Published OnlineFirst January 20, 2014; DOI: 10.1158/2159-8290.CD-13-0646

RESEARCH ARTICLE

Inhibition of KRAS -Driven Tumorigenicity by Interruption of an Autocrine Cytokine Circuit

Zehua Zhu 1 , 4, Amir R. Aref 2, Travis J. Cohoon 1 , Thanh U. Barbie 7 , Yu Imamura 1, Shenghong Yang 1 , 4, Susan E. Moody 1 , Rhine R. Shen 1 , Anna C. Schinzel 1 , Tran C. Thai 1 , 4, Jacob B. Reibel 1 , Pablo Tamayo 4 , Jason T. Godfrey 5 , Zhi Rong Qian 1 , Asher N. Page 2 , 3, Karolina Maciag 4 , 6, Edmond M. Chan 1 , 4, Whitney Silkworth 4 , Mary T. Labowsky 1 , Lior Rozhansky 1 , Jill P. Mesirov 4 , William E. Gillanders 7 , Shuji Ogino 1 , Nir Hacohen 4 , 6, Suzanne Gaudet 2 , Michael J. Eck 2 , 3, Jeffrey A. Engelman 5 , Ryan B. Corcoran 5 , Kwok-Kin Wong 1 , William C. Hahn 1 , 4, and David A. Barbie 1 , 4

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ABSTRACT Although the roles of mitogen-activated protein kinase (MAPK) and phospho- inositide 3-kinase (PI3K) signaling in KRAS -driven tumorigenesis are well estab- lished, KRAS activates additional pathways required for tumor maintenance, the inhibition of which are likely to be necessary for effective KRAS-directed therapy. Here, we show that the IκB kinase (IKK)– related kinases Tank-binding kinase-1 (TBK1) and IKKε promote KRAS-driven tumorigenesis by regu- lating autocrine CCL5 and interleukin (IL)-6 and identify CYT387 as a potent JAK/TBK1/IKKε inhibitor. CYT387 treatment ablates RAS-associated cytokine signaling and impairs Kras -driven murine lung cancer growth. Combined CYT387 treatment and MAPK pathway inhibition induces regression of aggressive murine lung adenocarcinomas driven by Kras mutation and p53 loss. These observations reveal that TBK1/IKKε promote tumor survival by activating CCL5 and IL-6 and identify concurrent inhibition of TBK1/IKKε, Janus-activated kinase (JAK), and MEK signaling as an effective approach to inhibit the actions of oncogenic KRAS.

SIGNIFICANCE: In addition to activating MAPK and PI3K, oncogenic KRAS engages cytokine signaling to promote tumorigenesis. CYT387 , originally described as a selective JAK inhibitor, is also a potent TBK/ IKKε inhibitor that uniquely disrupts a cytokine circuit involving CCL5, IL-6, and STAT3. The effi cacy of CYT387-based treatment in murine Kras -driven lung cancer models uncovers a novel therapeutic approach for these refractory tumors with immediate translational implications. Cancer Discov; 4(4); 1–14. ©2014 AACR.

INTRODUCTION associated IL-8, CXCL1, and CXCL2 expression also pro- motes senescence ( 14 ), tumor cell survival, and angiogenesis Oncogenic mutations in KRAS and receptor tyrosine ( 15, 16 ). How oncogenic RAS activates these cytokines and kinases (RTK) drive tumor growth by engaging multiple their role in RAS-dependent cancers remains incompletely downstream mitogenic pathways, including RAF–MAPK and characterized. PI3K–AKT (1 ). KRAS also activates RAL–GEF (2, 3) and Activation of RALA and RALB by RAL–GEF enhances infl ammatory signals such as NF-κB ( 4–6 ). Although concur- cancer cell proliferation and survival (17 ). A specifi c RALB– rent MAPK–PI3K pathway inhibition is under clinical evalu- SEC5 complex engages the innate immune signaling kinase ation, multiple approaches are likely necessary to identify Tank-binding kinase-1 (TBK1) to promote cell survival (18 ). effective KRAS -targeted therapy. TBK1 is required for transformation by oncogenic KRAS, Oncogenic RAS induces infl ammatory cytokines that acti- sustains KRAS -dependent cancer cell viability, and regulates vate NF-κB and STAT3. For example, KRAS-driven non– basal autophagy (18–22 ). The TBK1 homolog inhibitor of small cell lung cancers (NSCLC; ref. 7 ) and pancreatic ductal IκB kinase ε (IKKε; encoded by IKBKE ) also promotes NF-κB adenocarcinomas (PDAC; ref. 8 ) engage cell autonomous activation downstream of KRAS (23 ), substitutes for AKT interleukin (IL)-1 signaling. Similarly, oncogenic RAS- to drive cell transformation (24 ), and is induced by RAS- induced IL-6 promotes both oncogene-induced senescence associated cytokines such as IL-1 and IL-6 (25 ). Thus, TBK1/ ( 9 ) and cell transformation ( 10 ), and STAT3 is required IKKε signaling is coopted by oncogenic KRAS and facilitates for Kras-driven PDAC development in mice (11–13 ). RAS- tumorigenesis. Following viral infection, TBK1 and IKKε amplify IFN-β production via an autocrine loop ( 26 ). Here, we identify a Authors’ Affi liations: Departments of 1 Medical Oncology and 2 Cancer Biol- similar circuit involving CCL5 and IL-6 required for KRAS - ogy, Dana-Farber Cancer Institute; 3 Department of Biological Chemistry driven lung tumorigenesis and potently suppressed by 4 and Molecular Pharmacology, Harvard Medical School, Boston; Broad CYT387, a novel TBK1/IKKε and Janus-activated kinase Institute of Harvard and MIT, Cambridge; 5 MGH Cancer Center, 6 Center for Immunology and Infl ammatory Diseases, Massachusetts General Hospital, (JAK) inhibitor. Charlestown, Massachusetts; and 7 Department of Surgery, Division of Biol- ogy and Biomedical Sciences, Washington University, St. Louis, Missouri Note: Supplementary data for this article are available at Cancer Discovery RESULTS Online (http://cancerdiscovery.aacrjournals.org/). TBK1-Regulated Cell Survival Involves Autocrine A.R. Aref and T.J. Cohoon contributed equally to this work. CCL5 and IL-6 and STAT3 Signaling Corresponding Authors: David A. Barbie, Dana-Farber Cancer Institute, 450 Brookline Avenue D819, Boston, MA 02215. Phone: 617-632-6049; Fax: Expression of Tbk1 is required for transformation by onco- 617-632-5786; E-mail: [email protected] ; William C. Hahn, William_ genic KRAS ( 18 , 20 , 21 ). Although Tbk1 −/− mouse embryonic [email protected]; and Kwok K. Wong, [email protected] fi broblasts (MEF) proliferate in standard culture, we noted doi: 10.1158/2159-8290.CD-13-0646 marked impairment of Tbk1−/− MEF proliferation in a clono- ©2014 American Association for Cancer Research. genic assay compared with wild-type (WT) littermate control

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RESEARCH ARTICLE Zhu et al.

A E –/– F 1,600 Tbk1 MEFs 35 WT MEFs WT 1,400 30 1,200 25 1,000 Tbk1–/– MEFs 800 20 600 15 ** 400 –/– 10

Tbk1 MEFs # Colonies Tbk1–/– 200 5 *

CCL5 levels (pg/mL) 0 0

EGFP EGFP CM IL-6 MEFs –/– CCL5 –/– WT CM TBK1-KD CXCL10 TBK1-WT WT MEFs Tbk1 CCL5+IL-6 TBK1 Tbk1 B Conditioned β-Actin CCL5+IL-6+CXCL10 media WT Tbk1–/– G12V + + G KRAS –/– –/– –/– H Tbk1 MEFs Tbk1 MEFs WT MEFs Tbk1 MEFs WT Tbk1–/– Serum: 0 h 0.5 h 1 h 4 h 0 h 0.5 h 1 h 4 h Serum: 0 h 1 h 0 h 1 h pSTAT3 pSTAT3 (Y705) (Y705) STAT3 STAT3 C TBK1 KRAS –/– WT Tbk1 TBK1 CXCL10 CCL5 CXCL10 CCL5 β-Actin β-Actin

D 1.2 I WT Tbk1–/– Tbk1–/– + CCL5 EGF 1 Ccl5 (min): 0 5 10 20 30 60 90 120 0 5 10 20 30 60 90 120 0 5 10 20 30 60 90 120 Cxcl10 pSTAT3 0.8 Il6 (Y705) 0.6 STAT3

