Lactate Dehydrogenase B Is Required for the Growth of KRAS-Dependent Lung Adenocarcinomas

Published OnlineFirst December 6, 2012; DOI: 10.1158/1078-0432.CCR-12-2638

Clinical Cancer Human Cancer Biology Research

Mark L. McCleland1, Adam S. Adler1, Laura Deming1, Ely Cosino2, Leslie Lee2, Elizabeth M. Blackwood2, Margaret Solon1, Janet Tao1,LiLi3, David Shames4, Erica Jackson5, William F. Forrest6, and Ron Firestein1

Abstract Purpose: This study is aimed to identify genes within the KRAS genomic amplicon that are both coupregulated and essential for cell proliferation when KRAS is amplified in lung cancer. Experimental Design: We used an integrated genomic approach to identify genes that are coamplified with KRAS in lung adenocarcinomas and subsequently preformed an RNA interference (RNAi) screen to uncover functionally relevant genes. The role of lactate dehydrogenase B (LDHB) was subsequently investigated both in vitro and in vivo by siRNA and short hairpin RNA (shRNA)–mediated knockdown in a panel of lung adenocarcinoma cells lines. LDHB expression was also investigated in patient tumors using microarray and immunohistochemistry analyses. Results: RNAi-mediated depletion of LDHB abrogated cell proliferation both in vitro and in xenografted tumors in vivo. We find that LDHB expression correlates to both KRAS genomic copy number gain and KRAS mutation in lung cancer cell lines and adenocarcinomas. This correlation between LDHB expression and KRAS status is specific for lung cancers and not other tumor types that harbor KRAS mutations. Consistent with a role for LDHB in glycolysis and tumor metabolism, KRAS-mutant lung tumors exhibit elevated expression of a glycolysis gene signature and are more dependent on glycolysis for proliferation compared with KRAS wild-type lung tumors. Finally, high LDHB expression was a significant predictor of shorter survival in patients with lung adenocarcinomas. Conclusion: This study identifies LDHB as a regulator of cell proliferation in a subset of lung adenocarcinoma and may provide a novel therapeutic approach for treating lung cancer. Clin Cancer Res; 19(4); 1–12. 2012 AACR.

Introduction (33%; ref. 1). The importance of these mutations is Lung cancer is one of the most prevalent cancer forms, highlighted by the development of genetically engineered responsible for more than one million annual deaths mouse models that harbor KRAS and p53 mutations and are worldwide. In the clinical setting, lung cancer is classified able to drive lung adenocarcinoma initiation and progres- according to two main histologic types, small-cell lung sion (2). In recent years, molecular and cancer genomic cancer (SCLC) and non–small cell lung carcinoma (NSCLC; approaches have increased our understanding of the path- ref. 1). Eighty-five percent of all lung cancers are attributable ogenesis of NSCLC and have led to therapies that directly to NSCLC, of which lung adenocarcinoma is the most target the precise genetic alterations that drive tumor frequent histologic subtype (1). On a molecular level, lung growth. adenocarcinomas frequently harbor mutations in the KRAS The molecular genotyping of NSCLC into discrete molec- oncogene (25%) and the tumor suppressor protein p53 ular classes has redefined therapeutic approaches. Onco- genic alterations in the kinases EGF receptor (EGFR) and anaplastic lymphoma kinase (ALK) are found in 15% to 25% of NSCLC (3, 4). Patients with tumors harboring the Authors' Affiliations: Departments of 1Pathology, 2Translational Oncol- ogy, 3Bioinformatics & Computational Biology, 4Molecular Diagnostics & activated EGFR and ALK kinase respond to the tyrosine Cancer Cell Biology, 5Research Oncology, and 6Biostatistics, Genentech, kinase inhibitors gefitinib and erlotinib (5, 6) and crizoti- Inc., South San Francisco, California nib (7), respectively. Importantly, ALK and EGFR activation Note: Supplementary data for this article are available at Clinical Cancer is mutually exclusive of KRAS mutation, underscoring the Research Online (http://clincancerres.aacrjournals.org/). need for identifying and characterizing therapeutic options M.L. McCleland, A.S. Adler, and L. Deming contributed equally to this work. for KRAS-driven lung cancer. Corresponding Author: Ron Firestein, Genentech, Inc., 1 DNA Way, South KRAS is one of the most commonly mutated oncogenes San Francisco, CA 94080. Phone: 650-225-8441; Fax: 650-467-2625; in lung cancer (1), exhibiting activating alterations in about E-mail: [email protected] 25% to 30% of lung adenocarcinomas. While point muta- doi: 10.1158/1078-0432.CCR-12-2638 tions in codon 12 or 13 account for the majority of KRAS 2012 American Association for Cancer Research. mutations (8), recent studies have reported that KRAS also

www.aacrjournals.org OF1

McCleland et al.

