Targeting DDX3 with a small molecule inhibitor for lung cancer therapy

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1 Research rticle Targeting X3 with a small molecule inhibitor for lung cancer therapy Guus M ol 1,2, Farhad Vesuna 1, Min Xie 1, Jing Zeng 3, Khaled ziz 3, ishant Gandhi 3, nne Levine 1, shley Irving 1, orian Korz 1, Saritha Tantravedi 1, Marise R Heerma van Voss 1,2, Kathleen Gabrielson 4, Evan ordt 5, rian M Polster 5, Leslie Cope 6, Petra van der Groep 2, tul Kondaskar 7, Michelle Rudek 6, Ramachandra S Hosmane 7, Elsken van der Wall 8, Paul J van iest 2,6, Phuoc T Tran 3,6 & Venu Raman 1,2,6,* bstract Lung cancer is the most common malignancy worldwide and is a focus for developing targeted therapies due to its refractory nature to current treatment. We identified a R helicase, X3, which is overexpressed in many cancer types including lung cancer and is associated with lower survival in lung cancer patients. We designed a first-in-class small molecule inhibitor,, which binds to X3 and abrogates its activity. Inhibition of X3 by caused G1 cell cycle arrest, induced apoptosis, and promoted radiation sensitization in X3-overexpressing cells. Importantly, in combination with radiation induced tumor regression in multiple mouse models of lung cancer. Mechanistically, loss of X3 function either by shr or by impaired Wnt signaling through disruption of the X3 b-catenin axis and inhibited nonhomologous end joining the major repair pathway in mammalian somatic cells. verall, inhibition of X3 by promotes tumor regression, thus providing a compelling argument to develop X3 inhibitors for lung cancer therapy. Keywords X3; repair; lung cancer; radiation-sensitizing agent; small molecule inhibitor Subject Categories Cancer; Respiratory System I /emmm Received 25 June 214 Revised 9 February 215 ccepted 12 February 215 Published online 27 March 215 EM Mol Med (215) 7: Introduction Lung cancer is the most common cancer worldwide, and it claims more lives than prostate, colon, and breast cancer combined (Siegel et al, 213). epending on tumor type and stage, the treatment for lung cancer patients typically consists of surgery or chemoradiation. For most patients, current treatments do not cure the disease and are associated with substantial toxicity. lthough surgical resection offers the best long-term survival for lung cancer patients, only a subset of these patients are considered operable and chemoradiation is the only option for the majority of patients (Manser et al, 25). Recent advances in radiation therapy such as stereotactic body radiation therapy (SRT) or stereotactic ablative radiation therapy (SR) have shown increased efficacy to reduce lung tumor burden and offer a new therapeutic modality to non-surgical patients. Clinical experiences with SRT in early-stage lung cancer and oligometastatic cancer have demonstrated excellent local control of greater than 9% (Timmerman et al, 21). ecause of increased toxicity with delivery of SRT to large treatment targets or following re-treatment, there has been an ongoing search for tumor-selective radiation sensitizers that would enable the use of lower dose per fraction with increased efficacy (Senthi et al, 212). In our quest to characterize cellular pathways that are essential for the oncogenic state, we have identified X3, an R helicase, which is dysregulated in many cancer types including lung cancer. X3 is a member of the E-box family which is involved in a number of cellular processes like transcription, R splicing, mr export, and translation initiation (Lorsch, 22; Rocak & Linder, 24). X3 has also been associated with cancer biogenesis (Hu et al, 24). Previously, we identified X3 in a microarray screen of breast cancer cells exposed to cigarette smoke and demonstrated its role in cancer progression (otlagunta et al, 28). X3 promotes proliferation and cellular transformation (Hu et al, 24; Shih et al, 27; Lee et al, 28), has anti-apoptotic properties (Li et al, 26; Sun et al, 28, 211), modulates cell adhesion and motility (Chen et al, 214), and responds to hypoxia via HIF-1a (otlagunta et al, 211; ol et al, 213). esides the oncogenic role of X3 in cancer biogenesis, there is a report that indicates loss of 1 epartment of Radiology and Radiological Science, Johns Hopkins University School of Medicine, altimore, M, US 2 epartment of Pathology, University Medical Center Utrecht, Utrecht, The etherlands 3 epartment of Radiation ncology, Johns Hopkins University School of Medicine, altimore, M, US 4 epartment of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, altimore, M, US 5 epartment of nesthesiology, University of Maryland School of Medicine, altimore, M, US 6 epartment of ncology, Johns Hopkins University School of Medicine, altimore, M, US 7 epartment of Chemistry & iochemistry, University of Maryland, altimore County, M, US 8 epartment of Internal Medicine, University Medical Center Utrecht, Utrecht, The etherlands *Corresponding author. Tel: ; vraman2@jhmi.edu 648 EM Molecular Medicine Vol 7 o5 215 ª 215 The uthors. Published under the terms of the CC Y 4. license