0.4 TBK1

0.2 β-Actin Relative mRNA expression Relative mRNA 0 WT Tbk1–/–

Figure 1. Autocrine CCL5 drives TBK1-dependent clonogenic proliferation. A, crystal violet stain and phase-contrast images (×10) of WT or Tbk1 −/− MEFs cultured in a clonogenic assay for 10 days. B, crystal violet stain of Tbk1−/− MEFs cultured clonogenically in conditioned medium from WT or Tbk1 −/− MEFs. C, cytokine antibody arrays of conditioned medium from WT or Tbk1−/− MEFs. D, mRNA levels of Ccl5, Cxcl10 , or Il6 in WT or Tbk1−/− MEFs. Relative expression normalized to WT MEF mRNA levels, mean and SEM of triplicate samples shown. P < 0.001 for each comparison by t test. E, CCL5 levels by ELISA 72 hours after enhanced GFP (EGFP), TBK1-WT, or TBK1-KD reexpression in Tbk1 −/− MEFs. Mean ± SD of duplicate samples shown. TBK1 and β-actin immunoblot below. F, colony number following media supplementation of Tbk1−/− MEFs, mean ± SEM of six replicates shown. **, P < 0.001 and *, P < 0.01 for comparison with untreated Tbk1−/− MEFs by t test. G, immunoblot of Y705 phospho-STAT3 (pSTAT3), STAT3, TBK1, and β-actin levels in WT or Tbk1 −/− MEFs serum-starved overnight and stimulated with 10% serum for the indicated times. H, immunoblot of Y705 pSTAT3, STAT3, TBK1, KRAS, and β-actin levels 48 hours following KRASG12V expression in serum-starved and stimulated WT or Tbk1−/− MEFs. I, WT or Tbk1−/− MEFs ± CCL5 (10 ng/mL) stimulated with EGF (100 ng/mL). Immunoblot shows Y705 pSTAT3, STAT3, TBK1, and β-actin.

MEFs (Fig. 1A ). To assess the role of cell contact versus a MEFs (Fig. 1D ), whereas others such as Cxcl1 were increased secreted factor, we plated Tbk1−/− MEFs in the clonogenic assay (Supplementary Fig. S1C and S1D). Reintroduction of WT with conditioned medium from WT or Tbk1 −/− MEFs propa- but not kinase-dead (KD) TBK1 restored CCL5 production gated at high density (Fig. 1B ). Conditioned medium from by Tbk1−/− MEFs, revealing kinase-dependent regulation of WT but not Tbk1 −/− MEFs rescued colony formation, revealing this chemokine (Fig. 1E ). that Tbk1 regulates secreted factors that promote cell prolif- To examine the contribution of CCL5, CXCL10, and/or eration and may contribute to KRAS -driven tumorigenesis. IL-6 to TBK1-regulated survival, we supplemented media Because TBK1/IKKε regulates cytokine production, we with each factor and measured Tbk1 −/− MEF colony for- assessed conditioned medium from WT or Tbk1 −/− MEFs mation. CCL5 (10 ng/mL) rescued Tbk1 −/− MEFs colonies using a cytokine antibody array. Tbk1−/− MEF conditioned comparably with WT MEF conditioned medium, whereas medium lacked CCL5 and exhibited decreased CXCL10 lev- IL-6 had a modest effect and CXCL10 failed to rescue colony els compared with WT MEF conditioned medium (Fig. 1C formation (Fig. 1F and Supplementary Fig. S1E). Adding IL-6 and Supplementary Fig. S1A). CCL5 and CXCL10 were also or CXCL10 to CCL5 did not increase Tbk1 −/− MEF colonies. absent in Tbk1 −/− MEF clonogenic culture media (Supple- Thus, autocrine CCL5 and IL-6 signaling promote TBK1- mentary Fig. S1B). Because TBK1 also regulates IL-6 ( 27 ), regulated proliferation/survival. we measured Ccl5, Cxcl10 , and Il6 mRNA levels and observed Because CCL5 and IL-6 induce prosurvival JAK–STAT sig- reduced expression of each cytokine/chemokine in Tbk1 −/− naling, we next measured Y705 pSTAT3 phosphorylation in

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Targeting Oncogenic KRAS via Cytokine Suppression RESEARCH ARTICLE

A D His-TBK1 + IKKε–Tide GST–IKKε + IKKε–Tide LPS-stimulated RAW macrophages 35

IC50 = 228 nmol/L IC50 = 201 nmol/L IC50 = 9.2 nmol/L 100 100 30 25 20 50 50 15 CYT387 CYT387 10 [ADP] (%control) [ADP] (%control) IC50 = 58 nmol/L IC50 = 42 nmol/L 5 –4 –3 –2 –1 0 –4 –3 –2 –1 0

Relative IFNB1 expression 0

Log concentration (µmol/L) Log concentration (µmol/L) 1 5 1 5 1 5 10 10 10 0.1 0.5 0.1 0.5 0.1 0.5 0.01 0.05 0.01 0.05 0.01 0.05 –LPS +LPS B 293T Cells DMSO TBK1-WT IKKε-WT MRT67307 CYT387 Ruxolitinib

Concentration (µmol/L)

18 IC50 = 60 nmol/L IC50 = 358 nmol/L IC50 > 10 μmol/L 16

EGFP EGFP DMSO MRT67307 CYT387 Ruxolitnib TBK1-KD DMSO MRT67307 CYT387 Ruxolitnib IKK ε -KD 14 pCYLD (S418) 12 10 CYLD 8 6 TBK1 4 2 ε 0

Relative CCL5 expression

IKK 1 5 1 5 1 5 10 10 10 0.1 0.5 0.1 0.5 0.1 0.5 0.01 0.05 0.01 0.05 0.01 0.05 –LPS

β +LPS

-Actin DMSO MRT67307 CYT387 Ruxolitinib C RAW macrophages Concentration (µmol/L) MRT67307 CYT387 Ruxolitinib 45 (μmol/L) 40 0.1 0.5 1.0 0.1 0.5 1.0 0.1 0.5 1.0 IC50 = 331 nmol/L IC50 = 63 nmol/L IC50 = 589 mol/L (–) LPS DMSO 35 30 pIRF3 (S396) 25 20 IRF3 15 10 pTBK1 (S172) 5 0 Relative IL-6 expression 1 5 1 5 5 1 10 10 TBK1 10 0.1 0.5 0.1 0.5 0.1 0.5 0.01 0.05 0.01 0.05 0.01 0.05 –LPS +LPS IKKε DMSO

MRT67307 CYT387 Ruxolitinib β-Actin Concentration (µmol/L)

Figure 2. CYT387 inhibits JAK and TBK1/IKKε signaling. A, in vitro kinase assay with His-TBK1 (4 nmol/L) or GST–IKKε (2 nmol/L), 100 μmol/L IKKε– Tide, 100 μmol/L ATP, and CYT387. Assays performed in duplicate, ADP generated normalized to dimethyl sulfoxide (DMSO) control, mean ± SD shown. B, immunoblot of S418 pCYLD, CYLD, TBK1, IKKε, and β-actin levels in 293T cells 24 hours following transient cotransfection of Myc-tagged CYLD with TBK1-WT, TBK1-KD, IKKε-WT, or IKKε-KD and 4-hour treatment with DMSO or 5 μmol/L MRT67307, CYT387, or ruxolitinib. C, immunoblot of S396 pIRF3, IRF3, S172 pTBK1, TBK1, IKKε, and β-actin levels in macrophages stimulated with lipopolysaccharide (LPS) (100 ng/mL) for 2 hours ± DMSO or inhibitor pretreatment at the indicated concentrations for 1 hour. D, mRNA levels of IFNB1, CCL5 , and IL-6 in macrophages stimulated with LPS (100 ng/mL) for 2 hours ± pretreatment with inhibitors for 1 hour. Mean and SEM of triplicate samples shown.