noma (KRAS amplified): NCI-H322T, NCI-H838; adeno- Translational Relevance carcinoma (KRAS mutant): HOP-62, NCI-H2122, NCI- There are many molecular subsets of cancer that have H1573, A427, NCI-H1734, NCI-H2030, NCI-H2009, historically been difficult to target, leaving patients with A549, NCI-H2126, NCI-H23; bronchioalveolar adenocar- few therapeutic options. One such example of this is lung cinoma (KRAS mutant): NCI-H358; non–small cell (KRAS adenocarcinomas that exhibit aberration in the KRAS mutant): SW1573; adenocarcinoma (Met amplification): oncogene. KRAS is altered, through activating mutation NCI-H1993; squamous cell carcinoma (Met amplification): and copy number gain, in nearly 30% of lung adeno- EBC1; adenocarcinoma: NCI-H1793, RERF-LC-OK, NCI- carcinomas. Using an RNA interference (RNAi) screen to H1568, VMRC-LCD, ABC-1, RERF-LC-KJ, NCI-H2126; knockdown genes within the KRAS amplicon, we find bronchioalveolar adenocarcinoma: NCI-H1666; adenos- that lactate dehydrogenase B (LDHB) is upregulated and quamous carcinoma: NCI-H596; large cell carcinoma: essential in KRAS-driven lung cancers. Targeting LDHB HOP-92. The following colon cell lines were used: SW48, with small-molecule inhibitors may provide a therapeu- SW1417, RKO, KM-12, HT55, HCA-7, C2BBe1, SW948, tic option for lung cancer patients with KRAS aberration. SW837, SW620, SW1463, SK-CO-1, LS1034, HCT-15, DLD-1, COLO 678, CL-40. Cell lines were cultured in RPMI-1640 or Dulbecco’s modified Eagle’s medium (DMEM; high glucose) medium, 10% FBS, and 1% peni- undergoes copy number gain at chromosome 12p12 in cillin–streptomycin (Invitrogen). RAS 11% to 15% of lung tumors (9, 10). genes are members The following antibodies were used for immunoblot of the small GTPase super family (11) and function analysis: LDHA (Santa Cruz Biotechnology; sc-133123), to propagate growth factor signaling through activation of LDHB (Epitomics; 2090-1), TUBULIN (Sigma; T6074), and c-RAF and phosphoinositide-3-kinase (PI3K) pathways. HRP-conjugated anti-mouse and anti-rabbit secondary Despite concerted efforts, KRAS mutation defines a genetic antibodies (Jackson ImmunoResearch). subtype of lung cancer that is currently not amenable to therapeutic intervention (12). Recent studies have used siRNA screen high-throughput chemical and genetic screens to identify An RNAi screen for genes that regulate lung cancer growth synthetic lethal phenotype in the context of KRAS muta- was carried out in 4 lung adenocarcinoma cell lines, 2 with tions (13–16). These screens have yielded promising chromosome 12p KRAS amplification (NCI-H838, NCI- kinases and other cellular machinery. Significant work is H322T) and 2 without KRAS amplification (RERF-LC-KJ, required to determine whether these interactions will trans- NCI-H1568). Genes were selected on the basis of 2 main late into therapies for KRAS-driven tumors. criteria: (i) they reside within the KRAS amplicon on chro- While previous studies have mostly focused on identi- mosome 12p (analysis completed with Tumorscape; http:// fying synthetic interactions in KRAS-mutant cell lines, www.broadinstitute.org/tumorscape) and (ii) their gene here we set out to identify genes within the KRAS genomic expression and copy number were significantly upregulated amplicon that are both coupregulated and critical for cell in 10% or more of lung adenocarcinoma tumors and were proliferation when KRAS is amplified. We characterized correlated (P < 0.05; analysis completed with The Cancer the 12p11/12 amplicon where KRAS resides, and report Genome Atlas Data Portal; http://tcga-portal.nci.nih.gov). the presence of 18 additional genes that are coamplified The heatmap of 735 NSCLC tumors and cell lines was and overexpressed in lung adenocarcinomas. Using a loss made with Integrative Genomics Viewer using data from of function RNA interference (RNAi) screen on these 18 Tumorscape. genes, we identified lactate dehydrogenase B (LDHB) as Dharmacon siGENOME siRNAs were individually being specifically required in KRAS amplified lung cancer reverse transfected using DharmaFECT 4 (Dharmacon) in for cell proliferation. We find that LDHB expression 96-well format in triplicate. Four independent siRNAs were significantly correlates to both KRAS copy number gain used for each gene. Cell number was determined with and KRAS mutation in lung adenocarcinoma cell lines CellTiter-Glo (Promega) 6 days after transfection. Values and tumors. LDHB knockdown reduced cell growth in were normalized to a nontargeting control siRNA, and then in vitro in vivo KRAS-mutant lung cancer both and . Finally, Z scores were calculated using the following formula: (gene LDHB was a prognostic indicator of poor outcome in lung value plate average)/plate SD. To reduce the Z scores for adenocarcinomas. Our work identifies LDHB and more each gene to a single comparable value, a DZ score was broadly, lactate metabolism, as a potential therapeutic calculated: average of the Z scores from the two 12p-ampli- target for KRAS-driven lung cancer. fied cell lines average Z scores of the nonamplified cell lines. Genes with a DZ score less than 1 were considered Materials and Methods hits in the 12p-amplified cell lines. Only genes with two or Cell lines and antibodies more siRNA oligos with a DZ score less than 1 were The following lung cell lines were used: adenocarcinoma considered further. Two independent siRNAs from the (ALK fusion): NCI-H2228; adenocarcinoma (EGFR siGENOME pool were used for subsequent LDHB mutant): NCI-H1650, NCI-H1975, HCC827, NCI-H820, knockdown analysis (Dharmacon D-009779-01 and D- HCC2279, HCC2935, HCC4011, HCC4006; adenocarci- 009779-02).