2 Guus M ol et al Targeting X3 in lung cancer EM Molecular Medicine X3 via p53 inactivation can promote tumor malignancy in nonsmall cell lung cancer (Wu et al, 214). lso, recent evidence has identified that X3 acts as an allosteric activator of casein kinase 1 in the Wnt/b-catenin pathway (Cruciat et al, 213). Initially, the Wnt/b-catenin pathway was described in colon cancer. ctivating mutations of X3 were also shown to be involved in pathogenic Wnt pathway activation in medulloblastoma (Jones et al, 212; Pugh et al, 212; Robinson et al, 212) and chronic lymphatic leukemia (CLL) (Wang et al, 211). Recently, it has been shown that activated Wnt signaling predicts decreased survival in lung cancer patients (Xu et al, 211; Shapiro et al, 213) and decreases sensitivity to radiation therapy (Woodward et al, 27; Zhang et al, 21). In the present study, we synthesized a X3 inhibitor, (diimidazo[4,5-d:4,5 -f]-[1,3]diazepine) (Kondaskar et al, 21) which can potentially be used in cancer treatment. inding of to X3 impedes the function of X3, resulting in activation of cell death pathways, inhibition of the Wnt-signaling pathway, and abrogation of non-homologous end-joining (HEJ) activity. In combination with radiation, synergistic cell death effects were observed both in vitro and in multiple preclinical lung cancer models. Results X3 overexpression correlates with aggressive lung cancer X3 is expressed in lung cancer cell lines (H23,, H46, 549, and H3255) but not in the normal lung cell line HEC (Fig 1). To assess the effect of X3 on malignant growth, we generated two cell lines with reduced X3 expression shx3 and 549shX3. Parental and 549 cells, transfected with vector control, efficiently form colonies and grow rapidly. However, knockdown of X3 significantly reduced colony formation (Fig 1 and C) and proliferation (Fig 1) and resulted in a higher percentage of cells undergoing senescence (Fig 1E). To corroborate our findings in lung cancer patients, we analyzed 95 lung cancer samples for X3 expression. In normal lung parenchyma, we saw little or no expression of cytoplasmic X3 (herein X3 expression) (Fig 1F). However, almost all (94 out of 95) lung cancer samples expressed X3, of which 63 samples (66%) expressed high levels of X3 (Fig 1G J). High X3 expression was equally distributed among different histological subtypes of lung cancer including SCLC and SCLC (Fig 1J). Patients whose lung cancer samples expressed high levels of X3 died on an average 18 months earlier as compared to patients with low X3-expressing tumors (Fig 1K). The hazard ratio (HR) for death was 2.1 (95% CI; ). Furthermore, X3 was found to be a predictor of overall survival, independent of tumor size, grade, and histological type by multivariable analysis (Table 1 and ). In addition, analysis of gene signatures in human cancers indicates that high X3 expression correlates with shorter overall survival in SCLC (Supplementary Fig S1) (ild et al, 26). These results indicate that X3 is essential for cancer cell proliferation and survival, especially in aggressive subtypes of lung cancer, and may be an important molecular determinant of lung cancer survival. binds to X3 and decreases its helicase function ased on the role of X3 in proliferation and as a potential marker of aggressive cancer, we rationally designed small molecules to bind specifically to the TP-binding cleft of X3 (Kondaskar et al, 21). We identified a fused diimidazodiazepine molecule (; Fig 2) that exhibited promising cell death kinetics and has a computed binding affinity of 8 kcal/mol between and X3 (Fig 2 and C). To evaluate binding of to X3, we synthesized two biotinylated molecules (Fig 2 and E) and demonstrated that binds specifically to X3, but not to the closely related proteins X5 and X17 (Fig 2F). To establish whether can perturb the helicase activity of X3, we carried out helicase assays as described (Sengoku et al, 26, Fig 2G and H). significantly reduced the unwinding activity of ed1p (yeast homolog of X3), in a dose-dependent manner, starting with as little as 5 nm. inhibits cancer growth and radiosensitizes lung cancer cells in a X3-dependent manner To evaluate whether inhibition of X3 by would lead to cancer cell cytotoxicity, we assessed cell viability in various lung cancer cell lines (Fig 2I). Cancer cell lines with high levels of X3 expression (549,, H23, and H46) were more sensitive to (IC 5 = lm) as compared to H3255, a cell line with Figure 1. X3 expression and knockdown phenotype in lung cancer cell lines and in lung cancer patient samples. Immunoblot of X3 expression in lung cancer cell lines., C Colony-forming assays in () and 549 (C) lung cancer cells after knockdown by shr lentiviral constructs designed against X3 or vector control. Corresponding immunoblots displaying knockdown levels of X3. Mean from 3 replicates with S. Proliferation of 549 and cells after knockdown of X3. Mean from 3 replicates with S. (549 P =.11, P =.14; exponential curve fit, extra sum of squares F-test). E b-galactosidase staining in parental 549 cells and 549 X3 knockdown cells displaying senescent cells identified by the blue color. F Expression of X3 by immunohistochemistry in normal lung tissue. G X3 expression in squamous cell carcinoma. H X3 expression in adenocarcinoma. I X3 expression in small cell carcinoma. J Expression of X3 in different histological types of lung cancer. ll data sets were compared against each other (chi-square test, P =.481). K Survival analysis of lung cancer patients in low and high X3 expressing tumors (Kaplan Meier curve and log-rank test, P =.16). ata information: Scale bars: 25 lm. Source data are available online for this figure. ª 215 The uthors EM Molecular Medicine Vol 7 o

3 EM Molecular Medicine Targeting X3 in lung cancer Guus M ol et al H23 H H3255 HEC X3 ctin Colonies shcontrol shx3 shcontrol shx3 X3 ctin C Colonies shcontrol 549 shx3 shcontrol shx3 X3 ctin E shcontrol shx shControl 549shX3 (p=.11) shcontrol shx3 (p=.14) Cells ays F ormal lung G Squamous cell carcinoma J X3 expression in subtypes of lung cancer Cytoplasmic X3 Low (%) High (%) Histology (34) 63 (66) small cell carcinoma 6 2 (33) 4 (67) adenocarcinoma (46) 15 (54) squamous cell ca (28) 26 (72) other 22 7 (32) 15 (68) H denocarcinoma I Small cell carcinoma K 1 8 Lung cancer low X3 high X3 Survival (%) 6 =32 4 p =.16 = Follow up (month) Figure EM Molecular Medicine Vol 7 o ª 215 The uthors

4 Guus M ol et al Targeting X3 in lung cancer EM Molecular Medicine Table 1. Comparison of X3 expression with clinical parameters and survival analysis. Cytoplasmic X3 uclear X3 verage Low ( 1) High (2 3) P-value < 1% 1% P-value Mean age (range) 61.6 (36 78) 63.3 (38 79).443 # 62.4 (36 79) 67.8 (58 79).249 # Mean tumor size (range) 4.2 (1.3 1.) 3.8 (.9 1.).38 # 4. (.9 1.) 3.5 ( ).612 # Histological type Small cell carcinoma 33.3% (2) 66.7% (4).481 1% (6) % ().75 Squamous cell carcinoma 27.8% (1) 72.2% (26) 86.1% (31) 13.9% (5) denocarcinoma 46.4% (13) 53.6% (15) 1% (28) % () ther 31.8% (7) 68.2% (15) 1% (22) % () Grade 1 %() 1% (2).758 1% (2) % () % (12) 62.5% (2) 9.6% (29) 9.4% (3) % (12) 62.5% (2) 96.9% (31) 3.1% (1) 4 25%(3) 75% (9) 1% (12) % () Gender Male 35.1% (27) 64.9% (5) % (72) 6.5% (5).583 $ Female 31.2% (5) 68.8% (11) 1% (16) % () Stage (pathologic) I&II 38.9% (21) 61.1% (33) % (5) 7.4% (4).571 $ III & IV 28.6% (6) 71.4% (15) 1% (21) % () Univariate Multivariate Variables HR (95% CI) P-value HR (95% CI) P-value Tumor size 1.78 ( ).283 Histological type ( ) ( ).247 Grade ( ) ( ).12 Cytoplasmic X ( ) ( ).3 () aseline characteristics of differentially expressed cytoplasmic and nuclear X3 in lung cancer patient samples (P-values are determined by chi-square test unless otherwise indicated: # = t-test; $ = Fisher s exact test). () Univariate and multivariate cox regression analyses in lung cancer patient samples on clinically relevant variables related to aggressiveness (tumor size, histological type, and grade). Tumor size was not used in multivariate regression analysis as it has a univariate P-value >.2 and made the multivariate regression model less predictive. low X3 expression (IC 5 > 25 lm). Percentage of cells undergoing early apoptosis (nnexin V positive) and late apoptosis (PI positive) is shown in Supplementary Fig S2. Since radiation therapy is one of the mainstays for treatment of lung cancer, we assessed the combination effect of and radiation. We carried out colony-forming assays to establish the response of 549 cells (high X3 expression) and H3255 cells (low X3 expression) to radiation with or without (Fig 2J and K). ot only did cause cytotoxicity in 549 cells, but at low concentrations of 1 lm (P =.1) and 2 lm (P =.1), it also sensitized 549 cells to c-radiation. H3255 cells (low X3 expression), on the other hand, were not sensitized to c-radiation by (1 lm, P =.95; 2 lm, P =.65). X3 knockdown and perturb common gene regulatory pathways To confirm the inhibition of X3 by and determine specificity, we measured gene expression in M-M-231 cells by microarray analysis after treatment of or knockdown of X3. s shown in Fig 3, gene expression changes of - treated cells correlated with X3 knockdown cells (rho =.673, using genes altered in both classes at P <.5). This is supported by a Venn diagram displaying the overlap of gene expression between shx3- and -treated cells (Fig 3). This indicates that the functional activity of is via X3 inhibition and that it could be used as a small molecule inhibitor of X3. ª 215 The uthors EM Molecular Medicine Vol 7 o

