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JAK2 inhibition sensitizes resistant EGFR-mutantlung adenocarcinoma to tyrosine kinase inhibitorsSizhi P. Gao,1 Qing Chang,1 Ninghui Mao,1 Laura A. Daly,1 Robert Vogel,2

Tyler Chan,1 Shu Hui Liu,1 Eirini Bournazou,1 Erez Schori,1 Haiying Zhang,3

Monica Red Brewer,4,5 William Pao,4,5 Luc Morris,6 Marc Ladanyi,7,8 Maria Arcila,7

Katia Manova-Todorova,9 Elisa de Stanchina,10 Larry Norton,1,11 Ross L. Levine,1,8,11

Gregoire Altan-Bonnet,2 David Solit,1,8,11,12 Michael Zinda,13 Dennis Huszar,13*David Lyden,3,14,15† Jacqueline F. Bromberg1,11†

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Lung adenocarcinomas with mutant epidermal growth factor receptor (EGFR) respond to EGFR-targetedtyrosine kinase inhibitors (TKIs), but resistance invariably occurs. We found that the Janus kinase (JAK)/signal transduction and activator of transcription 3 (STAT3) signaling pathway was aberrantly increased inTKI-resistant EGFR-mutant non–small cell lung cancer (NSCLC) cells. JAK2 inhibition restored sensitivityto the EGFR inhibitor erlotinib in TKI-resistant cell lines and xenograft models of EGFR-mutant TKI-resistantlung cancer. JAK2 inhibition uncoupled EGFR from its negative regulator, suppressor of cytokine signaling5 (SOCS5), consequently increasing EGFR abundance and restoring the tumor cells’ dependence on EGFRsignaling. Furthermore, JAK2 inhibition led to heterodimerization of mutant and wild-type EGFR subunits,the activity of which was then blocked by TKIs. Our results reveal a mechanism whereby JAK2 inhibitionovercomes acquired resistance to EGFR inhibitors and support the use of combination therapy with JAKand EGFR inhibitors for the treatment of EGFR-dependent NSCLC.

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INTRODUCTION

Lung cancer is the most frequent cause of cancer death (1), and non–smallcell lung cancer (NSCLC) is the most common subtype. Somatic activat-ing mutations of the tyrosine kinase domain of the epidermal growthfactor receptor (EGFR) are found in about 26% of all patients with lungadenocarcinoma and confer sensitivity to first-generation EGFR tyrosinekinase inhibitors (TKIs), gefitinib and erlotinib (2, 3). Clinical responsesare variable, although most patients exhibit good response rates to theseinhibitors. However, acquired resistance to TKIs unfailingly occurs, inmost cases (>60%) due to the acquisition of “gatekeeper” mutations(T790M) in the EGFR, which is thought to alter kinase ATP (adenosine5′-triphosphate) affinity above that of gefitinib or erlotinib (4, 5). Progression-free survival with TKI treatment is only 9 to 12 months, and overall sur-vival is less than 20 months (2, 3). Notably, the acquisition of secondarymutations in EGFR emphasizes a continued dependence on EGFR

1Department of Medicine, Memorial Sloan Kettering Cancer Center (MSKCC),New York, NY 10065, USA. 2Computational Biology Program, MSKCC, NewYork, NY 10065, USA. 3Children’s Cancer and Blood Foundation Laboratories,Departments of Pediatrics, Cell and Developmental Biology, Weill CornellMedical College (WCMC), New York, NY 10021, USA. 4Division of Hematology/Oncology, Vanderbilt-Ingram Cancer Center (VICC), Nashville, TN 37232,USA. 5Personalized Cancer Medicine, VICC, Nashville, TN 37232,USA. 6Department of Surgery, MSKCC, New York, NY 10065, USA. 7Depart-ment of Pathology, MSKCC, New York, NY 10065, USA. 8Human Oncologyand Pathogenesis Program, MSKCC, New York, NY 10065, USA. 9MolecularCytology, MSKCC, New York, NY 10065, USA. 10Antitumor Assessment, MSKCC,New York, NY 10065, USA. 11WCMC, New York, NY 10021, USA. 12MetastasisResearch Center, MSKCC, New York, NY 10065, USA. 13Oncology iMED,AstraZeneca, Waltham, MA 02451, USA. 14Department of Pediatrics, MSKCC,New York, NY 10065, USA. 15Drukier Institute for Children’s Health, MeyerCancer Center, WCMC, New York, NY 10021, USA.*Present address: Takeda Pharmaceutical Company Ltd., Cambridge, MA02139, USA.†Corresponding author. E-mail: dcl2001@med.cornell.edu (D.L.); bromberj@mskcc.org (J.F.B.)

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signaling in these cancers. The need to overcome both innate and acquiredresistance has been a major therapeutic challenge.

EGFR is a member of the ERBB/human epidermal growth factor re-ceptor (HER) family of membrane-bound receptor tyrosine kinases (RTKs)(6). Aberrant regulation of EGFR, including gain-of-functionmutations andoverexpression, is a common feature of many epithelial malignancies,which has led to the development of EGFR TKIs (7). We previously de-scribed that signal transduction and activator of transcription 3 (STAT3)is persistently tyrosine-phosphorylated or activated (pSTAT3) in NSCLC(cell lines and primary tumors) due to EGFR-driven up-regulation ofinterleukin-6 (IL-6) expression, leading to a feed-forward IL-6/Janus kinase(JAK)/STAT3 loop. Furthermore, JAK inhibition abrogates proliferationin NSCLC cell lines, including those that are TKI-resistant (8). JAK1/2 in-hibitors have shown promise in preclinical models of NSCLC (9–15). In-hibitors of JAKs were developed for immunologic suppression for organtransplantation and for the treatment of myeloproliferative neoplasms inpatients with activating mutations in the JAK2 pathway (16, 17) and arein early-phase clinical trials for lymphomas and solid tumors on the basisof promising preclinical studies (11–15, 18–20).

Our present study investigated the mechanisms by which JAK inhibi-tion represses cell growth in NSCLC cells, alone or in combination withTKIs. Here, we found that JAK2 inhibition overcame acquired resistanceto TKIs in EGFR-mutant lung adenocarcinoma in vitro and in vivo.