OF2 Clin Cancer Res; 19(4) February 15, 2013 Clinical Cancer Research

LDHB Is a Regulator of KRAS-Dependent Lung Cancer

Gene expression analysis gene sets for Glucose Metabolic Process (21). The expres- For cell lines, RNA was harvested from 96-well plates 3 or sion level of all glycolysis signature genes was averaged in 4 days after siRNA transfection using the TaqMan Gene each tumor and either separated by KRAS gene mutation Expression Cells-to-CT kit (Applied Biosystems). For mouse status or directly compared with the average expression of tissues/tumors, RNA was harvested using Qiagen RNeasy the RAS signature genes in the same tumor. kit. Quantitative RT-PCR was carried out with the TaqMan One-Step RT-PCR Master Mix Reagents kit using Taqman Survival analysis Gene Expression Assays according to manufacturer protocol A lung adenocarcinoma tumor microarray was obtained (Applied Biosystems). All samples were normalized to a from the Leeds collection. Patient samples were collected glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with approval from the Yorkshire and Humber East Ethics control. Committee (approval number: NRES 05/Q1206/147). In total, 170 formalin-fixed, paraffin embedded (FFPE) lung Xenograft tumors adenocarcinomas were stained by immunohistochemistry NCI-H2122 cells with doxycycline-inducible LDHB for LDHB and correlated to clinical outcome. Kaplan–Meier knockdown were constructed using the lentivirus pHush- survival curves were plotted for survival until death or shRNA system (17). Two independent short hairpin RNAs censored from the study and for 5-year survival or censored. (shRNA) sequences were cloned for LDHB knockdown P values were calculated by a log-rank test. Fitting the data (shLDHB-1 target sequence: GGATATACCAACTGGGCTA with a Cox proportional hazards model lead to nearly and shLDHB-2 target sequence: GTACAGTCCTGATTG- identical P values (data not shown). For survival analysis CATC). Hairpins were cloned into the pSHUTTLE-H1 vector of LDHB gene expression, LDHB expression and survival and then Gateway (Invitrogen) recombined into a puro- data were previously published (Gene Expression Omnibus mycin-selectable lentiviral vector (17). A nontargeting GSE11969; ref. 22). LDHB expression was mean-centered shRNA (pHush-shNTC) was used for control experiments across all tumors then separated by above or below the and was obtained from David Davis (Genentech). Lentivi- mean. All patients were censored from the study after more rus production and infections were carried out as previously than 5 years of follow-up, thus the 5-year survival is shown. described (18). Cells were selected for stable integration P value was calculated by a log-rank test. with 2 mg/mL puromycin. For each shRNA cell line, 5 106 cells were injected Immunohistochemistry subcutaneously into the backs of female NCr nude mice Immunohistochemistry was conducted on 4 mmthick (Taconic) to initiate tumor growth. After tumors reached FFPE tissue sections mounted on glass slides. All immu- 200 to 300 mm3 in size, the animals from each cell line were nohistochemistry steps were carried out on the Ventana split into 2 groups and fed either 5% sucrose or 5% sucrose Discovery XT autostainer (Ventana Medical Systems). þ 1 mg/mL doxycycline to induce hairpin expression. Pretreatment was done with Cell Conditioner 1 with the Tumors with a starting volume less than 200 mm3 at the standard