5 EM Molecular Medicine Targeting X3 in lung cancer Guus M ol et al C Gln27 Me Me Tyr2 Me K-298 H S H H E Me SK-153 Me R2 R3 H H S F X3 iotin K-298 SK-153 G 5 3 R13 R H H X5 ed1p TP 5 X17 p82 p72 ed1p R41 3 R H ed1p negative ed1p without TP ed1p + 5nM ed1p + vehicle control ed1p + 1nM ed1p + 2nM R duplex (R41:R13) I Cell viability (%) H3255 H46 H23 Unwound R (R13) (μm) J K 1 H3255 Survival Fraction.1.1 1μM RK33 (p=.1) 2μM RK33 (p=.1) Survival Fraction.1.1 1μM RK33 2μM RK Radiation(Gy) Radiation (Gy) Figure EM Molecular Medicine Vol 7 o ª 215 The uthors

6 Guus M ol et al Targeting X3 in lung cancer EM Molecular Medicine Figure 2. Specific binding of to X3 and induction of radiosensitization in lung cancer cell lines. Chemical structure of. predicted molecular model of docked into the TP-binding cleft of X3. is displayed in pink, the surface of the Ec domain is in green, and the surface of the HELICc domain is in red. C Hydrogen bond interactions between and X3. lpha helices are displayed in green, and b-sheets are shown in maroon., E Chemical structures of biotin-linked at R3 position with ethylene amine linker (K-298) and biotin-labeled at R2 position with (PEG)2 ethylene amide linker (SK-153). The two structural differences of K-298 and SK-153 are the length of the biotin linker and attachment position at. F Immunoblots of pull-down assay of X3 with biotin, K-298, and SK-153. Lower panels display results using X5 and X17 antibodies. G Schematic representation of helicase assay. H Immunoblot displaying increasing concentrations of (5, 1, 2 nm) resulting in increased inhibition of unwinding of oligomer products (lanes 4 6). I MTS viability assay of various lung cancer cell lines treated with for 72 h. Mean from 3 replicates with S. J, K Colony-forming assay of 549 and H3255 cells treated with and with various doses of radiation 4 h later. Curves were fitted with a quadratic polynomial equation. Mean from 2 replicates with S. P-values were determined by the extra sum of squares F-test. Source data are available online for this figure. To further elucidate the cytotoxic mechanism of, we explored gene expression patterns by microarray analysis of X3 knockdown cells and cells treated with. We found that the mechanism behind the decreased cellular proliferation perhaps could be assigned to reduced cell cycle progression and inhibition of the MPK pathway (Fig 3C and ). To assess the effect of on a wide variety of cell lines, we tested the CI-6 panel of cell lines (Shoemaker et al, 1988; Shoemaker, 26) for a decrease in cellular growth (Fig 4 and ). ext, we compared the growth inhibition of the CI-6 cell lines by with that of 12 common F-approved drugs using network analysis (Fig 4C). well-connected sub-network in the middle of 4 3 shx3 log 2 fold change Common genes Rho =.67 Genes with p < log 2 fold change C Pathways affected by shx3 Pathways affected by MPKinase signaling p53 signaling TM signaling p38 MPK signaling Signal transduction IL1R Cell cycle: G1/S check point one remodelling poptosis through R3, R4/5 gene regulation via PPRalpha EGF signaling LS permutation -log(p-value) Caspase cascade in apoptosis CK regulation Cell cycle: G1/S check point Hypoxia TG family Prion pathway P53 signaling Cyclins and cell cycle regulat HIV-1 ef MPK signaling LS permutation -log(p-value) Figure 3. ioinformatics analysis of X3 knockdown and treatment. Scatter plot of the gene expression log2 fold change in X3 knockdown and -treated M-M-231 cells. Each red dot represents a gene, which was significantly perturbed after treatment with and after knockdown of X3. The Venn diagram depicts the number of common genes dysregulated by both shx3 and treatments. C, iocarta pathway analysis of gene expression in X3 knockdown and -treated cells. Pathways are ranked on LS permutation P-values from top to bottom. ª 215 The uthors EM Molecular Medicine Vol 7 o