RESULTS

JAK2 inhibition resensitizes TKI-resistant cells andxenograft models to erlotinibWe previously demonstrated by immunohistochemistry that pSTAT3 ispresent in 42% of NSCLC cells that have wild-type EGFR and in 88%of NSCLC cells that have mutant EGFR, mediated through increased

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IL-6/JAK signaling (8). Further examination of this cohort of samplesrevealed that 31% of EGFR-mutant NSCLC tumors had high expres-sion (immunohistochemistry) of pSTAT3. Here, we sought to determinethe relevance of JAK/STAT3 activation in tumors that had developed re-sistance to TKIs. Patients with EGFR-mutant NSCLC had their tumorsrebiopsied upon development of acquired resistance to erlotinib or ge-fitinib (hereafter referred to collectively as TKI) (5). We examined the

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abundance of pSTAT3 in 10 TKI-resistant tumors, 4 of which werematched against the untreated primary tumor. We determined that theabundance of pSTAT3 was high (score 2 to 3+) in 68% (4 of 6) of un-matched samples and either similar or increased in all four matched spe-cimens compared to the respective pre-TKI samples (fig. S1A) (21, 22).These results led us to hypothesize that pSTAT3 may be a relevant targetin TKI-resistant disease.

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Fig. 1. Synergistic antiproliferative effects of combined EGFR blockadewith JAK inhibition. (A) MTT-based proliferation assay in H1975, PC-9R,

in the MTT assay are boxed. (B) Western blotting in lysates from H1975,PC-9R, and H1650 cells treated with JAKi (AZD1480, 1 mM), TKI (erlotinib,

and H1650 cells treated with the TKI erlotinib (Ti) in combination with theJAKi AZD1480 (Ji). Data are means ± SEM from five replicates in threeindependent experiments. *P < 0.05; **P < 0.001, versus AZD1480 alone(two-tailed Student’s t test). Below each graph are the representativecombination indices (CIs) of erlotinib (TKI) in combination with AZD1480(JAKi): CI < 0.9 indicates synergy, CI between 0.9 and 1.1 is additive,and CI > 1.1 indicates antagonism. The drug dosage combinations used

0.2 mM), or the combination for 1 hour. Blots are representative of threeexperiments and are quantified in fig. S1D. (C) Tumor volume trackingin mice bearing xenografts of H1975, PC-9R, or H1650 cells and treatedwith vehicle (C), AZD1480, erlotinib, or the combination (Ji + Ti) for 12 to 25days. Doses are provided inMaterials andMethods. Data aremeans ± SEM(n=5 to7miceper group). *P<0.05,AZD1480versus thecombination (two-tailed Student’s t test).

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We tested this hypothesis by treating TKI-resistant, pSTAT3+

NSCLC cell lines (H1975, PC-9R, and H1650) and xenografts with aJAK inhibitor (JAKi; AZD1480) alone or in combination with a TKI(erlotinib) as a negative control. Treatment with JAKi reduced the abun-dance of pSTAT3 and inhibited the proliferation of cultured cells, withmedian inhibitory concentrations in the range of 0.25 to 1.50 mM (Fig. 1,A and B, and fig. S1B) (8, 10, 12, 23). Furthermore, in vivo studies dem-onstrated a significant inhibitory effect of JAKi as a single agent on thegrowth of NSCLC xenograft tumors (Fig. 1C). In contrast, TKI (erlotinib)alone did not inhibit the proliferation of TKI-resistant cell lines H1975and PC-9R and partially inhibited the proliferation of semiresistantH1650 in vitro and in vivo (Fig. 1, A and C), as previously demonstrated(24–26). However, the TKI-resistant cell lines (H1975 and PC-9R) andthe TKI-semiresistant cell line (H1650) were rendered sensitive to TKIby the addition of JAKi, evidenced by decreased cell viability and in-creased apoptosis in cultured cells (Fig. 1A and fig. S1C). The abundanceof pSTAT3, phosphorylated EGFR (pEGFR), and the downstream ef-fector phosphorylated extracellular signal–regulated kinase (pERK)in these cultured TKI-resistant cell lines were markedly reduced uponcombination treatment with JAKi and TKI, whereas expectedly, TKIalone had no effect (Fig. 1B and fig. S1D). In vivo, dual blockade ofJAK and EGFR led to the greatest inhibition of tumor growth when com-pared to either JAKi or TKI alone in H1975, PC-9R, and H1650 xenografts(Fig. 1C). The enhanced inhibitory effect of combination treatment wasaccompanied by decreased pSTAT3, pEGFR, and pERK abundance andreduced proliferation (by Ki67 staining) (fig. S1E). These data indicatethat combined JAKi/TKI treatment is superior to monotherapy and canovercome resistance to EGFR inhibitors in EGFR-mutant NSCLC.

JAK2 inhibition increases EGFR signalingHaving determined that dual JAK and EGFR inhibition overcameacquired TKI resistance, we next sought to define the mechanisms un-derlying this phenomenon. Treating cell lines (H1975, PC-9R, and H1650),

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xenografts (H1975 and PC-9R), and transgenic EGFR-mutant, TKI-resistant NSCLCmousemodels [EGFRL858R +T790M (27)] with JAKireduced the abundance of pSTAT3 in the tumor cells (Figs. 1B and 2, Aand B, and figs. S1E and S2, A and B). However, JAK inhibition led to anincrease in EGFR signaling. Specifically, the abundance of EGFR,pEGFR, and pERK was increased with no apparent effect on the abun-dance of phosphorylated AKT or phosphorylated S6 (Fig. 2 and fig. S2,A to E). Additionally, JAK inhibition enhanced the EGFR-mediated RASactivation in cell lines (fig. S3A). Treating cell lines with JAKi resulted inincreased pERK abundance after 10 min, which slowly returned to base-line as that of pSTAT3 reappeared over 12 to 24 hours (fig. S3B). The shorttime scale required for the JAK inhibition–mediated increase in pERKabundance suggested an effect on signaling rather than de novo transcrip-tion or translation. A similar phenomenon was observed in vivo.We treatedtumor-bearing mice with a single dose of JAKi and observed a rapid in-crease in pERK abundance with a reciprocal reduction in pSTAT3 abun-dance 4 to 6 hours after administration of the drug. Twenty-four hourslater, as pSTAT3 abundance returned, we observed a concomitant reduc-tion in ERK activation (assessed by pERK staining) (fig. S3C).

