updated version Access the most recent version of this article at: doi:10.1158/1541-7786.MCR-13-0642

Supplementary Access the most recent supplemental material at: Material http://mcr.aacrjournals.org/content/suppl/2014/04/22/1541-7786.MCR-13-0642.DC1

Visual A diagrammatic summary of the major findings and biological implications: Overview http://mcr.aacrjournals.org/content/12/7/1055/F1.large.jpg

Cited articles This article cites 35 articles, 14 of which you can access for free at: http://mcr.aacrjournals.org/content/12/7/1055.full#ref-list-1

Citing articles This article has been cited by 3 HighWire-hosted articles. Access the articles at: http://mcr.aacrjournals.org/content/12/7/1055.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at Subscriptions [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://mcr.aacrjournals.org/content/12/7/1055. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.