time. Primary antibodies were used at the fol- time of treatment were excluded from further analysis. lowing concentrations: MCT1 (Santa Cruz Biotechnolo- After 7 days, 3 mice from each group were euthanized and gy; sc-365501) at 1 mg/mL, MCT4 (Santa Cruz Biotech- the tumors were harvested for LDHB knockdown analysis. nology; sc-50329) at 0.1 mg/mL, LDHA (Cell Signaling Tumor measurements were carried out on the remaining Technologies; 3582) at 0.081 mg/mL, and LDHB (Epi- mice every 3 to 4 days until day 18 postdoxycycline admin- tomics; 2090-1) at 0.04 mg/mL. Slides were incubated istration. Mouse body weight was also recorded and show- with primary antibody for 60 minutes at 37oC. Ventana ed no major changes (data not shown). Tumor growth Mouse or Rabbit OmniMap was used as the detection inhibition values (TGI) were determined with the follow- system. Ventana DAB and Hematoxylin II were used for ing calculation: 100 1 [(tumor volumefinal tumor chromogenic detection and counterstain. Immuohisto- volumeinitial for doxycycline treated group)/(tumor chemical (IHC) analyses were conducted by a board volumefinal tumor volumeinitial for sucrose treated certified pathologist (R. Firestein). group)]. Results Data analysis of lung adenocarcinoma microarray data Oncogenic activation of KRAS is typically achieved Previously published microarray data of lung adenocar- through single residue point mutations that generate a cinomas was used (Gene Expression Omnibus GSE31210; constitutively active form of the protein. In lung adenocar- ref. 19). For Figs. 2D, 5B, and 5D, we used the previously cinomas, the KRAS locus is amplified in 10% to 15% of lung defined signature of RAS activation (20). Tumors (n ¼ 226) tumors, suggesting an alternative mechanism of KRAS acti- were separated on the basis of high or low expression of vation in this tumor type (Supplementary Table S1; ref. 9). LDHB, separated by mean LDHB expression across all To further characterize the KRAS locus in lung adenocarci- tumors. The expression level of all RAS signature genes was noma, we conducted a GISTIC analysis on copy number averaged in each group of tumors. For Fig. 5A, we used a data derived from 735 NSCLC tumors and cell lines. While defined glycolytic gene signature that was adapted from the KRAS is the most significantly amplified gene (Q value ¼ Broad Institute Molecular Signatures Database (MSigDB) 10 22), the region of copy number gain is relatively broad

www.aacrjournals.org Clin Cancer Res; 19(4) February 15, 2013 OF3

McCleland et al.

and harbors 18 additional genes that are both amplified and genes in the screen was confirmed by quantitative RT-PCR in overexpressed (Fig. 1A and B). NCI-H838 cells (Supplementary Fig. S1). Comparing the Z To functionally interrogate the candidate genes residing scores between the amplified and disomic cell lines, genes in the 12p12 region of copy number gain, we conducted a with a differential cell proliferation effect were identified loss of function RNAi screen in lung adenocarcinoma cell (Fig. 1C). In addition to KRAS, 2 additional genes, LDHB lines that harbored either 12p amplification (NCI-H838, and MED21, met our significance criteria of at least 2 NCI-H322T) or were disomic at this region (RERF-LC-KJ independent siRNAs scoring at a DZ score of 1 or below and NCI-H1568). Gene expression and knockdown for all (Z score chromosome 12p-amplified lines Z score