To determine whether the effect of JAK inhibition on ERK phos-phorylation was specifically mediated through JAKs, we reducedJAK2 expression in NSCLC lines using small interfering RNAs (siRNAs).Depletion of JAK2 increased the abundance of total EGFR, pEGFR,and pERK (Fig. 2C and fig. S3, D and E). Additionally, we found thatseveral JAK2 inhibitors increased the amount of pEGFR and pERK inNSCLC cell lines, suggesting that the effect was not reagent-specific(fig. S3F). Conversely, we asked whether overexpression of a consti-tutively active form of JAK2 (JAK2V617F) (28) could decrease pEGFRabundance in NSCLC cell lines. Transient transfection of JAK2V617F

into PC-9R cells led to a reduction in pEGFR and pERK abundance(fig. S3G). Together, our data demonstrate that JAK2 inhibition en-hances EGFR signaling in NSCLC cell lines, xenografts, and trans-genic mice.

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JAK2 siRNA. Blots are representative of three experiments and are quantified in fig. S3E.

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Fig. 3. JAK2 inhibition increases surface EGFR expression throughSOCS5. (A) Detection of cell surface–bound EGFR on PC-9R cells usingAlexa Fluor–EGF at 4°C after a 1-hour treatment with AZD1480 or con-trol. Scale bars, 50 mm. (B) Serum-starved H1975, H1650, and PC-9Rcells were pretreated with AZD1480 or control for 1 hour. Surface pro-teins were biotinylated, precipitated with avidin resin beads, and ana-lyzed by Western blot for EGFR and c-MET. Blots are representativeof three experiments and are quantified in fig. S4A. (C) Detection ofJAK2-EGFR and SOCS5-EGFR interactions by Duolink staining in PC-9R cells treated with AZD1480 or control for 1 hour. Scale bars, 50 mm.(D) Cell lysates from H1650 and PC-9R cells treated with control orAZD1480 were immunoprecipitated with an antibody against EGFR orJAK2 and analyzed by Western blot for EGFR, JAK2, and ubiquitin.Loading controls were the heavy-chain (H-chain) immunoglobulin G (IgG)for the co-IP, and tubulin for the input. Blots are representative of three

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experiments and are quantified in fig. S4D. (E) PC-9R cells expressingJAK2 shRNA (JAK2sh) or vector control (Csh) constructs analyzed forSOCS5 and EGFR interactions by Duolink staining. Scale bars, 50 mm. Celllysates were analyzed for JAK2 and tubulin. (F) Western blot for SOCS5and tubulin in lysates from H1975 cells expressing scrambled control orSOCS5 shRNA (SOCS5sh). Control or SOCS5sh cells were treated withcontrol, AZD1480 (1 mM), or erlotinib (0.2 mM) for 1 hour and analyzedfor pEGFR, EGFR, and tubulin by Western blot. Representative blots areshown (n = 3). (G) Tumor volumes in mice bearing H1975-SOCS5Sh xeno-grafts and treated with vehicle or TKI (25 mg/kg per day) for 9 days. Dataare means ± SEM (n = 5 to 7 mice per group). **P < 0.01, control versusTKI (two-tailed Student’s t test). (H) Schematic depicting NSCLC cellsexpressing EGFR proteins, wherein JAK2 bridges SOCS5-dependentEGFR degradation, and inhibition or reduction of JAK2 uncouples SOCS5from EGFR, effectively increasing EGFR abundance on the cell surface.

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JAK2 inhibition increases the surface abundance ofEGFR by decreasing the association of EGFRwith SOCS5We hypothesized that altered EGFR turnover could account for the in-crease in EGFR abundance and signaling.We first examined the effect ofJAK inhibition on the levels of membrane-associated EGFR using flu-orescently conjugated EGF. JAKi treatment of NSCLC cells led to an in-crease in the surface staining of EGF-bound EGFR compared to control(Fig. 3A). We obtained similar results using a cell surface EGFR bio-tinylation assay, which revealed an increase in membrane-associatedEGFR in response to JAKi, but no effect on the surface expression ofc-MET (another RTK) (Fig. 3B and fig. S4A). These experiments were

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done in the absence of ligand (after extensive washing of the cells), whichsuggests that JAK2 inhibition decreased ligand-independent turnoverof EGFR, thus resulting in an increase in the steady-state cell surfaceEGFR/pEGFR abundance.

The turnover of EGFR occurs in a ligand-dependent and ligand-independent manner and is regulated through a physical complex withproteins that modify EGFR, leading to its ubiquitination by E3 ubiquitinligases (29). Ligand-independent degradation of EGFR is regulated in partthrough suppressor of cytokine signaling (SOCS) proteins SOCS4 andSOCS5. These highly homologous SOCS boxes containing proteins bindconstitutively to EGFR, together with elongins B/C, the cullin family ofubiquitin ligases and ring box proteins, to enhance EGFR ubiquitination

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Fig. 4. Enhancement of EGFR-ERK signaling by JAK inhibition is mediatedthrough heterodimerization between wild-type/mutant EGFR, which is abro-gated by TKI. (A) Serum-starved H1975 cells were pretreated with AZD1480or control (C) for 1 hour, and EGF ligand was added for the indicated times.Surface proteins were biotinylated, precipitated with avidin resin beads, andanalyzed by Western blot for surface EGFR (sEGFR) and c-MET (sMET).(B) Duolink staining (left) for wild-type (WT) EGFR (Myc) and mutant L858REGFR (MUT) interaction in H1975 cells expressing Myc-tagged WT EGFRprotein and treated with AZD1480 or control for 1 hour. Scale bars, 50 mm.Western blot (right) in lysates from H1975 parental cells (control) and cellsexpressing Myc-tagged WT EGFR were analyzed for EGFR, Myc-taggedprotein, and tubulin. (C) Top, experimental schematic in which H1975 cellsexpressing both theWT (blue sphere) and EGFR-L858R/T790M gatekeeper

mutant (red sphere) proteins are depicted as a single cell expressing varia-ble amounts of each. H1975 cells were treated with either TKI (0.2 mM) or theT790M-specific inhibitor WZ4002 (WZ; 25 nM) for 30 days, and selectedpopulations are depicted by their relative expression of WT and mutantEGFR per cell. Extracts fromWZ4002- and TKI-selected cells treated witheither control or AZD1480 were then analyzed by Western blot as indicated(note that EGFR detects both WT and mutant). The growth inhibitory effectsof AZD1480 (JAKi) in combination with erlotinib (TKI) are shown below asrepresentative CIs. (D) Western blotting as indicated in lysates from serum-starved PC-9R cells treated with AZD1480 for 1 hour and then EGF for30 min in the presence of erlotinib at the indicated concentrations. Blots in(A), (C), and (D) are representative of three experiments each and are quan-tified in figs. S5, A and C, and S6, respectively.