7 EM Molecular Medicine Targeting X3 in lung cancer Guus M ol et al GI5 (μm) 4 3 GI5 (μm) LX IMVI MLME-3M M14 M-M-435 SK-MEL-2 SK-MEL-28 SK-MEL-5 UCC-257 UCC-62 Melanoma IGRV1 VCR-3 VCR-4 VCR-5 VCR-8 CI/R-RES SK-V-3 varian CH CKI-1 RXF 393 S12C TK-1 U-31 Renal MCF7 M-M-231 HS 578T T-549 T-47 M-M-468 reast CCRF-CEM HL-6(T) K-562 MLT-4 RPMI-8226 SR 549 HP-62 HP-92 CI-H226 CI-H23 CI-H322M CI-H46 CI-H522 PC-3 U-145 CL25 HCC-2998 HCT-116 HCT-15 HT29 KM12 SW-62 SF-268 SF-295 SF-539 S-19 S-75 U251 Leukemia Lung Prostate Colon CS C Cyclophosphamide Letrozole ilotinib Sorafenib Toremifene Temsirolimus ltretamine Lenalidomide rsenic trioxide minolevulinic acid Everolimus Thioguanine 6 Mercaptopurine elfinavir Procarbazine 5 azacytidine Vincristine sulfate Gefitinib Paclitaxel elfinavir mesylate Fulvestrant exrazoxane Lapatinib acarbazine Imiquimod Tretinoin (TR) ecitabine ortezomib Estramustine asatinib Vorinostat Vinorelbine tartrate Ethinyl estradiol llopurinol Tamoxifen Romidepsin Vinblastine sulfate ctinomycin Calcium leucovorin Pentostatin Megestrol acetate Carboplatin Mesna leomycin ThioTEP Pipobroman Topotecan Hydroxyurea Idarubicin usulfan HCl Mitoxantrone Etoposide CCU (Lomustine) oxorubicin Teniposide aunorubicin itrogen mustard Epirubicin endamustine Zolendronic acid elta 1 testololactone Exemestane ocetaxel Estramustine Erlotinib Celecoxib Fludarabine romostanol Sunitinib Clofarabine Raloxifene imethyltestoste Cladribine nastrozole Cisplatin Cytarabine HCl Temozolomide Chlorambucil Gemcitabine Cytarabine Thalidomide Floxuridine Mitramycin Levamisole Triethylenemelamine Melphalan xaliplatin Capcitebine Uracil Mitomycin nitrogen C mustard Irinotecan Methotrexate Valrubicin Imatinib 5 fluorouracil Methoxsalen Carmustine Pemetrexed Mitotane aldrolone Streptozoticin elarabine Quinacrine HCl Ixabepilone mifostine Ifosfamide Vorinostat Temozolomide 6 Mercaptopurine Thioguanine RK 33 acarbazine Clofarabine Cladribine Fludarabine CU (Carmustine) Streptozoticin Quinacrine HCl Ifosfamide Cyclophosphamide xaliplatin 5 fluorouracil Pemetrexed mifostine 5 azacytidine Tretinoin (TR) ilotinib Sorafenib Imiquimod Exemestane Raloxifene Imatinib CCU (Lomustine) elfinavir mesylate rsenic trioxide Sunitinib Tamoxifen nastrozole imethyltestosterone romostanolone propionate elfinavir Ethinyl estradiol Celecoxib Carboplatin endamustine Methoxsalen elarabine exrazoxane Mitoxantrone Teniposide usulfan Epirubicin oxorubicin Etoposide aunorubicin Idarubicin HCl Valrubicin Hydroxyurea Triethylenemelamine Chlorambucil Uracil nitrogen mustard ThioTEP Melphalan itrogen mustard Irinotecan Topotecan Mitomycin C Pipobroman Cytarabine Methotrexate Gemcitabine Cytarabine HCl Floxuridine leomycin Cisplatin asatinib Erlotinib Everolimus Calcium leucovorin Lapatinib Gefitinib llopurinol Fulvestrant ecitabine Pentostatin Procarbazine Mitotane Estramustine Ixabepilone Toremifene ltretamine elta 1 testololactone Temsirolimus aldrolone Capcitebine ortezomib Estramustine Mitramycin Letrozole Megestrol acetate Zolendronic acid Mesna Romidepsin ctinomycin Vinblastine sulfate Vinorelbine tartrate Paclitaxel Vincristine sulfate ocetaxel ocetaxel Vincristine sulfate Paclitaxel Vinorelbine tartrate Vinblastine sulfate ctinomycin Romidepsin Mesna Zolendronic acid Megestrol acetate Letrozole Mitramycin Estramustine ortezomib Capcitebine aldrolone Temsirolimus elta 1 testololactone ltretamine Toremifene Ixabepilone Estramustine Mitotane Procarbazine Pentostatin ecitabine Fulvestrant llopurinol Gefitinib Lapatinib Calcium leucovorin Everolimus Erlotinib asatinib Cisplatin leomycin Floxuridine Cytarabine HCl Gemcitabine Methotrexate Cytarabine Pipobroman Mitomycin C Topotecan Irinotecan itrogen mustard Melphalan ThioTEP Uracil nitrogen mustard Chlorambucil Triethylenemelamine Hydroxyurea Valrubicin Idarubicin HCl aunorubicin Etoposide oxorubicin Epirubicin usulfan Teniposide Mitoxantrone exrazoxane elarabine Methoxsalen endamustine Carboplatin Celecoxib Ethinyl estradiol elfinavir romostanolone propionate imethyltestosterone nastrozole Tamoxifen Sunitinib rsenic trioxide elfinavir mesylate CCU (Lomustine) Imatinib Raloxifene Exemestane Imiquimod Sorafenib ilotinib Tretinoin (TR) 5 azacytidine mifostine Pemetrexed 5 fluorouracil xaliplatin Cyclophosphamide Ifosfamide Quinacrine HCl Streptozoticin CU (Carmustine) Fludarabine Cladribine Clofarabine acarbazine RK 33 Thioguanine 6 Mercaptopurine Temozolomide Vorinostat Figure 4. Comparison of the GI 5 values of with F-approved drugs on the CI-6 panel of cell lines., The graph depicts the growth inhibitory properties (GI5) of for the CI-6 panel of cell lines. The CI-6 is a panel of 6 extensively characterized human cell lines derived from nine distinct tumor types: melanoma, ovarian, renal, breast, leukemia, lung, prostate, colon, and CS. C etwork analysis of 12 F-approved drugs and based on GI 5 in the CI-6 cell line panel. Unsupervised cluster analysis of the 12 F-approved drugs based on the correlation structure of the GI 5 levels. The result is shown as a symmetric heat map with positive associations depicted in yellow and negative associations shown in blue. ata information: Error bars represent S and all experiments were done in replicates. the plot indicates that all of these drugs have similar patterns of sensitivity across the cell lines. and several other agents are not connected to networks, indicating that none of these have nearneighbors among F-approved drugs in cancer. We also performed an unsupervised cluster analysis of the 12 Fapproved drugs based on the correlation structure of the GI 5 levels (Fig 4). sits in the bottom right corner in a small cluster of weak-to-moderately correlated agents including dacarbazine, thioguanine, temozolomide, and vorinostat, supporting the distinctive working mechanism of as compared to other drugs. 654 EM Molecular Medicine Vol 7 o ª 215 The uthors

8 Guus M ol et al Targeting X3 in lung cancer EM Molecular Medicine Cerebellum Kidney Liver Control C Treated C -Molecular layer -Purkinje cells C-Granular cell layer -White matter -Glomerulus -Tubules Centrilobular area circled Concentration (ug/g) rgan t½e (hours) plasma 7.31 ± 1.16 kidney 6.7 ± 1.17 liver 1.1 ± 1.24 spleen 8.92 ± 1.45 breast 7.48 ± 1.18 lung 7.25 ± 1.25 brain 6.72 ± 1.23 muscle 6.25 ± 1.19 Spleen elimination (hours) lood smear C C -Monocytes -Platelet C-eutrophils -RC Retention Time (min) [M+H] + Structure C Mouse Microsomes Human Microsomes Inc. Time + PH - PH + PH - PH 1% 1% 1% 1% 3 11% 8% 49% 85% 6 4% 69% 35% 73% Peak Retention Time + (min) [M+H] Postulated Metabolite M Unable to determine Unable to determine M Loss of ethyl phenyl ether M Unable to determine E 4 Vehicle (1μM) F 3 p=.1 CR (%baseline) H14-1 rug or vehicle ligo FCCP TP (nmol/gram protein) Time (min) Control ligo Figure 5. ª 215 The uthors EM Molecular Medicine Vol 7 o