Figure 1. RNAi screen for genes coamplified with KRAS that regulate lung cancer cell growth. A, Heatmap of 735 NSCLC tumors and cell lines showing copy number of a 12 Mb region of chromosome 12 surrounding the KRAS gene. B, shown are Q values (significance of gene amplification in NSCLC data from A) of eighteen genes coamplified with KRAS. These genes had increased copy number and expression in primary lung tumors. C, RNAi screen of genes coamplified with KRAS. Black and red bars represent effects on cell growth for each individual siRNA oligos, as measured by DZ score (Z score KRAS amplified cell lines Z score disomic cell lines). Red bars indicate individual siRNA oligos that had a stronger growth inhibition in KRAS-amplified lung cancer cell lines compared with nonamplified lung cancer cell lines. Genes with 2 or more individual siRNAs specifically inhibiting growth in KRAS-amplified lines are highlighted in red.

OF4 Clin Cancer Res; 19(4) February 15, 2013 Clinical Cancer Research

LDHB Is a Regulator of KRAS-Dependent Lung Cancer

Figure 2. LDHB is upregulated in KRAS-mutant lung adenocarcinomas. A and B, box plot of relative MED21 (A) and LDHB (B) log2 mRNA expression in KRAS wild-type or KRAS-mutant NSCLC cell lines. P values are Student t test. C, average LDHB IHC score SD in KRAS wild-type (n ¼ 31) or KRAS-mutant (n ¼ 24) lung adenocarcinoma tumors. P value is a Student t test. D, Relative Ldhb mRNA expression is shown for normal mouse lung or transgenic mouse models of lung cancer with KRASG12D-activating mutation with or without p53Dlox/Dlox deletion. P values between normal and each tumor type are Student G12D Dlox/Dlox t tests. E, Representative images of LDHB IHC protein staining in normal lung or KRAS ; p53 lung cancer mouse model. F, average log2 expression RAS signature genes (20) SEM in lung adenocarcimoma tumors with low (n ¼ 97) or high (n ¼ 129) LDHB expression (below or above mean LDHB expression across all tumors, respectively). P value is a Student t test. disomic lines). MED21 (hSrb7) is a member of the mediator signature (ref. 20; Fig. 2F). To determine whether the complex and is a general regulator of transcription (23). relationship between KRAS activation and LDHB extended LDHB is a metabolic enzyme that catalyzes the intercon- to other tumor types, LDHB expression was also examined version of lactate and pyruvate (24, 25). in colon cancer, which frequently harbors KRAS mutations Because LDHB and MED21 are frequently coamplified (26). LDHB was not differentially expressed in colon cancer with KRAS, we first examined whether expression of these cell lines harboring KRAS mutation, suggesting that the genes also correlate to KRAS mutation status. We assessed correlation between LDHB and KRAS may be specific to LDHB and MED21 gene expression in both KRAS wild-type lung adenocarcinomas (Supplementary Fig. S2). We con- and mutant lung cancer cell lines and found that both genes clude that LDHB is upregulated in lung cancer cell lines and are upregulated in KRAS-mutant cell lines (Fig. 2A, B). The tumors that are characterized by RAS pathway activation. correlation was significantly stronger for LDHB, which To determine whether LDHB is required for the prolif- prompted us to look further into LDHB expression and eration of KRAS-mutant cancer cells, we examined a panel function in the context of KRAS activity. LDHB protein and of 24 lung adenocarcinoma cell lines for LDHB expression. RNA are overexpressed in KRAS-mutant human lung Consistent with our expression data in lung tumors, LDHB tumors as well as in 2 mouse lung adenocarcinoma models was specifically upregulated in both KRAS-amplified and driven by mutant KRAS (ref. 2; Fig. 2C–E). Consistent with KRAS-mutant lung cancer cell lines when compared with this finding, we also observed that LDHB was upregulated in KRAS wild-type cell lines (Fig. 3A and Supplementary Table lung cancers that contain a "RAS activated" gene expression S2). In contrast, LDHA, the main LDH isoform, was

www.aacrjournals.org Clin Cancer Res; 19(4) February 15, 2013 OF5

McCleland et al.