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and degradation of the receptor in a c-Cbl–independent manner (30–32).Additionally, SOCS5 can bind to JAK1 and JAK2 (33).We hypothesizedthat JAK2 may form a complex between SOCS4/5 and EGFR as hasbeen described for JAK2-SOCS3-gp130 (34–37). To examine the effectof JAK inhibition on the interaction between SOCS4/5 and EGFR, we usedthe in situ proximity ligase assay (PLA or Duolink), which can measureassociations between proteins including those that are weak and transient.Notably, this approach does not allow one to differentiate between direct orindirect associations. We chose this technique because the SOCS4/5 pro-teins are labile and difficult to detect by standard biochemical methods.We found that JAK2 and both SOCS4 and SOCS5 are constitutively asso-ciated with the EGFR, but these interactions were abrogated with JAK in-hibition (Fig. 3C and fig. S4B). We then asked if EGFR activity wasrequired for an association between SOCS5 and JAK2 with the EGFR.We treated PC-9R cells with a T790M mutant–specific TKI, and byPLA, we demonstrated that both SOCS5 and JAK2 were no longer asso-ciatedwithEGFR(fig. S4C).Additionally, by coimmunoprecipitation (co-IP)using antibodies against EGFR and JAK2, we found that JAK2 inter-acted with EGFR. Inhibition of JAK1/2 activity with a JAKi reduced theassociation between JAK2 and EGFR (Fig. 3D and fig. S4D). Additionally,by depleting JAK2 abundance through the use of short hairpin RNA(shRNA),we observed a reduced association between SOCS4/5 andEGFR,as assessed by PLA (Fig. 3E and fig. S4E). In agreement with a previousstudy showing that SOCS5 mediates the ubiquitination of EGFR and en-hances its degradation (31), we found that disrupting the SOCS5-EGFR as-sociation with a JAKi reduced the amount of ubiquitinated EGFR in H1650cells (Fig. 3D). To determine whether SOCS5 played a role in regulatingEGFR abundance, we reduced SOCS5 levels by siRNA in H1975 andPC-9R cells.We observed an increase in total EGFR and pEGFRabundance,which was not further increased by JAK inhibition (fig. S4F). Similarly, re-ducing SOCS5 abundance with shRNA increased the abundance of totalEGFR and pEGFR in H1975 cells compared to control cells (H1975-Csh). Compared to that in controls, treating H1975-SOCS5sh cells withTKI (erlotinib) reduced the abundance of pEGFR and enhanced growth in-hibition both in cultured cells and in xenografts (Fig. 3, FandG, and fig. S4,G to I). Thus, a reduction of SOCS5was sufficient to reverse TKi resistance,similar to that seen with the combination of JAKi and TKI. Finally, the as-sociation between JAK2 and EGFR was intact in H1975-SOCS5sh cellswhen assessed by PLA (fig. S4J). In summary, these data suggest that ac-tivated JAK2 reduces EGFR abundance by coupling the negative regu-latory SOCS4/5 proteins to EGFR (Fig. 3H).

JAK2 inhibition promotes erlotinib-sensitiveheterodimers between wild-type and mutant EGFRGiven that JAK inhibition (and/or a reduction in SOCS5) increased EGFRabundance and downstream signaling (as assessed by the abundance ofpERK), we next asked how this increase could paradoxically restore sen-sitivity to TKIs rather than promote resistance. It has been shown thatligand-induced EGFR internalization is more rapid for wild-type EGFRthan mutant EGFR (38–40). Here, we examined EGF-mediated EGFRinternalization and determined that it occurred more rapidly in JAKi-treated cells compared to vehicle-treated cells (Fig. 4A and fig. S5A), sug-gesting that JAKi may increase the abundance of specifically wild-typeEGFR. We hypothesized that JAKi promotes the formation of hetero-dimers between wild-type and mutant EGFR, the activity of which can berepressed by TKI; thus, we next determined whether wild-type EGFRcould indeed form heterodimers with mutant EGFR. Unfortunately, noantibodies exist that discriminate wild-type from mutant EGFR; there-fore, to test our hypothesis, we used H1975 (L858R + T790M mutant)cells that overexpressed a Myc-tagged wild-type EGFR (41) and found

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that JAKi increased the association between Myc-tagged wild-type EGFRand the L858R + T790M EGFR in these cells (Fig. 4B). Furthermore, weshowed that JAK inhibition increased the abundance of markers indicat-ingwild-type EGFR signaling in anNSCLC line (H292) that overexpressedonly wild-type EGFR (fig. S5B). Next, we selected H1975 cells enrichedfor wild-type EGFR (“wild type–dominant subclone”) by treating cellswithWZ4002 (42), a T790Mmutant–specific TKI. Conversely, we selectedfor H1975 cells enriched for the L858R/T790M EGFR (“mutant-dominantsubclone”) by treating cells with erlotinib to target wild-type EGFR, as pre-viously described (43) (Fig. 4C). Treatment of wild type–dominant cellswith a JAKi led to an increase in the abundance of pEGFR, EGFR, andpERK (Fig. 4C, fig. S5C). Cotreatment of EGFR wild type–dominantcells with JAKi and erlotinib led to a greater drug synergism as comparedto the parental H1975 cells (CI, 0.26 versus 0.40) (Fig. 4C). On the otherhand, cotreatment of mutant-dominant cells with JAKi and erlotinib didnot overcome resistance to erlotinib (CI, 0.91 versus 0.40) (Fig. 4C). Inconclusion, these data support our hypothesis that JAK inhibition leadsto enhanced heterodimer formation between wild-type and TKI-resistantmutant EGFR.

We next asked whether the JAKi-dependent increase in wild-typeEGFR abundance and activity could be blocked by the TKI erlotinib. Al-though wild-type EGFR is sensitive to TKI, it requires higher concentra-tions to inhibit its activity compared to mutant EGFR. We postulated thatthe wild-type/mutant EGFR heterodimers would thus require higher con-centrations of erlotinib as compared to cells expressing erlotinib-sensitivemutant EGFR to inhibit signaling. pEGFR and pERK abundance in con-trol TKI-resistant PC-9R and H1975 cells was unaffected by erlotinib,whereas in JAKi-treated cells, pEGFR and pERK abundance was mark-edly attenuated by increasing concentrations of TKI (Fig. 4D and figs.S5D and S6A). In summary, our data suggest that JAK2 inhibition in-duced an increase in EGFR abundance through the loss of SOCS4/5-mediated degradation, leading to the formation of wild-type/mutant,TKI-sensitive heterodimers (Fig. 5).