−/− −/− = ε = WT or Tbk1 MEFs. Tbk1 MEFs exhibited low pSTAT3 lev- (IC50 58 nmol/L) and IKK (IC50 42 nmol/L) kinase activ- els following release from serum starvation ( Fig. 1G ). Onco- ity in the presence of 100 μmol/L ATP (Fig. 2A ). In contrast, genic KRAS G12V expression or stimulation of RAS activity another JAK1/2 inhibitor, ruxolitinib, failed to inhibit TBK1 −/− ε > μ with EGF failed to rescue STAT3 signaling in Tbk1 MEFs or IKK in this assay (IC50 1 mol/L for both; Supplemen- ( Fig. 1H and I ). Supplementation of Tbk1 −/− MEF media with tary Fig. S2A). MRT67307 was comparable with CYT387 in = ε CCL5 completely restored pSTAT3 activation under basal the TBK1 assay (IC 50 40 nmol/L), but inhibited IKK less = conditions and following EGF stimulation (Fig. 1I ). Thus, potently (IC50 242 nmol/L; Supplementary Fig. S2A). To TBK1-regulated CCL5 promotes both clonogenic prolifera- confi rm these observations in intact cells, we examined the tion and autocrine STAT3 activation. effect of inhibitor treatment on TBK1/IKKε S418 CYLD phos- phorylation, which mediates IKKε-induced transformation CYT387 Is a Potent TBK1/IKKe Inhibitor (30 ). Treatment with CYT387 abrogated TBK1/IKKε–induced MRT67307 has been characterized as a potent TBK1 inhibi- CYLD phosphorylation in 293T cells, similar to MRT67307 tor ( 28 ). We noted that CYT387, a JAK1/2 kinase inhibi- and in contrast to ruxolitinib ( Fig. 2B ). These fi ndings estab- tor in clinical trials for myelofi brosis, also inhibited TBK1 lished CYT387 as a potent TBK1/IKKε inhibitor. with low nanomolar potency ( 29 ). To explore the TBK1- and To determine activity of these inhibitors in a physiologic IKKε-specifi c activities of CYT387, we fi rst measured TBK1/ setting, we next measured IFN-γ–induced JAK activity or IKKε–mediated phosphorylation of a substrate motif peptide lipopolysaccharide (LPS)-induced TBK1/IKKε signaling in (IKKε-Tide) in vitro (30 ). CYT387 potently inhibited TBK1 murine RAW macrophages. As expected, ruxolitinib treatment

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RESEARCH ARTICLE Zhu et al.

potently suppressed IFN-γ–induced Y701 pSTAT1, in con- KRAS-dependent cell lines secreted CCL5 and IL-6 into the trast to MRT67307 (Supplementary Fig. S2B). CYT387 was media over time in culture, in contrast to KRAS -independent less potent than ruxolitinib in these assays, but suppressed cells ( Fig. 3D and E ). Extracellular signals in the tumor = the STAT1 target gene Gbp1 (IC50 587 nmol/L) like rux- microenvironment, such as IL-1 and EGF, further enhanced = olitinib (IC 50 20 nmol/L) and in contrast to MRT67307 TBK1 activation as well as CCL5 and IL-6 production in A549 > μ (IC50 10 mol/L; Supplementary Fig. S2C). Similar results cells (Supplementary Fig. S3C and S3D). To study the func- were obtained for IFN-γ–induced Cxcl10 mRNA expression tional consequences of this autocrine cytokine production, (Supplementary Fig. S2D). CYT387 treatment potently inhib- we assessed CCL5 and IL-6 neutralization in two-dimensional ited LPS-induced S396 IRF3 phosphorylation at concentra- (2D) culture or in a microfl uidic three-dimensional (3D) tions < 1 μmol/L, similar to MRT67307 and in contrast cell culture system in which A549 lung tumor spheroids are to ruxolitinib (Fig. 2C ). As previously reported, MRT67307 cocultured in collagen with human vascular endothelial cells treatment paradoxically induced TBK1 S172 activation loop (HUVEC) or growth factors such as EGF (Fig. 3F and Sup- phosphorylation ( 28 ), which was less pronounced follow- plementary Fig. S3E; ref. 34 ). This 3D system more closely = ing CYT387 treatment in this assay. MRT67307 (IC 50 228 recapitulates cancer cell behavior in the extracellular matrix, = nmol/L) or CYT387 (IC50 201 nmol/L) treatment also sup- captures features of the tumor microenvironment, and also pressed expression of the IRF3 target gene Ifnb1 (Fig. 2D). enables inhibitor studies. Incubation of A549 cells with MRT67307 or CYT387 further impaired LPS-induced expres- CCL5- and IL-6–neutralizing antibodies in 2D culture had sion of Ccl5 and Cxcl10, whereas ruxolitinib failed to suppress a minor effect on proliferation (Supplementary Fig. S3F). In Ifnb1, Ccl5 , or Cxcl10 expression ( Fig. 2D and Supplementary contrast, combined CCL5 and IL-6 blockade completely sup- = Fig. S2E). MRT67307 (IC 50 331 nmol/L) or ruxolitinib pressed A549 cell proliferation in response to EGF in 3D cul- = (IC 50 589 nmol/L) each partially suppressed LPS-induced ture, compared with neutralization of either cytokine alone Il6 , whereas CYT387 strongly inhibited Il6 mRNA levels (Fig. 3G ). We also observed that KRAS-independent H1437 = (IC 50 63 nmol/L; Fig. 2D ). These fi ndings confi rmed spheroids failed to disperse and proliferate in response to CYT387 as a multitargeted JAK and TBK1/IKKε inhibitor EGF even at 48 hours (Fig. 3H ). Together, these fi ndings (Supplementary Fig. S2F) that potently suppresses multiple demonstrate that TBK1 signaling and CCL5 and IL-6 pro- cytokines, including CCL5 and IL-6. duction not only promote clonogenic proliferation of MEFs, but also KRAS -dependent lung cancer cell proliferation and CCL5 and IL-6 Promote Human KRAS -Dependent migration. Lung Cancer Transformation To determine the role of these cytokines in human KRAS - CYT387 Treatment Impairs KRAS -Dependent driven lung cancer, we fi rst measured TBK1 and STAT3 NSCLC Viability activation in a panel of human NSCLC lines. Because KRAS We next assessed the capacity of CYT387 to target this dependency does not strictly correlate with KRAS mutation sig naling pathway and impair the tumorigenic potential status ( 19, 20 , 31 ), we examined specifi c cell lines for their of KRAS-dependent lung cancer cells. Treatment of A549 dependence on KRAS expression (Supplementary Fig. S3A). cells with MRT67307 or CYT387 suppressed the clonogenic Although one group reported that A549 cells are KRAS growth and induced cytotoxicity, in contrast to ruxolitinib independent (31 ), we observed sensitivity of this cell line to ( Fig. 4A and Supplementary Fig. S4A). Both MRT67307 and KRAS and TBK1 suppression, consistent with other reports CYT387 preferentially impaired the viability of KRAS -depend- ∼ μ (19 , 22 , 32, 33). The majority of other KRAS-dependent ent A549 and HCC44 cells (IC50 2 mol/L) compared with NSCLC cell lines also required TBK1 for survival (Supple- KRAS -independent H1437 and H1568 cells, whereas rux- mentary Fig. S3A). We then compared activation of TBK1 olitinib had a negligible effect (Fig. 4B and Supplementary and cytokine signaling in a panel of KRAS -mutant/depend- Fig. S4B and S4C). ent cell lines (A549, HCC44, H23, H1792, H460, H2009, and To extend these fi ndings, we treated a panel of 12 NSCLC H1944 cells) to KRAS-WT/independent cells (Fig. 3A and cell lines with 18 different kinase inhibitors targeting multiple Supplementary Fig. S3B; ref. 19 ). We measured TBK1 activity pathways. Cell lines dependent on KRAS were also dependent on by S172 phosphorylation and STAT3 activity by Y705 phos- TBK1, with the exception of H1819 cells (19, 20), which instead phorylation 24 hours after plating, and normalized values to required IKBKE expression for survival (Supplementary Figs. total TBK1 or STAT3 levels, respectively. Although phospho- S3A and S4D). As expected, cell line sensitivity to MRT67307 rylated (p) TBK1 levels were low at baseline, KRAS -dependent and the related TBK1 inhibitor BX-795 was correlated, similar to NSCLC cells exhibited higher levels of both pTBK1 and the concordance between the MAPK/ERK kinase (MEK) inhibi- pSTAT3 compared with KRAS -independent cells ( Fig. 3A and tors AZD6244 and CI-1040 (Fig. 4C ). KRAS-dependent cell sen- Supplementary Fig. S3B). Activation of pTBK1 and pSTAT3 sitivity to these TBK1 inhibitors was also comparable with MEK was more pronounced in KRAS -dependent NSCLC cells after inhibitors. Cell line sensitivity to CYT387 signifi cantly correlated 72 hours in culture ( Fig. 3B ). Suppression of KRAS in A549 with MRT67307, but not AZD6244 (Fig. 4C ). Indeed, the near- cells reduced levels of both pTBK1 and pSTAT3 (Fig. 3C ), est neighbor analysis showed correlation of CYT387 sensitivity consistent with direct engagement of RALB–TBK1 signaling with both BX-795 and MRT67307, despite different off-target by oncogenic KRAS (18 ). effects ( Fig. 4D ). Similarly, CI-1040 was the nearest neighbor to These fi ndings suggested that autocrine cytokine signaling AZD6244 and the AKT inhibitor MK-2206 was closely related downstream of oncogenic KRAS contributes to the prolif- to the phosphoinositide 3-kinase (PI3K) inhibitor GDC0941 eration of KRAS -dependent cells. Indeed, A549 and other (Supplementary Fig. S4E and S4F), confi rming the specifi city