9 EM Molecular Medicine Targeting X3 in lung cancer Guus M ol et al Figure 5. Toxicity studies of in mice. Following injection of 2 mg/kg of, twice a week for 7 weeks, extensive histopathological examination was carried out following necropsy. Identical patterns were observed both in the control and in the treated mice (n = 2). Samples were stained with H&E. Scale bar is 5 lm. Pharmacokinetics of in SCI mice at various time intervals. Results are mean S from 5 mice. LC-MS/MS method was used to determine concentration of in mouse plasma and tissue. C Liquid chromatography mass spectrometry (LC-MS/MS) analysis was performed to determine different metabolites of. and metabolites characterized by LC-MS/MS in human liver microsomes using the scan mode function of the LC-MS/MS. E HPI cells were treated with (1 lm), H14-1 (25 lm), or vehicle, followed by oligomycin (oligo,.5 lg/ml), FCCP (3 lm), and antimycin (, 1 lm) while oxygen consumption rate (CR) was measured. Pyruvate (1 mm) was added in combination with FCCP to ensure that substrate supply was not rate-limiting for maximal CR. ata are mean S from 2 to 3 wells and representative of independent experiments performed with two different HPI passages. CR is baseline-normalized to the point prior to drug or vehicle addition. F HPI microglial cells were incubated for 1 h in glucose-free XF24 assay medium that was supplemented with 2-deoxyglucose (5 mm) and pyruvate (1 mm). (1 lm), oligomycin (.5 lg/ml), or vehicle control was additionally present as indicated. Results are mean S from 12 replicates pooled from experiments using two consecutive passages. Significance was assessed by two-sided, unpaired t-test. Toxicology, biodistribution, pharmacokinetics, and metabolism of Prior to initiating the animal experiments, we carried out toxicology, biodistribution, and metabolism assessments of. Toxicology studies indicated that, at the dose used, was non-toxic in SCI mice. s shown in Fig 5, histopathology of the different tissues from control () and -treated mice did not exhibit any discernable morphological changes. iodistribution studies revealed that was able to accumulate at therapeutic dose in various organs, thus enhancing the clinical relevance for the use of as chemotherapeutic agent (Fig 5). Moreover, PHindependent and PH-dependent metabolism was observed when was incubated with human liver and mouse microsomes (Fig 5C). Subsequent chromatography analysis identified three potential metabolites (Fig 5). In addition, liver and kidney function tests, as well as the blood and lipid profiles, were not altered between the control and -treated groups (Table 2). does not perturb mitochondrial functions To test whether therapeutically relevant concentrations of interfere with mitochondrial function, (5 or 1 lm) or vehicle was added to the highly aggressive proliferating immortalized Table 2. lood toxicity studies. lood samples of -treated SCI mice lood cells S S ormal range RC (M/ll) Hb (g/dl) MCV (fl) WC (K/ll) Thrombocytes (K/ll) ,972 Liver biochemical values LT (U/l) ST (U/l) LP (U/l) Kidney biochemical values U (mg/dl) Creatinine (mg/dl) Calcium (mg/dl) lbumin (g/dl) Lipids and other biochemical values Cholesterol (mg/dl) Triglycerides (mg/dl) mylase (U/l) ,63 1,4 Glucose (mg/dl) The table displays blood, liver, kidney, and lipid toxicity data from -treated SCI mice. RC, red blood cells; Hb, hemoglobin; MCV, mean corpuscular volume; WC, white blood cells; LT, alanine aminotransferase; ST, aspartate transaminase; LP, alkaline phosphatase; U, blood urea nitrogen. 656 EM Molecular Medicine Vol 7 o ª 215 The uthors

10 Guus M ol et al Targeting X3 in lung cancer EM Molecular Medicine (HPI) cell line (Cheepsunthorn et al, 21), while cellular oxygen consumption was measured. The TP synthase inhibitor oligomycin (oligo), the uncoupler FCCP, and the electron transport chain inhibitor antimycin () were added subsequent to. rugs that uncouple oxidative phosphorylation from electron transport increase TP synthesis-independent oxygen consumption measured in the presence of oligomycin. rugs that inhibit electron transport impair maximal respiration measured in the presence of the uncoupler FCCP. at 5 (not shown) or 1 lm (Fig 5E) failed to alter baseline oxygen consumption rate (CR), CR measured in the presence of oligomycin (oligo), or CR measured in the presence of the uncoupler FCCP. s a positive control, H14-1 (25 lm), a cl-2 inhibitor, which is known to cause both mitochondrial uncoupling and respiratory inhibition (Milanesi et al, 26), elevated oligomycininsensitive oxygen consumption and decreased CR measured in the presence of FCCP. xygen consumption in the presence of all drugs was potently inhibited by antimycin, indicating that it was primarily mitochondrial in origin. To additionally demonstrate that a therapeutically relevant concentration does not interfere with mitochondrial function, we evaluated TP levels in HPI cells incubated with the cell-permeable mitochondrial complex I substrate pyruvate (1 mm) in the absence of glucose and the presence of the glycolysis inhibitor 2-deoxyglucose (5 mm). Under these conditions, the majority of cellular TP was generated by mitochondria, as demonstrated by addition of the TP synthase inhibitor oligomycin, which significantly decreased total cellular TP by approximately 7%. In contrast to oligomycin, (1 lm) failed to significantly decrease TP. Thus, has no effect on either mitochondrial respiration or TP generation. in combination with radiation promotes tumor regression in preclinical models of X3-overexpressing lung cancer To assess whether could be a clinically useful radiosensitizer, we evaluated in combination with different radiation doses in both an immune competent Twist1/Kras G12 autochthonous lung tumor model (Fig 6) (Tran et al, 212) and an orthotopic human xenograft model for lung cancer. This autochthonous model harbors a Kras G12 mutation and overexpresses Twist1 and dx3, which made it a suitable model to test efficacy of (Fig 6). Following tumor formation, the mice were treated either individually or with a combination of, carboplatin, and radiation as depicted in Fig 6C. Tumor progression was followed by microcomputed tomography (micro-ct) imaging before treatment and 1 week after treatment, after which tumor volume was measured and confirmed by H&E staining of lung sections (Fig 6). In addition, we carried out immunohistochemistry analysis for Ki67 expression in these tumors (Supplementary Fig S3). Similar to our in vitro results, enhanced the radiation effect by 3.7-fold (P =.1), which was 1.5-fold more than the radiation sensitization caused by carboplatin (P =.259) (Fig 6E). To validate the radiation sensitization by, we generated an orthotopic lung cancer model by injecting 549 cells into the tail vein of athymic Cr-nu/nu mice. The treatment schedule was similar to that described in Fig 6C. verall, significantly enhanced radiation-induced tumor regression (2.6-fold; P =.1) in the orthotopic human lung cancer model (Fig 6F and G). ext, we evaluated the effect of in our Twist1/Kras G12 lung cancer model with a fractionated treatment regimen (Fig 6H). uring the 3 weeks following treatment, we saw a modest decrease in tumor growth with radiation and even more so with the combination of and radiation; this was however not significant (Fig 6I and J). verall, this demonstrates that in combination with hypofractionated radiation, such as used with stereotactic ablative radiation (SR), effectively decreases lung tumor load in two distinct preclinical lung cancer models and performs significantly better than the commonly used radiosensitizer carboplatin. induces G1 arrest and causes apoptosis s loss of X3 altered proliferation, we performed cell cycle analysis by flow cytometry, following biological knockdown of X3 by shx3 and by treatment. We found a 13.7% (P =.6) decrease in S-phase and a 14.1% (P =.7) increase in G1-phase in X3 knockdown cells (Fig 7), consistent with a G1 arrest. Similarly, treatment with also resulted in a G1 arrest of 549 and cells in a dose-dependent manner (Fig 7 and C). Subsequently, we assessed proteins involved in G1/S cell cycle transition (cyclin 1 and E1), apoptosis, and MPK pathway by immunoblotting. We found a substantial reduction of cyclin 1, which was especially evident 2 h after treatment with in Figure 6. induces radiosensitization in preclinical mouse models of lung cancer. Schematic describing the Twist1/KrasG12-inducible mouse model. Confirmation of high expression of Twist1 and X3 in the lung tumors of the transgenic Twist1/Kras G12 mouse. Scale bar is 1 lm. C Treatment schedule for mice receiving hypofractionated radiation (SR). Stars are intraperitoneal (i.p.) injections with. Micro-CT images of transgenic Twist1/Kras G12 mice treated as in (C), before treatment and 1 week after treatment. Tumors are indicated by arrows and confirmed by H&E staining of lung sections (lower panel). Scale bar is 25 lm. E Quantification of tumor volume measured by micro-ct in Twist1/Kras G12 mice, as shown in (). Significance was assessed by two-sided, unpaired t-test. Error bars represent S. F n orthotopic lung tumor model was generated using 549 human lung cancer cells and treated as in (C). Figure displays H&E staining of lung sections from radiation-treated (upper panel) and - and radiation-treated mice (lower panel). Scale bar is 2 mm. G Quantification of tumor burden (as tumor surface divided by total lung surface) in orthotopic 549 lung cancer mouse model, as shown in (F). Significance was assessed by two-sided, unpaired t-test. Error bars represent S. H Treatment schedule for mice receiving fractionated radiation in 1 fractions. Stars indicate i.p. injections with. ownward lightning bolts indicate 3-Gy radiation fractions. I Micro-CT images of transgenic Twist1/Kras G12 mice treated as in (H), before treatment and 1 week after treatment. J Quantification of tumor volume measured by micro-ct in Twist1/Kras G12 mice, as shown in (I) and expressed as relative tumor size. Significance was assessed by two-sided, unpaired t-test. Error bars represent SEM. ª 215 The uthors EM Molecular Medicine Vol 7 o