DISCUSSION

Constitutive activation of oncogenic signaling pathways in cancers has ledto the development of targeted inhibitors. Erlotinib and gefitinib, TKIs ofEGFR, both induce clinical responses in up to 90% of patients with EGFR-mutant lung adenocarcinoma, yet these responses endure, on average, lessthan 1 year (2). Efforts to understand the mechanisms of resistance haverevealed significant cross-inhibition betweenmitogenic pathways, such thatblockade of one pathway relieves the negative feedback on another result-ing in a relative “insensitivity” to the targeting pharmacologic agent (44–48).In the case of EGFR-mutant NSCLC, an estimated 60% of tumors acquirea gatekeeper mutation that impairs TKI binding (5). To date, clinical trialsof drugs intended to restore sensitivity to tumors with acquired resistancehave been unsuccessful.

Aberrant regulation of the IL-6–JAK–STAT3 signaling pathway is acritical mediator of tumorigenesis, which has similarly led to the devel-opment of targeted inhibitors (17). Here, we found that high pSTAT3abundance persists in TKI-resistant NSCLC primary tumors, suggestingthat pSTAT3 signaling may play a role in mediating TKI resistance.Consistent with our observations, Yao et al. showed that IL-6–activatedgp130/JAK/STAT3 signaling decreased TKI sensitivity in resistantNSCLC H1650 cells that have no T790M gatekeeper mutation or otherknown resistance mechanisms (10). Another publication by Li et al. sug-gested that a STAT3/Bcl2/Bcl-XL survival pathway may be required forearly adaptive and late acquired TKI resistance, in an erlotinib-selectedresistant NSCLC cell line (49). Additionally, we showed that JAK inhibition

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alone reduced the growth of NSCLC xenografts (14). These data supportthe therapeutic potential of JAKi for the treatment of solid tumors asmonotherapy (12–14, 18, 20).

Although JAK inhibition alone could decrease the growth of TKI-resistant cells, we were surprised that combined treatment with JAKi andTKI could overcome resistance in TKI-resistant cells and xenografts. Spe-cifically, PC-9R and H1975 cells expressing the TKI-resistant gatekeeperEGFR and semi–TKI-resistant H1650 cells were rendered sensitive to aTKI when treated in combination with a JAKi. We then examined themechanisms underlying this synergism.

We first determined that either JAK2 depletion or inhibition of activ-ity enhanced EGFR/RAS/MEK/ERK signaling through an increase inmembrane EGFR protein abundance in the absence of ligand. Severalnon–ligand-dependent inhibitors of EGFR have been identified, includingleucine-rich repeats and immunoglobulin-like domains protein 1 (LRIG1),receptor-associated late transducer (RALT; also known as MIG6 andERRFI1), SOCS4, and SOCS5. Ectopic overexpression of SOCS5 andSOCS4was shown to induce the degradation ofEGFR in a ligand-and c-Cbl–independent manner and through an elongin B/C–dependent process(6, 30, 31, 50). Although structural studies have suggested that theSH2 domains of SOCS4 and SOCS5 proteins may bind to the phospho-rylated Tyr1068 of the EGFR, no functional studies have demonstratedthis (6, 30, 31, 36). More recently, SOCS5 was shown to inhibit JAK1 andJAK2 through its N-terminal domain (33). Additionally, Shc-1 was identi-fied as a potential substrate for SOCS5, suggesting that this protein can

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negatively regulate multiple tyrosine kinases (33). Here, our data suggestthat JAK2 bridges a ternary complex between SOCS5 and EGFR. Spe-cifically, pharmacologically inhibiting JAK2 or knocking down its proteinabundance led to a loss of SOCS5 association with the EGFR, resultingin attenuated ubiquitination of EGFR and the consequent up-regulationof EGFR surface expression and downstream RAS-pERK signaling. Sim-ilarly, a reduction of SOCS5 in TKI-resistant H1975 cells also led to an in-crease in pEGFR, which was inhibited by TKI (specifically, erlotinib).Notably, the loss of SOCS5 had no impact on the association betweenJAK2 and EGFR. Conversely, enforced expression of activated JAK2could attenuate EGFR signaling possibly through a mechanism involvingJAK2-mediated increased SOCS4/5 recruitment to the EGFR. In support ofthis hypothesis, Harada et al. observed that in a TKI-resistant PC-9 cellclone, increased pJAK2 abundance was associated with reduced totalEGFR and pEGFR abundance compared to that in the parental line (23).Finally, the JAK2-EGFR association is likely dependent on activated EGFRbecause pharmacological inhibition of EGFR resulted in the loss of bothEGFR-JAK2 and EGFR-SOCS5 interactions. In summary, these data sug-gest that activated EGFR is negatively regulated by activated JAK2 inpart by bringing SOCS5 to EGFR, leading to its degradation (51, 52).

The resultant increase in EGFR expression and activity translatedinto restoration of sensitivity to TKIs was surprising.We determined thatJAK inhibition led to increased levels of heterodimers between mutantEGFR and wild-type EGFR, which were sensitive to TKIs. Inhibition ofspecifically wild-type EGFR in these heterodimers could explain how

MUT MUT

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MEK

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JAKinhibitor

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JAK2 inhibition sensitizes resistant EGFR mutant lung adenocarcinoma to tyrosine kinase inhibitors

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Fig. 5. Working model: JAK inhibition enhances TKI-sensitive EGFR sig-naling and growth inhibition in NSCLC. Schematic depicting TKI-resistantNSCLC cells expressing both WT and gatekeeper mutant EGFR pro-teins, which form homodimers or heterodimers. At steady state, JAK2 pro-motes SOCS5-dependent EGFR degradation. TKI-resistant, homodimericmutant EGFR is the principle driver of the RAS–MEK (mitogen-activatedprotein kinase kinase)–ERK signaling cascade (in bold). Inhibition or re-

duction of JAK2 uncouples SOCS5 from EGFR, effectively increasingTKI-sensitive, wild-type EGFR homodimers/heterodimers and signaling(in bold). Although the role of EGFR WT:MUT heterodimer signalinghas not been clearly defined, we hypothesize that it may regulate TKIsensitivity. This working model may explain the synergistic actions ob-served between the EGFR-targeted TKI and JAKi on NSCLC tumorgrowth.