Figure 3. LDHB is required for lung cancer cell line proliferation when highly expressed. A, immunoblot analysis of LDHB and LDHA in lung adenocarcinoma cell lines that are KRAS wild-type, mutant, or amplified. B, relative cell proliferation (using CellTiter-Glo assay) of lung adenocarcinoma cell lines using 2 independent LDHB siRNA oligos compared with siNTC control. Immunoblot analysis of LDHB and LDHA are shown below each cell line. KRAS status refers to the mutation or amplification status of KRAS. KRAS-dependent cells (denoted as þ) have reduced cell proliferation after KRAS RNAi (see Supplementary Fig. S3). C, cell lines were categorized as either high or low LDHB expressers following mean centered normalization (cell lines above the mean were considered high and below the mean considered low). Plot shows relative cell growth (using CellTiter-Glo assay) of each cell line following LDHB knockdown (values were normalized to an siNTC control for each cell line and represent the average of 2 independent siLDHB oligos). Each dot or square denotes an individual cell line and error bars represent SD. Colored circles and boxes highlight oncogenic mutation associated with each particular cell line. P value indicates a statistically significant difference in cell growth between the 2 groups, Student t test.

ubiquitously expressed in all lung cell lines examined. To elevated LDHB levels and are highly dependent on its explore the functional consequences of LDHB expression in expression for proliferation. KRAS aberrant cell lines, we used 2 independent siRNAs to To determine whether LDHB itself is a downstream KRAS knockdown LDHB in KRAS amplified, mutant, or wild-type target, we examined LDHB gene expression after KRAS lung adenocarcinoma cell lines. Both KRAS-mutant and knockdown. We observed that LDHB gene expression did KRAS-amplified cell lines were generally more sensitive to not directly change after KRAS knockdown (Supplementary LDHB knockdown (Fig. 3B). To ensure that these effects Fig. S3B), implying that LDHB is required in KRAS-mutant were not due to technical differences in knockdown levels, cells, but is not directly regulated by KRAS activity. loss of LDHB was confirmed by immunoblot in each cell Because LDHB expression does not seem to be directly line tested. Moreover, no changes in LDHA protein levels regulated by KRAS, we considered whether LDHB is upre- were observed upon LDHB knockdown (Fig. 3B). Because gulated in lung cancers that contain mutations in other KRAS mutation does not always confer dependence on oncogenic drivers (i.e., EGFR mutation, c-Met amplifica- KRAS for growth (27), we examined the sensitivity of each tion, and ALK fusion). Therefore, we immunoblotted a cell line to KRAS RNAi-mediated knockdown (Fig. 3B and panel of cell lines that were annotated for mutation status Supplementary Fig. S3). With minor exception, KRAS- (Supplementary Fig. S4A). LDHB was also upregulated mutant cell lines were dependent on KRAS for growth. in other lung cancer subtypes, in particular those driven These results show that KRAS-dependent cell lines express by c-MET (2/2 cell lines) and EGFR (3/8 cell lines). The level