11 EM Molecular Medicine Targeting X3 in lung cancer Guus M ol et al CCSP rtt Twist1 dx3 + ox ox rtt ox rtt Twist1 tet 7 Luc tet Kras G12 C 15Gy SR SR + SR 2mg/kg week week 1 week 2 week 3 efore Established lung tumors confirmed by microct microct E fter H&E Relative tumor size (%) SR + SR Carbo Carbo + SR p=.1 F G H p=.259 p=.1 SR + SR Tumor burden SR + SR 2mg/kg XRT 3Gy week Established lung tumors confirmed by microct week 1 week 2 week 3 microct I XRT + XRT J efore fter Relative tumor size (%) XRT + XRT Figure EM Molecular Medicine Vol 7 o ª 215 The uthors

12 Guus M ol et al Targeting X3 in lung cancer EM Molecular Medicine Cells (%) p=.35 1 p=.7 p=.6 p=.65 6 vector shx Cells (%) 549 p=.35 p=.73 2μM 4μM 6μM C Cells (%) p= p=.9 2μM 4μM 6 p=.69 6μM p=.23 4 p=.12 p=.41 p=.19 2 G1 S G2/M Cell cycle phase G1 S G2/M Cell cycle phase G1 S G2/M Cell cycle phase 549 E F 2 hours 8 hours 24 hours 2 hours 8 hours 24 hours 549 H3255 Cyclin 1 Cyclin E1 Caspase 7 ERK1/2 perk1/2 ctin cleaved Caspase 7 cleaved Caspase 9 ctin Figure 7. Effect of X3 knockdown and on cell cycle progression and apoptosis. Cell cycle analysis of cells treated with shx3 and processed by flow cytometry. Knockdown of X3 led to a decrease of cells in S-phase and an increase of cells in G1-phase, indicative of a G1 arrest. Significance was assessed by two-sided, unpaired t-test. Error bars represent S., C Cell cycle analysis of 549 and cells by flow cytometry after treatment with (, 2, 4, and 6 lm). induced a G1 cell cycle arrest in both cell lines. Significance was assessed by two-sided, unpaired t-test. Error bars represent S., E Immunoblot of cell cycle-related proteins (Cyclin 1 and Cyclin E1) and cell death-related proteins (cleaved caspase 7, cleaved caspase 9) in549 and cells after treatment with (1 lm). Initially, a strong decrease of Cyclin 1 was observed. fter 8 and 24 h, cleaved caspases 9 and 7 were apparent. F Immunoblot of MPK pathway-related proteins ERK1/2 and phosphorylated ERK1/2 in 549,, and H3255 ( resistant) cells 24 h after treatment with (7.5 lm or1 lm). ERK2 and especially ERK1 become dephosphorylated after treatment with in 549 and cells but not in H3255 cells. utlined boxes indicate spliced lanes. Source data are available online for this figure. 549 and cells (Fig 7 and E). lso, we found cleavage of caspases 7 and 9 in 549 cells (Fig 7) and of caspase 9 in cells (Fig 7E). Moreover, we found a reduction of phosphorylated ERK2 and ERK1 in 549 and lung cancer cells (high X3 expression) and an increase of ERK1/2 phosphorylation in H3255 cells (low X3 expression) (Fig 7F). Collectively, these results suggest that curbs proliferation and induces apoptosis in a X3-dependent fashion. Wnt signaling is mediated by X3 and inhibited by s X3 is implicated in Wnt signaling (Cruciat et al, 213) and Wnt signaling is an important regulator of proliferation and can cause radiation resistance (Woodward et al, 27; Zhang et al, 21), we evaluated the relation between X3/ and the Wnt pathway. To determine the spatial pattern of X3 and b-catenin expression, we carried out immunofluorescence staining for X3 and b-catenin ª 215 The uthors EM Molecular Medicine Vol 7 o