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JAK inhibition conferred sensitivity to otherwise resistant tumors. About30% of tumors resistant to erlotinib lack known alterations (namely,T790M mutation or MET amplification); this phenotype is exemplifiedin the H1650 cell line (5). We observed the greatest growth inhibitorysynergy between the JAK2 inhibitor and erlotinib in H1650 cells. Wehypothesize that the JAK2-regulated increase in EGFR expression andactivity enhances the dependence or “addiction” to this signaling path-way. We propose that increased EGFR signaling activates negative feed-back loops suppressing other pathways that normally participate inpromoting survival and proliferation. The chronic inhibition of compensa-tory pathways leads to the EGFR-addicted phenotype. This hypothesis hasbeen proposed for other diseases such as estrogen receptor (ER)– andHER2-positive breast cancers, in which the expression and activity of thesedrivers of disease are positively correlated with response to targeted inhibi-tors. For example, modifiers of ER abundance such as inhibitors of thephosphatidylinositol 3-kinase–mammalian target of rapamycin pathway re-sult in synergistic antiproliferative effects when given in combination withendocrine therapy, leading to a survival advantage in patients (53–55). Ourresults demonstrate crosstalk between the JAK and EGFR oncogenicsignaling pathways, thus providing a molecular rationale for combinationJAK- and EGFR-targeted tyrosine kinase inhibitor therapies in patients thatexhibit innate or acquired resistance to EGFR-targeted TKIs, a group thatultimately includes all patients with EGFR-mutant lung adenocarcinoma.

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MATERIALS AND METHODS

Human tumor specimensBiopsy specimens were obtained from patients starting TKI therapy andthose who had developed progression of disease on continuous TKI ther-apy. These specimens were subsequently analyzed for EGFR kinase–activating mutations/second T790M mutation, and assessment of METgene copy number alterations was carried out by standard protocols aspreviously described (22).

DrugsThe JAK1/2 inhibitor AZD1480 was provided by AstraZeneca. The type IIJAK2 inhibitor BBT594was provided byNovartis. INCB18424 and EGFRTKI erlotinibwere purchased fromChemietek. Pan-JAKi P6was purchasedfromCalbiochem. The EGFRT790M–specific inhibitorWZ4002 was pur-chased from Selleck Chemicals. For in vitro experiments, the inhibitorswere dissolved in 100% dimethyl sulfoxide (DMSO) to prepare a 10mMstock and stored at −20°C. For in vivo experiments, the indicated inhibitorswere formulated daily in purified, sterile water supplemented with 0.5%methyl cellulose and 0.1% Tween 80.

Cell linesCell lines were grown in RPMI 1640 medium. PC-9 and PC-9R cell lineswere previously described (43); H1975, H1650, and H292 cell lines werepurchased from the American Type Culture Collection (ATCC). H1975cells overexpressing Myc-tagged wild-type EGFR were generated by ret-roviral infection of pMSCVpuro-IRES-EGFR-Myc/His into H1975 cells,and stable clones were isolated by puromycin selection (41, 56).

AnimalsC57/B6 and athymic nude micewere purchased fromTaconic and Harlan.Mice harboring the CCSP-rtTA and tet-regulated EGFRL858R+T790M

transgenes (“C/L858R + T790M”) were developed by W. Pao. All ex-periments involving animals were approved by the Memorial Sloan Ket-tering Cancer Center Institutional Animal Care andUse Committee. Cell

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lineswere subcutaneously implanted in athymicmice for PC-9, H1975, andH1650 xenograft tumors in a 1:1 mixture of Matrigel (BD Biosciences)and culture medium. Tumor-bearing mice were randomized on the basisof tumor volume before the treatment, which was initiated when averagetumor volume was about 100 mm3. All inhibitors were given orally bygavage: AZD1480, 30 mg/kg, twice a day for H1975 and H1650, and20 mg/kg daily for PC-9R; erlotinib, 25 mg/kg, once a day for H1975,H1650, and PC-9R. Tumors were measured every 3 to 4 days, and tumorvolumes were calculated by the formula L ×W2 × p/6, where L is the tumorlength andW thewidth. Three hours before sacrifice, the animalswere giventhe last dose of drug treatment.

Cell lysate preparation, Western blotting, anddensitomeric analysisAdherent cells were lysed in cell lysis buffer, and Western blot analysiswas performed as previously described (8). Cells were treated with theproteasome inhibitor MG132 (N-carbobenzyloxy-L-leucyl-L-leucyl-L-leucinal) (5 mM) for 16 hours before the lysis of cells for the analysisof SOCS4 and SOCS5 proteins. Antibodies used for Western blottingwere as follows: anti-pSTAT3 (Y705), total STAT3, pERK (p44/42, T202/Y204), pAKT (S473), totalAKT, pS6 (S240/244), S6, pEGFR (Y1068), totalEGFR, L858R-EGFR– or del19-EGFR–specific antibodies, anti-JAK2(Cell Signaling Technology), and anti-JAK1 (BD Biosciences); anti–totalERK, EGFR, Myc, ubiquitin and horseradish peroxidase (HRP)–conjugatedanti-rabbit IgG, HRP-conjugated anti-mouse IgG, HRP-conjugated anti-rabbit IgG, SOCS4, and SOCS5 (Santa Cruz Biotechnology); anti–c-MET(Invitrogen); and anti–a-tubulin (Sigma-Aldrich). Densitometric quantifica-tion of high-resolution immunoblot imageswas performed using an analysisprogram, ImageJ (developed by W. Rasband, Research Services Branch ofthe National Institute of Mental Health). Analyses of the phosphorylationstatus of RTK and ErbB family members were performed using the RayBioHuman RTK and EGFR Phosphorylation Array kits according to the man-ufacturer’s instruction (RayBiotech).

RAS activation assayThe status of activated RAS was measured using the RAS Activation As-say Kit according to the manufacturer’s instruction (US Biological). Briefly,the cells were treated by AZD1480 as indicated, washed twice with ice-coldphosphate-buffered saline (PBS), and lysed with lysis buffer. The activated/guanosine 5′-triphosphate–bound RAS was then pulled down by agarosebead–conjugated RAS-binding domain of Raf-1, electrophoresed bySDS–polyacrylamide gel electrophoresis gel, and probed with an anti-RAS antibody.