OF6 Clin Cancer Res; 19(4) February 15, 2013 Clinical Cancer Research

LDHB Is a Regulator of KRAS-Dependent Lung Cancer

of overexpression was similar to that observed in KRAS- in fully formed tumors led to significant tumor growth mutant cancers. To functionally test whether LDHB over- inhibition when compared with the uninduced shLDHB expression reflected a cellular dependence on LDHB, we tumors or shNTC controls (Fig. 4A). LDHB knockdown in knocked down LDHB with 2 independent siRNA oligos in a the tumors was confirmed by immunohistochemistry and subset of these cell lines (Supplementary Fig. S4B and S4C). immunoblot at day 7 after doxycycline administration (Fig. We find that cell lines with high levels of LDHB are statis- 4B and C). These observations indicate that LDHB is tically more sensitive to loss of LDHB (P ¼ 0.00005) than required for growth of KRAS-mutant lung tumors in vivo. LDHB low-expressing lines (Fig. 3C). Taken together these data suggest that targeting LDHB may provide a broad KRAS-Mutant lung cell lines and adenocarcinomas are therapeutic option for patients with lung cancer that spe- dependent on glycolysis cifically overexpress LDHB. The "Warburg effect" is named after the observation that The metabolic and growth requirements of a three- many cancer cells generate energy using a high rate of dimensional tumor can be starkly different from cancer glycolysis, instead of oxidative phosphorylation (28). The cells grown in vitro. To test whether LDHB knockdown high rate of glycolysis occurs regardless of oxygen supply affects tumor growth in vivo, we used an inducible shRNA and produces an excess of pyruvate, which gets converted to lentiviral system to acutely deplete endogenous LDHB in lactate by LDHA or LDHB and subsequently exported from fully formed tumors. Two independent shRNAs to LDHB the cell. We hypothesized that LDHB upregulation may be (shLDHB) and a nontargeting control (shNTC) were intro- associated with a more general shift in cellular metabolism duced into a human KRAS-mutant lung cancer cell line toward glycolytic dependence. Because LDHB is overex- (NCI-H2122) and subsequently grown as xenografted pressed in KRAS-mutant tumors, we analyzed and com- tumors in mice. Doxycycline-induced LDHB knockdown pared the expression of a defined glycolytic gene signature

Figure 4. LDHB is required for lung tumor growth in vivo. A, tumor volumes from xenografted NCI- H2122 cells were measured over time (n ¼ 5–6 mice/group). shRNA expression was induced with doxycycline (sucrose was used as control). TGI values were determined for each shRNA (shLDHB-1 TGI ¼ 46%; shLDHB-2 TGI ¼ 77%). Error bars represent SEM. B, IHC staining for LDHB in representative shLDHB xenograft tumors after 7 days of sucrose or doxycycline treatment. C, immunoblot of LDHB and LDHA in xenograft tumors at day 7 postdoxycycline treatment.

www.aacrjournals.org Clin Cancer Res; 19(4) February 15, 2013 OF7

McCleland et al.

Figure 5. Activated RAS is associated with increased glycolysis in lung adenocarcinomas. A, average log2 expression glycolysis signature genes SEM is shown for lung adenocarcimoma tumors (left) that are KRAS wild-type (n ¼ 206) or KRAS-mutant (n ¼ 20). P value is a Student t test. Correlation

analysis between the average log2 expression of glycolysis signature genes and RAS signature genes in individual lung adenocarcinomas (right). P value for Pearson correlation is a one-tailed t test. B, sensitivity of 7 KRAS wild-type and 6 KRAS-mutant cell lines to 2-deoxyglucose (2-DG). Each dot or square denotes an individual cell line and error bars represent SD. P value indicates a statistically signiﬁcant difference in cell growth between the 2 groups, Student t test. C, average IHC scores SD for LDHB, MCT1, and MCT4 are shown for normal lung tissue or adenocarcinomas. P values are Student t tests.

D, average log2 expression RAS signature genes (20) SEM in lung adenocarcimoma tumors with low (n ¼ 118) or high (n ¼ 108) MCT4 expression (below or above mean MCT4 expression across all tumors, respectively). P value is a Student t test.

(see Methods) between KRAS wild-type and KRAS-mutant To explore the functional signiﬁcance of these gene lung tumors. Consistent with our hypothesis, KRAS-mutant expression changes, we tested the sensitivity of 7 KRAS tumors exhibited an elevated expression of the glycolytic wild-type (low LDHB expressing) and 6 KRAS-mutant (high gene signature compared with KRAS wild-type tumors (Fig. LDHB expressing) cell lines to the glycolysis inhibitor 2- 5A). Moreover, lung tumors that have increased expression deoxyglucose (2-DG). KRAS-mutant cell lines were signif- of the RAS gene signature also show increased expression of icantly more sensitive to 2-DG inhibition than KRAS wild- the same glycolysis genes (Fig. 5A). This effect seems to be type cells (Fig. 5B), suggesting that KRAS-mutant cells are speciﬁc to lung cancer, as a correlation between RAS signa- more addicted to glycolysis. Taken together, these data ture expression and glycolytic signature expression was not imply that KRAS-mutant tumors are associated with a shift observed in colon tumors (Supplementary Fig. S2C). This in tumor metabolism that is characterized by increased suggests that lung tumors with KRAS mutation and RAS dependence on glycolysis for energy demands. pathway activation are associated with a dependence on The dependence of KRAS-mutated lung tumors on gly- glycolysis. colysis and LDHB implies that these tumors are sensitive to