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Targeting Oncogenic KRAS via Cytokine Suppression RESEARCH ARTICLE

Baseline (24-h culture) 72-h culture

A B C 0.2 0.2 KRAS-WT KRAS-WT H28 H1437 H1568 A549 HCC44 H23 0.15 0.15 pTBK1 sh GFP sh LACZ sh KRAS -1 sh KRAS -2 (S172) pTBK1 TBK1 TBK1 0.1 0.1 pSTAT3 pSTAT3 0.05 0.05 (Y705) STAT3 Normalized intensity Normalized intensity STAT3 KRAS 0 0 β-Actin pTBK1/total TBK1 pSTAT3/total STAT3 β-Actin

D A549 cells E 72-h culture F Time in culture Time in culture 1,800 350 EGF or Spheroid 60 400 1,600 300 HUVECs Growth medium 50 1,400 300 1,200 250 40 1,000 200 30 200 800 150 20 600 1 mm 100 100 10 400

IL-6 levels (pg/mL) 50 IL-6 levels (pg/mL) 200 CCL5 levels (pg/mL) 0 CCL5 levels (pg/mL) 0 0 0 6 h 6 h 1cm1cm 12 h 24 h 36 h 48 h 60 h 12 h 24 h 36 h 48 h 60 h H23 H28 H23 H28 H522 H522 1 mm 1 cm H1944 H1437 H1568 H1944 H1437 H1568 HCC44 HCC44 G A549 spheroids Control αCCL5 αIL-6 αCCL5 + αIL-6 H EGF 0 h 0 h 0 h 0 h 35 H1437 spheroids 30 EGF 0 h 25 20 18 h 18 h 18 h 18 h 15 10 5 48 h 0 # Dispersed cells/spheroid

αIL-6 36 h 36 h 36 h 36 h ControlαCCL5

αCCL5+αlL-6

Figure 3. CCL5 and IL-6 promote RAS-driven lung cancer cell proliferation in 3D culture. A, baseline pTBK1/total TBK1 levels and pSTAT3/total STAT3 levels in NSCLC cell line panel 24 hours after plating. Blue, KRAS-mutant/dependent; red, KRAS-WT/independent. B, immunoblot of S172 pTBK1, TBK1, Y705 pSTAT3, STAT3, and β-actin levels in A549, HCC44, H23, H28, H1437, or H1568 cells after 72 hours of culture. Blue, KRAS -mutant/dependent; red, KRAS -WT/independent. C, immunoblot of pTBK1, TBK1, pSTAT3, STAT3, KRAS, and β-actin levels in A549 cells 72 hours following expression of control or KRAS-specifi c shRNAs. D, IL-6 and CCL5 ELISA from media during A549 cell culture. Assays performed in duplicate, mean, and SD shown. E, IL-6 and CCL5 ELISA after 72 hours in culture of additional KRAS-dependent (blue) or -independent (red) cell lines. Mean and SD of duplicate samples shown. F, schematic of microfl uidic 3D cell culture device. G, phase-contrast images of A549 spheroids at 0 (×20), 18, and 36 hours (×10) following EGF stimulation (20 ng/mL) ± neutralizing antibodies against CCL5 (100 ng/mL), IL-6 (100 ng/mL), or the combination. Mean ± SD number of dispersed cells per spheroid from tripli- cate devices shown. H, phase-contrast images H1437 spheroids 0 hours (×20) or 48 hours (×10) following EGF stimulation (20 ng/mL).

of this analysis. Together, these fi ndings demonstrated that to disrupt these cytokines in lung cancer cells and suppress CYT387 and other TBK1 inhibitors impair KRAS -dependent KRAS-driven tumorigenicity in vivo. cell viability in a distinct fashion compared with other targeted inhibitors. CYT387 Suppresses an Autocrine Cytokine Circuit To determine the sensitivity of cells in 3D culture, we next in KRAS -Dependent Lung Cancer Cells treated A549 spheroids with MRT67307, CYT387, or rux- We noted that IKKε overexpression promoted CCL5 and olitinib. Only CYT387 prevented HUVEC-induced spheroid IL-6 production even more potently than TBK1 (Supplemen- dispersal at 1 μmol/L concentration (Fig. 4E). Treatment tary Fig. S5A). IL6 mRNA expression also recovered during with 1 μmol/L CYT387 also abrogated A549 spheroid pro- the culture of Tbk1−/− MEFs, coinciding with inducible expres- liferation and migration in response to EGF stimulation, sion of IKKε (Supplementary Fig. S5B and S5C). Because in contrast to 1 μmol/L MRT67307 or ruxolitinib, whereas STAT3 induces IKKε (25 ), these fi ndings suggested that dis- combination of MRT67307 and ruxolitinib inhibited sphe- ruption of a circuit involving IKKε and JAK signaling was roid dispersal (Fig. 4F and Supplementary Fig. S4G). These required for effective suppression of IL6 . Indeed, treatment of fi ndings were consistent with the effects of CCL5 and IL-6 Tbk1 −/− MEFs with ruxolitinib disrupted this circuit and sus- neutralization, suggesting a unique potential for CYT387 tained inhibition of CCL5 and IL6 expression (Supplementary

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RESEARCH ARTICLE Zhu et al.

A A549 cells C Relative NSCLC cell line sensitivity D CYT387 nearest neighbors 150 150 Dependent Independent DMSO CYT387 100 100 Pearson MRT67307 Ruxolitinib 50 50 AZD6244 A549 HCC44 H23 H1792 H1819 H1944 H2009 H522 H1437 H1568 H1975 H2052 Inhibitor correlation (r) MRT67307 r = 0.70* r = 0.71* 0 0 CYT-387 (TBK1/JAK) 1 0 50 100 150 0 50 100 150 BX-795 (TBK1/PDK1) 0.8236* MRT-67307 (TBK1/SIK) 0.7823* B MRT67307 CYT387 BX795 CI-1040 JNK Inhibitor-1 (JNK) 0.3616 KRAS-mut/Dep VTX-11E (ERK) 0.3585 CI-1040 (MEK) 0.2029 100 100 % Control KRAS-WT/Indep AZD6244 (MEK) 0.1894 KRAS-WT/Dep MK-2206 (AKT) 0.1414 150 150 GDC0941(PI3K) 0.08016 LY294002 (PI3K) 0.04528 100 100 GDC0879 (RAF) –0.0747 A549 AG490 (JAK) –0.1156 50 50 Nifuroxazide (STAT) –0.1492 % Control CYT387

HCC44 50 CYT387 50 H1437 Flavopiridol (CDK) –0.1583 r = 0.78* r = 0.19 IKK-2 (TPCA) (IKKβ) –0.2686 H1568 0 0 Imatinib (ABL/KIT/PDGFR) –0.2906 0 50 100 150 0 50 100 150 BAY-11-11-7082 (NFκB) –0.4916 MRT67307 AZD6244 PLX-4720 (RAF) –0.5113 % Control Relative viability –0.5 –0.0 –0.5 1.0 –0.5 –0.0 –0.5 1.0 Log concentration (µmol/L) Log concentration (µmol/L) Row min Row max

E Control MRT67307 CYT387 Ruxolitinib F EGF 0 h 0 h 0 h 0 h 40 0 h 36 h 35 DMSO 30

HUVECs A549 25 0 h 36 h 18 h 18 h 18 h 18 h 20 CYT387 15 10 40 5 # Dispersed cells/spheroid 30 36 h 36 h 36 h 36 h 0 20

DMSO CYT387 10 Ruxolitinib # Dispersed MRT67307 cells/spheroid 0 DMSO CYT387

Figure 4. Inhibition of lung cancer cell proliferation and viability by CYT387. A, crystal violet stain of A549 cell clonogenic assay after 10-day treat- ment with dimethyl sulfoxide (DMSO), 2.5 μmol/L MRT67307, CYT387, or ruxolitinib. B, dose-response to MRT67307 or CYT387 treatment at 72 hours normalized to DMSO control, mean ± SEM of quadruplicate samples shown. Blue, KRAS-mutant/dependent cell lines; red, KRAS-WT/independent cell lines. C, cell viability of 12 KRAS-dependent (blue) or independent (red) lung cancer lines (purple, KRAS-WT/dependent) treated with 5 μmol/L inhibitors for 72 hours normalized to DMSO control. Plotted are relative cell line sensitivities for MRT67307/BX795 and AZD6244/CI-1040 (top), or CYT387/ MRT67307 and CYT387/AZD6244 (bottom). *, P < 0.05, Pearson correlation. D, normalized relative CYT387 sensitivity in the same NSCLC lines compared with 17 other targeted inhibitors at 5 μmol/L. Nearest neighbor analysis by Pearson correlation, *, P < 0.05. E, phase-contrast images (×20) of A549 spheroids cocultured with HUVECs and treated with DMSO, MRT67307, CYT387, or ruxolitinib (1 μmol/L) for 18 or 36 hours. Mean number and SD of dispersed cells per spheroid from triplicate devices shown. F, phase-contrast images (×20) of A549 spheroids stimulated with EGF and treated with DMSO or 1 μmol/L CYT387 for 36 hours. Mean number and SD of dispersed cells per spheroid from triplicate devices shown.