DNA constructspBabe-YFP-JAK2/V617F, an active form of human JAK2 expression con-struct, was generated by excising a wild-type JAK2 complementary DNAfrom pEF-YFP-JAK2 (57) usingAge I and Spe I, Klenow filled, and ligatedinto the SnaB I site of pBabe-puro. A polymerase chain reaction–basedmu-tagenesis of converting JAK2 amino acid 617 valine to phenylalanine wassubsequently performed using DNA polymerase Pfu and primer sets 5′-GAATTATGGTGTCTGTTTCTGTGGAGAGGAGAAC-3′ and 5′-GTTCTCCTCTCCACAGAAACAGACACCATAATTC-3′.

Cell transfection, RNA interference, andlentiviral infectionsH1975, H1650, and PC-9 cells were transfected by siRNAs (25 nM) withHiPerFect (Qiagen) according to the manufacturer’s protocol. Silencervalidated JAK2, SOCS4, and SOCS5 siRNAs and nonsilencing scramblesiRNAwere purchased from Qiagen. Cells were plated in a six-well plate

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and incubated for 36 hours after siRNA transfection, and cell lysates werecollected forWestern blotting. The plasmids pBabe-puro and pBabe-JAK2/V617F were transiently transfected into PC-9 cells with the SuperFect re-agent (Qiagen). Forty-eight hours later, cell lysates were collected for West-ern blotting. JAK2 shRNA, SOCS5 shRNA lentiviral, and control constructswere purchased from Sigma-Aldrich, and infections into H1975 followed byselection with puromycin (2 mg/ml) were performed according to the manu-facturer’s instructions.

Flow cytometryCells were resuspended in 0.5 ml of ice-cold 1% bovine serum albumin(BSA)–PBS. Cell apoptosis was determined by labeling with annexin Vand propidium iodide (PI). Samples were analyzed using a BD FACSCa-libur cytometer, and data were analyzed using FlowJo software. Underunfixed conditions, cells that were annexin V(−) and PI(−) wereconsidered viable cells. Cells that were annexin V(+) and PI(−) wereconsidered early-stage apoptotic cells. Cells that were annexin V(+)and PI(+) were considered late-stage apoptotic cells.

Duolink fluorescence stainingIn situ protein-protein interactions were measured by using the Duolink IIRed fluorescence staining kit according to the manufacturer’s instruction(Olink Bioscience). Cells were fixed in 4% paraformaldehyde (PFA), per-meabilized with 0.2% Triton X-100 in PBS, and probed with primary anti-bodies against the proteins of interest and isotype controls (1:100 to1:200) at 4°C overnight. The kit-provided PLA probes were subsequentlyadded, and signal amplification was performed. Imaging analyses wereconducted using a Zeiss fluorescence microscope. SOCS4 and SOCS5antibodies were purchased from Santa Cruz Biotechnology.

Cell viability assaysThe effects of AZD1480 and other inhibitors on in vitro cell growth wereassayed using the MTT Cell Proliferation Assay kit (ATCC). In brief, cellswere plated in 96-well plates in five replicates in RPMI plus 10% fetalbovine serum and allowed to attach for 24 hours before the addition ofDMSO as control or the indicated drugs. After 6 days, with medium re-plenishment at day 3, cells were first incubated with MTT until a purpleprecipitate was visible (in about 3 hours), which was then solubilized bydetergent and quantified by absorbance at 570 nm with a referencewavelength of 670 nm.

CoimmunoprecipitationThe cells treated with AZD1480 or control DMSO were first lysed bywhole-cell lysis buffer without detergent, followed by preclearing usingcontrol IgG/serum and protein A and GammaBind G Sepharose beads(protein A/G beads, Pharmacia). For EGFR IP, 1 mg of antibody was usedper 200 mg of protein extract; for JAK2 IP, 5 ml of antiserum was used. Co-IPs used 1 mg of precleared protein extract, which was incubated with theantibody/antiserum overnight at 4°C, before adding a 1:1 mix of proteinA/G beads for another 2 hours. The beads were washed five times withlysis buffer before boiling in sample buffer and being subjected to Westernblot analysis.

Biotinylation and precipitation of cell surface proteinsCells were serum-starved overnight and pretreated with AZD1480 or con-trol DMSO for 1 hour, and the cell surface proteins were then biotinylatedwith Sulfo-NHS-LC-Biotin (0.2 mg/ml; Pierce) in PBS for 30 min at 4°C.Unreacted biotin was quenched and removed by washing twice with ice-cold PBS containing 0.1 M glycine and twice with ice-cold PBS. For pre-cipitation of surface biotinylated proteins, cells were lysed with lysis

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buffer, and the cell lysate was incubated with NeutrAvidin Plus UltraLinkResin (Pierce) and equilibrated in PBS at 4°C for 1 hour. The resin wasthen washed twice with PBS, and the levels of cell surface EGFR andc-MET were analyzed by Western blotting.

Statistical and CI analysesThe effectiveness of the inhibitors used in this study, alone and in combi-nation, was analyzed by using CalcuSyn software (Biosoft). The CI wascalculated according to the Chou-Talalay method (58). CI < 0.9 indicatessynergy, CI between 0.9 and 1.1 is addictive, and CI > 1.1 indicates an-tagonism between the two drugs. The determination of the in vitro effectsof drugs on cell cycle and apoptosis was achieved by three independentexperiments. Data were analyzed by Student’s t test, and statistical signif-icance was set at P < 0.05.

Surface EGFR labeling using Alexa Fluor 488–EGFCells were seeded at 0.5 × 106 and grown on coverslips in six-well platesbefore being treated with 1 mM AZD1480 or control DMSO for 1 hour.The coverslips were then chilled on ice for 10 min and then incubatedwith Alexa Fluor 488–EGF (0.5 mg/ml; Molecular Probes) in serum-freemedium with 1% BSA at 4°C for 1 hour for surface EGFR labeling. Thecells were subsequently washed twice with cold PBS, fixed in PFA, andviewed by fluorescence imaging.

Immunohistochemistry and immunofluorescence analysesTumor xenografts were fixed immediately after removal in a 4% PFA so-lution for 24 hours at 4°C followed by standard paraffin embedding andsectioning. Slides underwent deparaffinization, antigen retrieval, and im-munohistochemistry and immunofluorescence analyses as previously de-scribed (8). Semiquantitative analysis of pSTAT3 was performed (forhuman specimens, xenografts, and mouse transgenic tumors) by consid-ering both the intensity (0, negative; 1, low; 2, moderate; 3, strong) and thepercentage of cells displaying a positive signal (0, negative; 1, 0 to 5%; 2,5 to 25%; 3, 25 to 50%; 4, 50 to 75%; 5, 75 to 100%), and the respectiveindices were multiplied to each other to calculate a score value of 0, 1+(low), 2+ (moderate), or 3+ (high).