13 EM Molecular Medicine Targeting X3 in lung cancer Guus M ol et al ß-catenin X3 merged C ß-cat: - IP: H3K4Me3 X3 X3 IP: H3K4Me3 ß-catenin ß-catenin W: ß-catenin W: X3 ß-cat: + E F G TCF activity p=.422 TCF activity TCF activity p=.29 p=.2 TCF activity p= ß-cat vector shx vector shx3 ß-cat ß-cat 1μM 2μM 3μM ß-cat 1μM 2μM 3μM H TCF activity p=.13 I TCF activity p=.154 p=.44 J relative mr expression vector shx3 vector 1.2 p=.24 p=.16 p=.48 xin-2 1. c-myc Cyclin 1.8 X3 shx3 vector shx3 vector shx ß-cat 1μM 2μM ß-cat 1μM 2μM K relative mr expression μM 1.2 p=.24 p=.399 xin-2 1. c-myc Cyclin 1.8 X3 2μM 2μM 2μM Figure EM Molecular Medicine Vol 7 o ª 215 The uthors

14 Guus M ol et al Targeting X3 in lung cancer EM Molecular Medicine Figure 8. Effect of on Wnt signaling via X3. b-catenin (red) and X3 (green) expression in cells. fter overexpressing b-catenin, both X3 and b-catenin accumulate in the nucleus. Scale bar is 1 lm. Immunoprecipitation with X3 or H3K4Me3 (control) and immunoblotted with b-catenin in 549 and cells. utlined boxes indicate spliced lanes. C Immunoprecipitation with b-catenin or H3K4Me3 (control) and immunoblotted with X3 in 549 and cells. utlined boxes indicate spliced lanes., E b-catenin/tcf4 activity was determined by the TP/FP reporter assay. Co-transfection with b-catenin is indicated below. F I and 549 cells were treated with (, 1, 2, and 3 lm) and co-transfected with b-catenin in (F, H). Treatment with decreased TCF4 activity in both cell lines. J, K ormalized mr expression of TCF4-regulated proteins (xin-2, c-myc, Cyclin 1) and X3 were measured by qrt PCR in cells after knockdown of X3 (J) and treatment with (K). ll experiments were repeated three times. ata information: Significance was assessed by two-sided, paired t-test. Error bars represent S. Source data are available online for this figure. in wild-type cells, as well as in b-catenin-overexpressing cells. In wild-type cells, we could detect X3 expression in the cytoplasm, but very little nuclear b-catenin staining (Fig 8, top panel). However, forced expression of b-catenin resulted in a relocalization of X3 from the cytoplasm to the nucleus (Fig 8, bottom panel). This indicates that X3 may act as a transporter protein to shuttle b-catenin in and out of the nucleus. To establish whether there is a physical interaction between X3 and b-catenin, we carried out co-immunoprecipitation with X3 and b-catenin. s shown in Fig 8 and C, X3 binds to b-catenin but not to H3K4Me3 (control), which was confirmed by immunoprecipitation using antibodies against both X3 and b-catenin. ased on the interaction between X3 and b-catenin, we quantified Wnt activity using a TCF reporter assay by transfecting and X3 knockdown cells with TP-FLSH or FP-FLSH constructs (van de Wetering et al, 22). Knockdown of X3 decreased TCF activity by 25% (P =.42) (Fig 8). However, in cells co-transfected with b-catenin, an increase in TCF activity was observed, but no significant difference was observed by knockdown of X3 (Fig 8E). reduced TCF activity significantly in and 549 cells, both in wild-type and in b-cateninoverexpressing cells (Fig 8F I). To confirm the results of the TCF reporter assay, we quantified transcript expression of common TCF-regulated genes xin-2, c-myc, and Cyclin 1 following knockdown of X3 as well as treatment with. s seen in Fig 8J, stable knockdown of X3 in cells reduced the expression of xin-2 (2.2-fold lower; P =.24), Cyclin 1 (2.7-fold lower; P =.2), and c-myc (1.4- fold lower; non-significant). Likewise X3 knockdown, treatment of cells reduced the expression of xin-2 (1.8-fold lower; P =.24) and Cyclin 1 (2.4-fold lower; P =.4) (Fig 8K). Radiation-induced double-strand break (S) repair is impaired by s promoted radiation sensitization, we evaluated the damage response following combination treatment with and c-radiation. Following c-radiation, 53P1 and c-h2x foci numbers increased within an hour and returned to pre-radiation foci numbers by 24 h. However, 53P1 and c-h2x foci persisted 24 h post- treatment, indicating reduced or delayed repair (Fig 9). The extent of the impaired damage repair was quantified by counting the number of severely damaged cells (> 1 foci per nucleus). This was determined for both 53P1 foci (Fig 9, top panel) and c-h2x foci (Fig 9, bottom panel). Most cells were severely damaged 1 h after radiation, but a large reduction in 53P1 and c-h2x foci after 6 h and normalization after 24 h, without significant cell death, indicated proficient damage repair in untreated 549 cells. Importantly, when these cells were pre-treated with, damage persisted well beyond 24 h (Fig 9). ext, we evaluated whether could impair homologous recombination (HR) and non-homologous end joining (HEJ), as these are the predominant repair mechanisms in double-strand break repair (Valerie & Povirk, 23). To determine this, we used two different stable cell lines with either a HR reporter construct (Chan et al, 28) or a HEJ reporter construct (Kriegs et al, 21). fter treatment with, we found no changes in HR activity but observed a significant 38% reduction of HEJ (Fig 9C and ). Moreover, we observed reduced HEJ activity after knockdown of X3 (Fig 9). ur mr analysis indicated that this could be due to decreased expression of MRE11 and PLM, two genes shown to be important for HEJ activity (Fig 9E). ext, we analyzed different proteins involved in HEJ like TR, and XRCC4 in -treated cells, with and without radiation. Treatment of 549 and cells with and radiation caused a decrease of TR but not XRCC4 (Fig 9F and G). lso, there was no difference in two other repair proteins, XRCC1 and Ku7. In conclusion, impairs radiation-induced damage repair by inhibiting HEJ activity. iscussion The concept of non-oncogene addiction postulates that certain nononcogenic genes are critical for survival of cancer cells but are not required, to the same degree, for the viability of normal cells and are therefore attractive targets for cancer drugs (Solimini et al, 27). ne such gene is X3, a member of the R helicase family, which we have shown to be dysregulated in breast cancer cell lines, up-regulated by HIF-1a, and involved in cancer maintenance and metastasis (otlagunta et al, 28, 211; ol et al, 213). Furthermore, we have published that X3 is an essential component for cellular proliferation and a decrease in its functional activity can lead to cellular stasis. Collectively, the function of X3 in cellular biogenesis, R metabolism, and translation has driven an interest to identify small molecule inhibitors of X3. In this paper, we show that abrogating X3 function leads to potent radiation sensitization in lung cancer, through inhibition of HEJ and Wnt signaling. ª 215 The uthors EM Molecular Medicine Vol 7 o