Fig. S5D). Thus, interruption of CCL5 and IL-6 together origenicity (7, 8), we focused on the consequences of inhibitor required inhibition of TBK1/IKKε and JAK signaling. treatment in this setting. Using single sample gene set enrich- We next assessed the effect of each inhibitor on this auto- ment analysis (ssGSEA) of KRAS and IL-1β signatures, we crine circuit in KRAS -dependent lung cancer cells. Treatment confi rmed coactivation of oncogenic KRAS and IL-1 signal- of A549 cells with 5 μmol/L MRT67307 transiently inhibited ing in primary human lung adenocarcinomas ( P = 0.02, nor- baseline pSTAT3 levels, but within 2 hours it led to reacti- malized mutual information statistic; refs. 19 , 35 ) and IKBKE vation of STAT3 consistent with the feedback we observed overexpression in these tumors (Fig. 5B ). In consonance with in Tbk1 −/− MEFs ( Fig. 5A ), as well as paradoxical induction this fi nding, IL-1 also promoted IKBKE mRNA and protein of TBK1 S172 activation. In contrast, treatment with rux- expression in A549 cells (Fig. 5C ). olitinib inhibited pSTAT3 but had no effect on pTBK1 levels Following short-term stimulation with IL-1 or IL-6 as ( Fig. 5A ). Uniquely, treatment of A549 cells with CYT387 a control, CYT387 treatment targeted both pTBK1 and inhibited pSTAT3 and induced pTBK1 to a lesser degree pSTAT3, in contrast to MRT67307 or ruxolitinib (Sup- than MRT673037 (Fig. 5A ). These fi ndings were consistent plementary Fig. S5E). CYT387 and MRT67037 treatment with our observations in macrophages and suggested dual but not ruxolitinib also inhibited basal and IL-1β–induced targeting of JAK and TBK1 signaling by CYT387 in KRAS - pCYLD levels, whereas CYT387 uniquely prevented IL-1β– dependent lung cancer cells. induced IKKε protein and mRNA expression ( Fig. 5D and Because IL-1 signaling potentiated TBK1 and CCL5/IL-6 Supplementary Fig. S5F). MRT67307 treatment failed to sup- activation in A549 cells and promotes KRAS-dependent tum- press IL-1β–induced CCL5/IL-6 expression, and ruxolitinib

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Targeting Oncogenic KRAS via Cytokine Suppression RESEARCH ARTICLE

A A549 cells D MRT67307 CYT387 Ruxolitinib DMSO MRT67307 CYT387 Ruxolitinib β DMSO 1 h 2 h 3 h 4 h 1 h 2 h 3 h 4 h 1 h 2 h 3 h 4 h IL-1 (h): 0 6 12 24 36 0 6 12 24 36 0 6 12 24 36 0 6 12 24 36 pSTAT3 IKKε

STAT3 pCYLD

pTBK1 CYLD TBK1 β-Actin

β-Actin E 300 300

B Human lung adenocarcinomas 250 250 KRAS mutation 200 200 KRAS signature NMI statistic 150 150 IL-1β signature 0.36 (P = 0.02) 100 100 IKBKE 0.34 (P = 0.03) IL-6 levels (pg/mL)

CCL5 levels (pg/mL) 50 50 –4 0 +4 0 0 Normalized enrichment score/expression DMSO DMSO CYT387 C A549 cells CYT387 Ruxolitinib 3 Ruxolitinib MRT67307 MRT67307 Unstimulated 2.5 IKBKE Unstimulated 2 F KRAS 1.5 β

IL-1 : 0 h 6 h 12 h 24 h 36 h 48 h 1 ε IL-1 Ruxolitinib 0.5 IKK RALB 0 β-Actin Relative RNA expression JAK/STAT3 IL-1β: 0 h 1 h 2 h 4 h 6 h 8 h 0.5 h MRT67307 TBK1/IKKε

CCL5 CYT387 IL-6

Figure 5. CYT387 treatment disrupts a TBK1/IKKε and JAK-dependent autocrine cytokine circuit. A, immunoblot of Y705 pSTAT3, STAT3, S172 pTBK1, TBK1, and β-actin levels in A549 cells treated with 5 μmol/L MRT67307, CYT387, or ruxolitinib. B, heatmap of KRAS/IL-1 signature expression and IKBKE gene expression in a panel of human lung adenocarcinomas. KRAS mutation status indicated by tick marks, scale refl ects ssGSEA normal- ized enrichment scores and IKKε gene expression. NMI, normalized mutual information statistic relative to KRAS signature. C, IKBKE mRNA levels and immunoblot of IKKε and β-actin levels in IL-1β–stimulated A549 cells. D, immunoblot of IKKε, S418 pCYLD, CYLD, or β-actin levels in IL-1β–stimulated A549 cells following dimethyl sulfoxide (DMSO), MRT67307, CYT387, or ruxolitinib (5 μmol/L) treatment. E, ELISA of CCL5 and IL-6 levels in A549 cells stimulated for 4 hours with IL-1β following pretreatment for 1 hour with DMSO, MRT67307, CYT387, or ruxolitinib (5 μmol/L). Mean and SD of duplicate samples shown. F, model of autocrine cytokine circuit involving TBK/IKKε, CCL5/IL-6, and JAK–STAT activation.

partially reduced these levels of these cytokines, whereas of TBK1 loss was dependent on its kinase activity, as IKK ε WT CYT387 treatment strongly inhibited CCL5/IL-6 production but not a kinase-dead allele restored A549 cell viability (Fig. 6B ). ( Fig. 5E and Supplementary Fig. S5G). These experiments con- We also treated A549 cells with escalating doses of CYT387 fi rmed that CYT387 targets TBK1/IKKε and JAK signaling in for 2 months and identifi ed two different clones (A549-C1 lung cancer cells and disrupts this autocrine cytokine circuit, and A549-C2) resistant to CYT387 at concentrations as high suppressing CCL5/IL-6 and inducible IKKε expression (Fig. 5F ). as 5 μmol/L ( Fig. 6C ). CYT387 or ruxolitinib treatment inhibited pSTAT3 in these clones, making it unlikely that IKK e Reactivation Promotes CYT387 Resistance drug effl ux or mutations in JAK were responsible for resist- In parallel to these studies, we conducted a kinase open ance (Supplementary Fig. S6A). Instead, each clone expressed reading frame (ORF) rescue screen to identify genetic modi- substantially increased levels of IKKε relative to parental fi ers of TBK1-regulated proliferation/survival in A549 cells. A549 cells (Fig. 6D ). CYT387-resistant A549-C1 cells were We expressed an arrayed library of 600 kinase ORFs in A549 resensitized to CYT387 treatment after IKKε suppression cells (36 ), then suppressed TBK1 using a TBK1-specifi c short ( Fig. 6E ), suggesting that IKKε reactivation contributes to hairpin RNA (shRNA) and measured cell viability. Although CYT387 resistance. we were unable to overexpress TBK1 stably, IKBKE was the To further assess the role of IKKε kinase signaling as a top-scoring ORF that rescued TBK1 suppression, whereas resistance mechanism, we mapped the JAK2 inhibitor resist- oncogenic KRAS enhanced dependency of A549 cells on TBK1 ance alleles Y931C and G935R ( 37 ) onto a model of the IKKε ( Fig. 6A and Supplementary Table S1). IKKε-mediated rescue kinase domain from the TBK1 structure ( 38 ). We noted

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RESEARCH ARTICLE Zhu et al.