SUPPLEMENTARY MATERIALSwww.sciencesignaling.org/cgi/content/full/9/421/ra33/DC1Fig. S1. STAT3 activation and inhibition in TKI-resistant NSCLC.Fig. S2. JAK2 inhibition increases the abundance of pEGFR and pERK in NSCLC.Fig. S3. JAK2 inhibition leads to rapid activation of EGFR/RAS/ERK signaling.Fig. S4. JAK2 links SOCS5 to EGFR, regulating sensitivity to TKI.Fig. S5. Quantification of Western blots in Fig. 4, A and C.Fig. S6. Quantification of Western blots in Fig. 4D.

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Acknowledgments:We thank T. Radimerski fromNovartis for BBT-594.We also thank themembers of our laboratories for helpful discussions and themembers of theMemorial SloanKettering Cancer Center Animal Imaging, Antitumor Assessment and Molecular Cytologycore facilities (in particular, H. Zhao, J. Qiu, A. Barlas, S. Fujisawa, and V. D. Gueorguiev).We would also like to thank L. Regales for providing assistance with the EGFR transgenicmice. We thank C. Haan for providing pEF-YFP-JAK2 and AstraZeneca for providingAZD1480. Funding: Our work was supported by NIH grants U54 CA148967 (J.F.B. andG.A.-B.), R01CA87637 (J.F.B.), and P30CA008748 (J.F.B.); theCharles andMarjorie HollowayFoundation (J.F.B.); the Sussman Family Fund (J.F.B.); the Lerner Foundation (J.F.B.); AstraZeneca (J.F.B.); the Manhasset Women’s Coalition Against Breast Cancer (J.F.B.); the NewYork State USBCWomen’s Bowling Association (J.F.B.); Uniting Against Lung Cancer grant(S.P.G.); the Children’s Cancer and Blood Foundation (D.L.); the Manning Foundation (D.L.);the Hartwell Foundation (D.L.); the Pediatric Oncology Experimental Therapeutics Investiga-tors Consortium (D.L.); the Stavros S. Niarchos Foundation (D.L.); the Champalimaud Foun-dation (D.L.); the Nancy C. and Daniel P. Paduano Foundation (D.L.); the Mary KayFoundation (D.L.); the American Hellenic Educational Progressive Association, 5th District(D.L.); the Malcolm Hewitt Wiener Foundation (D.L.); the George Best Costacos Foundation(D.L.); National Cancer Institute grant R01CA 098234-01 (D.L.); Susan G. Komen for theCure (D.L.); Physical Sciences-Oncology Centers training grant NCI-U54-CA143836 (D.L.);and the Beth C. Tortolani Foundation (J.F.B. and D.L.).Author contributions:D.L. and J.F.B.contributed to study design, data interpretation, andmanuscript preparation. S.P.G., L.A.D.,Q.C., G.A.-B., K.M.-T., M.L., N.M., T.C., S.H.L., R.V., E.B., D.S., E.d.S., and E.S.contributed to study design and data acquisition and analysis. S.P.G., L.A.D., Q.C., T.C.,and E.S. also contributed to manuscript preparation. M.A. and L.N. contributed to study designand manuscript preparation. R.L.L. and M.Z. contributed to study design. H.Z. characterizedthe wild-type allele in TKI-resistant cells. W.P. and M.R.B. produced H1975-EGFRmyccells. D.H. provided AZD1480. L.M. performed the statistical analysis. Competing in-terests: The authors declare that they have no competing interests. D.H. was an employeeof AstraZeneca and holds stock. W.P. is an employee of Roche. M.Z. is an employee ofAstraZeneca and has equity holdings. M.A. is on the advisory committee for AstraZeneca.M.L. is on the Afatinib targeted therapy advisory committee for National Comprehensive CancerNetwork/Boehringer Ingelheim. J.F.B. is a consultant for Roche/Genentech. Data andmaterials availability: A patent related to this manuscript is pending. A material transferagreement is required for AZD1480 (J.F.B.) and H1975-EGFRmyc cells (M.R.B.).

Submitted 6 July 2015Accepted 8 March 2016Final Publication 29 March 201610.1126/scisignal.aac8460Citation: S. P. Gao, Q. Chang, N. Mao, L. A. Daly, R. Vogel, T. Chan, S. H. Liu, E. Bournazou,E. Schori, H. Zhang, M. Red Brewer, W. Pao, L. Morris, M. Ladanyi, M. Arcila, K. Manova-Todorova,E. de Stanchina, L. Norton, R. L. Levine, G. Altan-Bonnet, D. Solit, M. Zinda, D. Huszar, D. Lyden,J. F. Bromberg, JAK2 inhibition sensitizes resistant EGFR-mutant lung adenocarcinomato tyrosine kinase inhibitors. Sci. Signal. 9, ra33 (2016).

8

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inhibitorsJAK2 inhibition sensitizes resistant EGFR-mutant lung adenocarcinoma to tyrosine kinase

Jacqueline F. BrombergStanchina, Larry Norton, Ross L. Levine, Gregoire Altan-Bonnet, David Solit, Michael Zinda, Dennis Huszar, David Lyden andHaiying Zhang, Monica Red Brewer, William Pao, Luc Morris, Marc Ladanyi, Maria Arcila, Katia Manova-Todorova, Elisa de Sizhi P. Gao, Qing Chang, Ninghui Mao, Laura A. Daly, Robert Vogel, Tyler Chan, Shu Hui Liu, Eirini Bournazou, Erez Schori,

DOI: 10.1126/scisignal.aac8460 (421), ra33.9Sci. Signal. 

inhibitors may overcome drug resistance in NSCLC patients.of mutant and wild-type receptors and were sensitive to EGFR inhibitors. Thus, combining JAK inhibitors with EGFRwhich reduced the ubiquitin-mediated degradation of EGFR. The EGFRs remaining at the cell surface were heterodimers the internalization and degradation of receptors. In the absence of functional JAK2, less SOCS5 interacted with EGFR,EGFR inhibitor sensitivity in NSCLC cells. JAK2 formed a complex with EGFR and the protein SOCS5, which promotes

. found that inhibiting the kinase JAK2 can restoreet altargeted inhibitors of the tyrosine kinase receptor EGFR. Gao small cell lung cancer (NSCLC) patients, resistance develops to−Although initially effective in treating some non

Reversing resistance in lung cancer

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