15 EM Molecular Medicine Targeting X3 in lung cancer Guus M ol et al 6μM 6μM Time (hours) P1 γh2x merged % nuclei > 1 foci % nuclei > 1 foci P Time (hours) 549 уh2x Time (hours) μm 2μM 4μM 6μM μm 2μM 4μM 6μM C Relative HR activity Relative HEJ activity p=.425 p=.231 p=.49 E Relative mr expression 1.5 MRE11 PLM p=.66 p= μM 6μM 4μM 6μM vector shx3 shcont shx3 shcont shx3 F hours 6 hours 24 hours G Gy 5Gy + Gy + 5Gy + Gy + 5Gy + Gy + 5Gy + Gy + 5Gy + Gy + 5Gy + Gy + 5Gy 2 hours 6 hours 24 hours 2 hours 6 hours 24 hours ptr X3 XRCC4 ctin Figure 9. esides the concept that X3 is essential for the cellular stress response, proliferation, and evasion of apoptosis, there are some data to indicate that X3 may have alternative functions and potential tumor suppressive functions (Chang et al, 25; Wu et al, 214). However, by mining the CSMIC database, we found only 7.7% of genetic abnormalities of the X3 gene typical for tumor suppressor genes (nonsense mutations, deletions or loss of heterozygosity), whereas 87.2% of X3 genetic abnormalities are more typical for a gain of function (substitution missense mutations). evertheless, it is possible that the functions of X3 662 EM Molecular Medicine Vol 7 o ª 215 The uthors

16 Guus M ol et al Targeting X3 in lung cancer EM Molecular Medicine Figure 9. Effect of on radiation-induced damage. Immunofluorescence images showing 53P1 and ch2x foci in 549 cells after 2-Gy radiation and 549 cells pre-treated with 6 lm, 12 h before radiation. verlap of 53P1 and ch2x is seen in the merged picture of the co-immunofluorescence staining. Scale bar is 2 lm. 549 cells were pre-treated with and radiated with 2 Gy, and 53P1 and ch2x foci were counted as a measure of damage. Cells with more than 1 foci 53P1 or ch2x were counted. More than 4 cells per sample were evaluated. C cells stably transfected with a homologous recombination (HR) reporter construct were treated with. Reporter constructs expressed GFP, which was quantified by flow cytometry. Experiments were repeated three times. cells, containing a stable non-homologous end-joining (HEJ) reporter construct, were treated with and knockdown of X3. Reporter construct expressed GFP, which was quantified by flow cytometry. ll experiments were repeated three times. E Microarray results from M-M-231 cells treated with and shx3 were validated by qrt PCR using HEJ Mechanisms of Ss Repair PrimePCR plates (io-rad) and performed in biological triplicates. F, G repair-related proteins (TR and XRCC4), X3, and actin were assessed by immunoblotting in 549 (F) and (G) cells. Cells were pretreated for 4 h with vehicle control or 6 lm and then radiated with or 5 Gy. utlined boxes indicate spliced lanes. ata information: Significance was assessed by two-sided, paired t-test. Error bars represent S. Source data are available online for this figure. are organ specific and may require cell-specific co-factors to determine its effect. Interestingly, a recent paper examined the role of Wnt/b-catenin signaling in Xenopus and C. elegans development and concluded that X3 is required for Wnt signaling (Cruciat et al, 213). Lung cancer patients are often treated with radiation therapy. Higher doses per fraction radiation can result in better local control but, dependent on tumor location or when re-treatment is needed, can be limited by toxicity (Senthi et al, 212). In our combination studies with and radiation, we saw a synergistic effect in vitro and greater than additive effects in two preclinical models of lung cancer. However, radiation sensitization of in combination with a fractionated radiation schedule had only limited effect in vivo. Given the radiosensitization we observed in vitro by clonogenic assays with standard doses of radiation (< 3 Gy), we propose that limited effect in vivo with standard fractionated radiation could be due to the relatively infrequent injections of in relation to radiation treatments. The combination effect of and radiation in vitro and in vivo was apparent in the reduction of damage repair following radiation and treatment. Mechanistically, Wnt/b-catenin signaling can mediate radiation resistance (Woodward et al, 27; Zhang et al, 21) but its precise mechanism is yet to be identified. To explore this, we assessed two of the most important repair mechanisms of Ss: HEJ and HR (Valerie & Povirk, 23) and found to inhibit HEJ activity but not HR. The impairment of HEJ results in reduced S repair, leading to genomic toxicity and thus potentiating cell death in X3- dependent cells. lthough not all molecular interactions of X3 in cellular biogenesis and adaption are known, it is clear that X3 is an essential component of Wnt signaling and is pivotal for the maintenance of the tumorigenic state. lso, as causes G1 arrest and HEJ is the predominant pathway for S repair in this phase of the cell cycle, this could result in profound radiosensitization. Thus, targeting the non-oncogene addiction of lung cancer cells to X3 by will shift their delicate balance toward tumor death. In summary, X3 is a hallmark of aggressive lung cancer and serves as a promising target for radioresistant lung cancer. Moreover, as a radiation sensitizer, because of its specificity to cancer cells and mild side effects, could lead to increased lung cancer patient survival and better quality of life. Materials and Methods Clinical samples Representative paraffin-embedded tissue blocks of 95 lung cancer patients were taken from the archive of the epartment of Pathology of the University Medical Center in Utrecht and routinely processed into a tissue microarray (TM). Intensity of cytoplasmic X3 was scored semi-quantitatively by two independent pathologists (r =.743). Survival statistics were obtained from the Comprehensive Cancer Center, The etherlands (IKL). Use of anonymous or coded left over material for scientific purposes is part of the standard treatment contract with patients in the UMCU (van iest, 22). Immunohistochemistry Sections of 4 lm were cut, mounted on SuperFrost slides (Menzel & Glaeser, runswick, Germany), deparaffinized, and rehydrated. Endogenous peroxidase was then blocked for 15 min with a buffer solution containing.3% hydrogen peroxide. ntigens were retrieved by boiling for 3 min in 1 mm citrate buffer (ph 6.) and cooled and washed in PS. Slides were subsequently incubated in a humidified chamber for 1 h with polyclonal rabbit anti-x3 (ngus et al, 21) diluted 1:1,. Subsequently, sections were washed in PS and incubated for 3 min with secondary antibodies (rightvision, Immunologic, uiven, The etherlands), washed with PS and developed with diaminobenzidine. Slides were counterstained with hematoxylin, dehydrated, and cover-slipped. ppropriate positive (validated breast cancer sample, thymus) and negative controls (normal lung tissue, normal liver tissue) were used throughout. Scoring of immunohistochemistry Scoring was done by two experienced pathologists (Paul J. van iest and Stefan Willems). Intensity of cytoplasmic X3 was scored semi-quantitatively from to 3, H-scores were determined, and percentages of cells with nuclear X3 expression were estimated. ut of three cores from the same patient, the maximum cytoplasmic X3 score was used for further analysis. X3 scores 1 and 2 were grouped as low X3 expression and evaluated against high X3 expression (scores 3). Cytoplasmic ª 215 The uthors EM Molecular Medicine Vol 7 o