ε ε A B LACZ IKK -WT IKK -KD C E shGFP shIKKε-1 shIKKε-2 shTBK1-1 rescue screen CYT387-resistant clones A549 cells A549 IKBKE 0.5 A549 sh LACZ sh TBK1 -1 sh LACZ sh TBK1 -1 sh LACZ sh TBK1 -1 2 SD IKKε 0.4 A549-C1 TBK1 DMSO 2 mos CYT387 × 2 0.3 Mean β-Actin A549-C1 A549-C1 A549-C2 shLACZ CYT387 1.4 0.2 shTBK1-1 D 2 SD 1.2 Relative viability A549 A549-C1

KRASG12V 1 -1 -2 -2 -1 ε ε ε 0.1 0.8 ε A549-C1 A549-C2 0.6 A549 IKKε sh IKK sh IKK sh GFP sh IKK 0.4 sh GFP sh IKK 0.0 0.2 β-Actin IKKε 0 200 400 600 Relative viability ORFs 0 LACZ IKKε-WT IKKε-KD β-Actin IKKε F G H A549-IKKε-Y88C spheroids * 450 EGF 160 400 0 h 36 h 35 350 140 30 300 120 DMSO 100 250 25 80 200 20 60 150 0 h 36 h 15 40 100 10 20 50 0 IL-6 levels (pg/mL) 0 CYT387 5 CCL5 levels (pg/mL) JAK2 0 # Dispersed cells/spheroid A549 * A549 36 h ε -Y88C

ε -Y88C 0 h α IL–6

CYT387 DMSO + α-CCL5 CYT387 A549+CYT387 A549+CYT387 + α-IL-6 α CCL5+ A549-IKK A549-IKK CYT387+ A549-IKK ε -Y88C+CYT387 A549-IKK ε -Y88C+CYT387

Figure 6. I K K ε-induced CCL5 and IL-6 promote CYT387 resistance. A, kinase ORF rescue screen of TBK1 suppression in A549 cells. Dotted lines mark 2 SD above and below the mean. Highlighted are IKBKE and KRASG12V . B, immunoblot of IKKε, TBK1, and β-actin levels in A549 cells 3 days following TBK1 suppression and IKKε expression. Relative viability after 6 days was normalized to control LACZ expression and suppression. Mean ± SD of 48 replicate samples shown. C, schematic of resistant cell line generation over 2 months of CYT387 treatment. D, immunoblot IKKε and β-actin levels in parental A549 cells, A549-C1, and A549-C2. E, crystal violet stain of A549 or A549-C1 cells stably infected with control shGFP , sh IKKε-1, or sh IKKε -2 and treated with dimethyl sulfoxide (DMSO) or CYT387 for 5 days. Immunoblot shows IKKε and β-actin levels. F, model of CYT387 binding to IKKε or JAK2. *, Homologous IKKε Y88 and JAK2 Y931 residues. G, ELISA of CCL5 and IL-6 levels in A549 or A549-IKKε-Y88C cells following 24 hours of treatment with DMSO or CYT387. Mean ± SD of duplicate samples shown. H, phase-contrast images (×20) of A549-IKKε-Y88C spheroids stimulated with EGF and treated with DMSO or 1 μmol/L CYT387 ± CCL5/IL-6 neutralization for 36 hours. Number of cells dispersed per spheroid from triplicate devices shown (mean ± SD).

homologous orientation of JAK2 Y931 and IKKε Y88 (Fig. 6F ), IKKε-Y88C cells dispersed despite 1 μmol/L CYT387 treat- suggesting that substitutions involving this residue would ment ( Fig. 6H ). Addition of CCL5- and IL-6–neutralizing block CYT387 binding to IKKε. Indeed, IKKε Y88C specifi - antibodies completely suppressed proliferation, confi rming cally retained kinase activity and rescued CCL5 and IL-6 pro- that IKKε-mediated CCL5 and IL-6 production was respon- duction following expression and CYT387 treatment in 293T sible for CYT387 resistance (Fig. 6H ). Together, these fi nd- cells (Supplementary Fig. S6B). We then stably expressed ings reveal that inhibition of this autocrine cytokine circuit IKKε Y88C, IKKε WT, IKKε KD, or enhanced GFP (EGFP) in contributes directly to CYT387 activity in KRAS -dependent A549 cells and cultured cells in 5 μmol/L CYT387. In contrast lung cancer cells. to EGFP or IKKε KD, both IKKε Y88C and to a lesser extent IKKε WT accelerated resistance (Supplementary Fig. S6C). Therapeutic Effect of CYT387 in Murine Sequencing of IKBKE in resistant A549-IKKε-Y88C cells that Kras -Driven Lung Cancers emerged confi rmed dominant expression of the Y88C allele CYT387 inhibits disease progression of JAK-dependent (Supplementary Fig. S6D). A549-IKKε-Y88C cells produced murine myeloproliferative neoplasms and suppresses pSTAT3 high levels of CCL5 and IL-6 insensitive to CYT387 treatment in vivo ( 29 ). Using the daily dose of CYT387 in these studies as (Fig. 6G ). When cultured as spheroids in 3D culture, A549- a starting point, we examined CYT387 treatment in a murine

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Targeting Oncogenic KRAS via Cytokine Suppression RESEARCH ARTICLE

A B KrasLSL-G12D/WT Baseline Week 16 120% 2 wks 120% 4 wks H H Vehicle Vehicle 80% Docetaxel 80% Docetaxel CYT387 CYT387 CYT387+ CYT387+ 40% docetaxel 40% docetaxel E 0% 0% 1.2 1.2 1.2 Ccl5 Il6 Ikbke 1 1 1 –40% –40% 0.8 0.8 0.8 Tumor burden (% change) Tumor Tumor burden (% change) Tumor 0.6 0.6 0.6 0.4 0.4 0.4 –80% 0.2 0.2 0.2 –80% n.s 0 0 0 * Relative mRNA expression Relative mRNA Relative mRNA expression Relative mRNA Relative mRNA expression Relative mRNA

C LSL-G12D/WT D Vehicle Vehicle Vehicle Vehicle Kras tumors LSL-G12D/WT Vehicle CYT387 CYT387 Kras tumors CYT387 Docetaxel Docetaxel pSTAT3 Docetaxel

Vehicle

Vehicle CYT387#1CYT387#2 CYT387+docetaxel CYT387+docetaxel μ CYT387+docetaxel 50 m pSTAT3 LSL-G12D/WT Flox/Flox STAT3 F Kras ;p53 G 120% Baseline Docetaxel Vehicle pCYLD AZD6244 H CYLD 80% CYT387 CYT387+ AZD6244 pTBK1 40% CYT387 TBK1 Week 8 0% β-Actin –40% H

CYT387 + burden (% change) Tumor docetaxel –80% n.s n.s **

Figure 7. Therapeutic effi cacy of CYT387 in Kras-driven murine lung cancer. A, percentage change in MRI tumor volume of Kras LSL-G12D/WT–induced lung cancer 2 and 4 weeks following vehicle, docetaxel, CYT387, or CYT387 + docetaxel treatment. *, P < 0.01, t test; n.s., not signifi cant. B, baseline and 16-week MRI images following CYT387 treatment. Arrows indicate baseline lung tumor burden. H, heart. C, immunohistochemistry for Y705 pSTAT3 levels in tumors from vehicle-, docetaxel-, CYT387-, or CYT387 + docetaxel–treated mice compared with hematoxylin and eosin (H&E) stain. D, immuno- blot of Y705 pSTAT3, STAT3, S418 pCYLD, CYLD, S172 pTBK1, TBK1, and β-actin levels in tumors from vehicle or two different CYT387-treated mice. E, mRNA levels of Ccl5, Il6 , and Ikbke in tumors from docetaxel-, CYT387-, or CYT387 + docetaxel–treated mice, normalized to vehicle control. Mean ± SEM of triplicate samples shown. F, percentage change in tumor burden of KrasLSL-G12D/WT ;p53 fl ox/fl ox lung cancer treated with vehicle, AZD6244, CYT387, or the combination at 2 weeks. Two mice treated with the combination were imaged at 3 weeks. **, P < 0.001, t test. G, baseline and 8-week MRI images following CYT387/AZD6244 therapy. Arrows indicate baseline lung tumor burden.

model of Kras G12D-driven lung cancer (39 ). We identifi ed mice from toxicity, whereas CYT387-treated mice exhibited no with established lung tumors by MRI (40 ) and treated them overt signs of toxicity. We observed potent synergy between with 100 mg/kg CYT387 daily by oral gavage, docetaxel 16 CYT387 and docetaxel (mean tumor volume reduction −62.8% mg/kg every other day by intraperitoneal injection, or combi- and −52.8% at 2 and 4 weeks, respectively, for the combina- nation of CYT387 and docetaxel. tion; Fig. 7A ), although this combination resulted in weight Single-agent CYT387 treatment reduced mean tumor vol- loss, which was not observed in mice treated with CYT387 ume on MRI as effectively as docetaxel at 2 weeks (−23.4% alone (Supplementary Fig. S7A). CYT387 vs. −13.0% docetaxel), whereas vehicle-treated ani- To determine whether CYT387 suppressed JAK and TBK1/ mals progressed similar to previous studies ( Fig. 7A ; ref. 40 ). IKKε signaling in vivo , we measured several markers of activity At 4 weeks, CYT387 treatment resulted in statistically signifi - in tumors from treated mice. CYT387 or CYT387/docetaxel cant and durable reduction in mean tumor burden (−30.5%, treatment specifi cally inhibited tumor pSTAT3 levels com- P < 0.01 vs. vehicle, t test) in contrast to docetaxel (+25.8%, pared with vehicle or docetaxel alone ( Fig. 7C and D ). TBK1 P = 0.626 vs. vehicle, t test; Fig. 7A ). CYT387-induced lung signaling was active in these tumors as measured by elevated tumor regression was durable even at 16 weeks ( Fig. 7B ). pCYLD and pTBK1 levels, and, in consonance with our fi nd- Furthermore, 2 of the docetaxel-treated mice died by 4 weeks ings in vitro , CYT387 treatment blocked CYLD phosphorylation

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RESEARCH ARTICLE Zhu et al.

and paradoxically increased pTBK1 ( Fig. 7D ). As a conse- JAK inhibitor, as a potent TBK1/IKKε inhibitor that disrupts quence, levels of Ccl5 , Il6 , and Ikbke were all preferentially this autocrine loop and causes regression of established murine reduced in CYT387 and CYT387/docetaxel–treated animals Kras -driven lung tumors in vivo . These observations elucidate (Fig. 7E ). Thus, response to CYT387 therapy correlated with a novel KRAS effector pathway emanating from RAL–GEF effective disruption of this cytokine circuit in vivo. through RALB and TBK1 that activates cytokine production Mitogen-activated protein kinase (MAPK) pathway inhibi- in an inappropriate context to promote tumor maintenance. tion is a central focus of current therapeutic strategies target- Because genetic and pharmacologic perturbation of these ing KRAS -driven cancers. Using a phospho-MAPK antibody kinases extinguishes this cytokine signaling network and inhib- array, we confi rmed that CYT387 treatment in vitro failed to its KRAS-driven tumorigenicity, targeting this effector pathway suppress MAPK pathway activation in KRAS-dependent A549 represents a promising new strategy for these treatment-refrac- cells, and instead increased pERK1/2 levels compared with tory cancers. ruxolitinib or with AZD6244, which inhibited MEK–ERK sig- naling as expected (Supplementary Fig. S7B). Treatment with Contributions of Infl ammatory Signaling to either MRT67307 or CYT387 rapidly induced feedback phos- KRAS-Induced Tumorigenesis phorylated extracellular signal—regulated kinase (pERK ) acti- Oncogenic KRAS constitutively activates multiple effectors, vation in A549 cells (Supplementary Fig. S7C), also reported including MAPK, PI3K, and RAL–GEF. Although most cancers following TBK1 suppression in this cell line (33 ). Inhibition of that harbor oncogenic KRAS mutations also depend on KRAS this feedback by cotreatment with AZD6244 and MRT67307 signaling, KRAS mutation and dependency are not strictly blocked cytokine expression, and the combination of AZD6244 correlated (19 , 31 ), indicating that RAS signaling is active and and CYT387 completely ablated basal IL-6 production (Sup- required other contexts as well. The fi nding that TBK1 links plementary Fig. S7D). Treatment of KRAS -dependent cell lines RALB to the generation of specifi c protumorigenic cytokines with AZD6244 and CYT387 together prevented pSTAT3 and provides mechanistic insights into the factors that drive KRAS- pERK1/2 activation, indicating that combined TBK1/IKKε, induced cell transformation. CCL5–CCR5 signaling promotes JAK, and MEK inhibition suppressed these multiple KRAS - chemotaxis and enhances metastasis of RAS-driven breast driven pathways (Supplementary Fig. S7E), resulting in signif- cancer cells ( 44 ). Multiple studies have established that IL-6– icantly greater cell death compared with either inhibitor alone and STAT3-induced survival signaling are required for RAS- (Supplementary Fig. S7F). Building on these experiments, mediated tumorigenesis (10–13 ). TBK1/IKKε–regulated CCL5 we next explored this combination in murine Kras LSL-G12D/WT ; and IL-6 infl uence the local infl ammatory microenvironment p53fl ox/fl ox (KP) lung cancer (41 ), an aggressive model with high and may also support tumorigenesis in a non–cell-autono- levels of pERK1/2 ( 40 ). In contrast to treatment with either mous fashion (45 ). Activation of this pathway is conversely agent alone, combined CYT387/AZD6244 therapy for estab- infl uenced by factors in the tumor microenvironment and lished KP tumors resulted in statistically signifi cant tumor thus may occur in other contexts, although our observations regression (−14.4%, P < 0.001 vs. vehicle, t test; Fig. 7F ). Fur- demonstrate that TBK1 is activated and required by lung can- thermore, we observed no overt drug-related toxicity and cer cells that depend on KRAS . KRAS-independent cancers that minimal disease progression in responding mice even at 8 exhibit little activation of TBK1 and cytokine signaling may weeks ( Fig. 7G and Supplementary Fig. S7G). Thus, the sup- maintain tumorigenesis via alternative means. pression of multiple RAS effector pathways by combination These studies identifi ed an autocrine cytokine circuit CYT387/AZD6244 treatment not only impairs the viability of involving TBK1/IKKε and JAK activity that sustained CCL5 KRAS -dependent NSCLC cells in vitro , but also inhibits tumor and IL-6 production in cancer cells, similar to IFN-β signaling growth in the treatment-refractory KP lung cancer model. (26 ). Like IFN-β, both CCL5 and IL-6 activate JAK–STAT sig- naling, inducing IKKε expression ( 25 ) and triggering further CCL5 and IL-6 production (Fig. 5F ). TBK1 inactivation and DISCUSSION JAK inhibition together were required to ablate CCL5 and In addition to their key roles in innate immunity, TBK1 IL-6 production in MEFs, and inhibition of TBK1/IKKε and and IKKε have also been implicated in malignant transfor- JAK by CYT387 was necessary to inhibit induction of IKKε, mation (42 ), basal autophagy (22 ), and obesity (43 ). These CCL5, and IL-6 in KRAS -dependent lung cancer cells. IKKε observations suggest that TBK1 and IKKε likely have multi- overexpression rescued TBK1 suppression, CYT387-resistant ple substrates whose phosphorylation regulates specifi c func- clones overexpressed IKKε, and an inhibitor-resistant IKKε tions in a context- and cell type–dependent manner. allele conferred resistance to CYT387, suggesting that these Oncogenic KRAS co-opts TBK1/IKKε signaling by activat- closely related kinases are key targets of CYT387. Although ing RALB ( 18 ) and inducing IKKε expression ( 23 ). Yet, the CYT387 inhibits IKKε kinase activity, IKKε may have a scaf- mechanism by which TBK1 and IKKε regulate transformation folding function or require maximal ATP-site occupancy and cancer cell survival remains incompletely characterized. for full inhibition, which is more diffi cult in the presence Here, we found that CCL5 and IL-6, key TBK1/IKKε–associated of excess kinase and has been observed for other enzymatic cytokines during innate immunity, are also aberrantly activated drug targets such as dihydrofolate reductase (DHFR; ref. 46 ). in KRAS -driven lung cancers. These cytokines, in turn, act in The requirement of CCL5 and IL-6 for IKKε-driven resistance an autocrine manner to activate JAK–STAT signaling, which confi rms the involvement of this autocrine signaling loop in amplifi es IL-6 production, promotes IKKε expression, and KRAS-dependent lung cancer proliferation. sustains growth factor–activated lung cancer cell prolifera- TBK1/IKKε also activate AKT ( 20, 21 ) and regulate NF-κB tion and migration. We identifi ed CYT387, a clinical-stage signaling components such as CYLD (19 , 30 ). Rescue of Tbk1−/−

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Targeting Oncogenic KRAS via Cytokine Suppression RESEARCH ARTICLE

MEF clonogenic proliferation by CCL5 or conditioned media In Vitro Enzyme Kinetic Assays was incomplete, suggesting that cell-intrinsic defects in AKT, Recombinant enzymes were incubated with ATP and IKKε tide NF-κB signaling, or autophagy ( 22 ) also contribute to TBK1- (ADDDYDSLDWDAKKK) as a specifi c TBK1/IKKε peptide substrate regulated cell survival. Indeed, MRT67307 treatment impaired as described previously (30 ). cell viability at higher concentrations, suggesting that TBK1 likely counterbalances oncogenic stress by additional means. Quantitative Real-Time PCR Given the emerging link between autophagy and RAS-driven Real-time PCR (RT-PCR ) was performed using LightCycler 480 tumorigenesis ( 47 ), it will be interesting to elucidate the SYBR Green I Master (Roche). The sequences of the primers used for interface between TBK1 signaling and this particular stress– RT-PCR are listed in Supplementary Table S3. response pathway in KRAS-dependent cancer cells. Implications for Therapy for KRAS -Driven Cancer Animal Studies Mouse experiments were conducted in agreement with a pro- CYT387 treatment of murine Kras -driven lung cancers was tocol approved by the Institutional Animal Care and Use Com- well tolerated, and suppression of tumor growth was more mittee (IACUC) of Dana-Farber Cancer Institute. Lung tumors in durable than standard docetaxel chemotherapy. Combined Kras LSL-G12D/WT or KrasLSL-G12D/WT ; p53 Flox/Flox mice were induced by CYT387 and AZD6244 treatment exhibited signifi cant thera- inhalation of adenoviral Cre recombinase and monitored by serial peutic activity in KP-induced murine lung cancers, a treat- MRI scanning as described previously (40 ). ment-refractory model. Because CYT387 and MEK inhibitors are in advanced stages of clinical development, these obser- Immunohistochemical Analysis vations provide a rationale for testing this combination in Immunohistochemical staining was performed on formalin-fi xed human KRAS -driven cancers. and paraffi n-embedded tissues from treated mice, sectioned at 5-μm Although kinase inhibitor selectivity is desirable, the complex- thickness. ity of solid tumors and cancer cell signal transduction neces- sitates concomitant inhibition of several signaling pathways Disclosure of Potential Confl icts of Interest (48 ). For example, multitargeted RTK inhibitors are clinically effective against a variety of tumors. Clinical trials of CYT387 S.E. Moody is employed as a consultant at N-of-One Therapeu- in myelofi brosis have also demonstrated safety and effi cacy tics. R.R. Shen is employed as a scientist at Agensys, Inc. P. Tamayo is employed as a consultant at Oracle Corporation. S. Gaudet is uniquely among JAK inhibitors in improving transfusion a consultant/advisory board member of Merrimack Pharmaceuti- dependence ( 49 ). Thus, multitargeted kinase inhibition need cals. J.A. Engelman has received commercial research grants from not correlate with enhanced toxicity. Finally, because RALB– Novartis, Sanofi -Aventis, Amgen, and AstraZeneca and is a consult- TBK1 signaling is distinct from MAPK and PI3K, the observa- ant/advisory board member of Novartis, Sanofi -Aventis, Genentech, tion that CYT387 treatment synergizes with MEK inhibition GlaxoSmithKline, Cell Signaling Technology, Agios, Janssen, and suggests that further combination strategies may be effective in Endo. K.-K. Wong has received commercial research support from treating KRAS -driven cancers and overcoming resistance. AstraZeneca, Infi nity, and Millennium. W.C. Hahn has received a commercial research grant from Novartis and is a consultant/advi- sory board member of Novartis and Blueprint Medicines. D.A. Barbie METHODS is a consultant/advisory board member of N-of-One Therapeutics. No potential confl icts of interest were disclosed by the other authors. Detailed protocols for all sections are described in the Supplemen- tary Methods. Authors’ Contributions Cell Culture Conception and design: Z. Zhu, L. Rozhansky, M.J. Eck, J.A. Engelman, Cells were cultured at 37°C in a humidifi ed incubator with 5% R.B. Corcoran, K.-K. Wong, W.C. Hahn, D.A. Barbie Development of methodology: Z. Zhu, A.R. Aref, T.U. Barbie, CO2 . NSCLC cell lines were obtained from the American Type Cul- ture Collection (ATCC) or the DFCI-84 collection, and cell identity S. Yang, A.C. Schinzel, K. Maciag, E.M. Chan, W. Silkworth, J.P. Mesirov, by KRAS mutation status was previously validated by sequencing S. Gaudet, K.-K. Wong, W.C. Hahn, D.A. Barbie (19 ). Murine RAW 264.7 gamma NO(−) cells and HUVECs were from Acquisition of data (provided animals, acquired and managed ATCC. Tbk1 −/− and WT MEFs were kindly provided by K . Fitzgerald. patients, provided facilities, etc.): Z. Zhu, T.J. Cohoon, Y. Imamura, Tumor spheroids were generated from A549 or NCI-H1437 cells S. Yang, S.E. Moody, T.C. Thai, J.B. Reibel, J.T. Godfrey, Z.R. Qian, using low attachment plates and embedded in a 3D microfl uidic A.N. Page, K. Maciag, M.T. Labowsky, L. Rozhansky, S. Ogino, culture system (34 ). N. Hacohen, R.B. Corcoran, K.-K. Wong, D.A. Barbie Analysis and interpretation of data (e.g., statistical analysis, biosta- Gene Transduction tistics, computational analysis): Z. Zhu, T.J. Cohoon, R.R. Shen, P. Tamayo, Z.R. Qian, L. Rozhansky, J.P. Mesirov, W.E. Gillanders, Cells nucleofection/transfection ( 38 ) or introduction of shRNAs/ S. Ogino, N. Hacohen, R.B. Corcoran, K.-K. Wong, W.C. Hahn, D.A. Barbie ORFs by lentiviral transduction was performed as described pre- Writing, review, and/or revision of the manuscript: Z. Zhu, viously ( 19 , 36 ). shRNA sequences are listed in Supplementary S.E. Moody, Z.R. Qian, J.P. Mesirov, S. Ogino, M.J. Eck, R.B. Corcoran, Table S2. K.-K. Wong, W.C. Hahn, D.A. Barbie Administrative, technical, or material support (i.e., reporting or Immunoblotting, Antibodies, and ELISA organizing data, constructing databases): Z. Zhu, T.U. Barbie, T.C. Immunoblotting was performed as described previously ( 19 ). Thai, Z.R. Qian, W.C. Hahn Cytokines, ELISA kits, and cytokine antibody arrays were purchased Study supervision: Z. Zhu, W.E. Gillanders, S. Gaudet, J.A. Engel- from R&D Systems. man, W.C. Hahn, D.A. Barbie

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RESEARCH ARTICLE Zhu et al.

Acknowledgments 16. Wislez M , Fujimoto N , Izzo JG , Hanna AE , Cody DD , Langley RR, et al. High expression of ligands for chemokine receptor CXCR2 in The authors thank C. Yu, J. Boehm, A. Stern, T. Sim, Q. Liu, and alveolar epithelial neoplasia induced by oncogenic kras. Cancer Res N. Gray for helpful discussions, and R. Sharifi Sedeh for assistance 2006 ; 66 : 4198 – 207 . with 3D culture analysis. 17. Bodemann BO , White MA . Ral GTPases and cancer: linchpin support of the tumorigenic platform. Nat Rev Cancer 2008 ; 8 : 133 – 40 . Grant Support 18. Chien Y , Kim S , Bumeister R , Loo YM , Kwon SW , Johnson CL , This work is supported by U.S. NIH K08 CA138918-01A1 (to et al. RalB GTPase-mediated activation of the IkappaB family kinase D.A. Barbie), P01 CA154303 and R01 CA130988 (to W.C. Hahn), TBK1 couples innate immune signaling to tumor cell survival. Cell and R01 CA122794, R01 CA166480, R01 CA163896, R01CA140594, 2006 ; 127 : 157 – 70 . and U01CA141576 (to K.-K. Wong). D.A. Barbie is supported by V 19. Barbie DA , Tamayo P , Boehm JS , Kim SY , Moody SE , Dunn IF , et al. Scholar, Gloria Spivak Faculty, GTM Fund for Lung Cancer Research, Systematic RNA interference reveals that oncogenic KRAS-driven and Friends of the Dana-Farber Cancer Institute Awards. Z. Zhu and cancers require TBK1. Nature 2009 ; 462 : 108 – 12 . K.-K. Wong are supported by Uniting Against Lung Cancer Grants. 20. 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 – 70 . Received September 17, 2013; revised January 13, 2014; accepted 21. Xie X , Zhang D , Zhao B , Lu MK , You M , Condorelli G , et al. IkappaB January 14, 2014; published OnlineFirst January 20, 2014. kinase epsilon and TANK-binding kinase 1 activate AKT by direct phosphorylation. Proc Natl Acad Sci U S A 2011 ; 108 : 6474 – 9 . 22. 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Targeting Oncogenic KRAS via Cytokine Suppression RESEARCH ARTICLE

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Inhibition of KRAS-Driven Tumorigenicity by Interruption of an Autocrine Cytokine Circuit

Zehua Zhu, Amir R. Aref, Travis J. Cohoon, et al.

Cancer Discovery Published OnlineFirst January 20, 2014.

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