OF8 Clin Cancer Res; 19(4) February 15, 2013 Clinical Cancer Research

LDHB Is a Regulator of KRAS-Dependent Lung Cancer

perturbations in the end-stage of glycolysis, namely lactate more global shift in metabolic requirements upon KRAS production. In addition to LDH isoforms, which generate mutation. Finally, we show that LDHB overexpression in lactate, the monocarboxylate transporters (MCT1-4) are key patients with lung adenocarcinoma have a poorer transmembrane proteins that regulate the import/export of prognosis. lactate (29). While MCT1-4 all have similar structure and Carcinogenesis is a complicated, multistep process that function, MCT1 and MCT4 are differentially upregulated in involves combinatorial alterations in oncogenes, tumor various tumor types and have garnered the most attention in suppressors, and metabolic pathways. It is important to cancer (30). Therefore, we examined the expression of note that these alterations in intracellular signaling path- MCT1 and MCT4 in lung adenocarcinomas. IHC analysis ways are a type of evolution that conforms to the changing revealed that MCT4, but not MCT1, was overexpressed in tumor microenvironment and bioenergetic requirements lung adenocarcinomas (Fig. 5C). Similar to LDHB, MCT4 that allows uncontrolled cell growth. Since Warburg’s expression was associated with RAS pathway activation (Fig. observation more than 50 years ago, in which tumor cells 5D). We suggest that KRAS-mutant adenocarcinomas have shift from oxidative phosphorylation to aerobic glycolysis, upregulated components of the lactate machinery to accom- we are only now beginning to understand the molecular modate an increased dependence on glycolysis. details of this transformation. In the last decade, there has been accumulating evi- LDHB expression in lung adenocarcinomas correlates dence that oncogenes, such as PI3K, AKT, mTOR, EGFR, with poor clinical outcome KRAS, MYC, along with hypoxia-inducible factor 1 We next examined whether the expression of LDHB in (HIF1) stimulate the transcription of key genes that lung cancer correlated with clinical outcome. We scored and mediate glycolysis and related metabolic pathways analyzed LDHB protein levels by immunohistochemistry (31). For example, PI3K/AKT activation facilitates cellular on a cohort of 383 lung adenocarcinomas (Fig. 6A and B), of glucose uptake by upregulating glucose transporters and which nearly half (n ¼ 170) had associated survival data boosting glycolysis through increasing hexokinase levels (Supplementary Table S3). In Kaplan–Meier analysis, 5- and stimulating phosphofructokinase activity (32). Like- year patient survival was significantly lower in LDHB high wise, genes involved in the metabolism of glutamine, (2 or 3 staining) as compared with LDHB low (0 or 1) which tumor cells need for the production of various tumors (33% vs. 48%, log-rank P ¼ 0.005). When we metabolic intermediates, are downstream targets of the assessed the independent prognostic effect of LDHB using MYC oncogene (33, 34). Direct regulatory inputs on a multivariate Cox model that adjusted for additional pre- metabolism are not restrictedtooncogenes,asdeletion dictors of survival, such as sex, age, and tumor grade, high of tumor suppressors, such as p53 and PTEN, also con- LDHB expression was associated with reduced 5-year sur- tribute to the metabolic shift (31). Taken together, the vival [HR ¼ 1.67; 95% confidence interval (CI), 1.06–2.62; concerted upregulation of oncogenes and downregula- P ¼ 0.027; Fig. 6C and D; and Supplementary Fig. S5]. tion of tumor suppressors directly alters the transcrip- Because we observed that LDHB is upregulated in a subset of tional controls of the metabolic circuitry. cancers containing other oncogenic drivers, we also exam- The direct role of KRAS in regulating tumor metabolism is ined if LDHB expression is predictive of lifespan in KRAS less well defined. Tumors with oncogenic RAS correlate with wild-type tumors using a microarray dataset that contained numerous metabolic aberrations, including increased con- associated outcome data. Interestingly, KRAS wild-type lung sumption of glucose and glutamine, increased production tumors with high expression of LDHB mRNA trended with a of lactic acid, altered expression of mitochondrial genes, poorer clinical outcome (Supplementary Fig. S5C). These and reduced mitochondrial activity (35–38). Our findings, data indicate that LDHB is upregulated in a significant that a glycolysis gene signature is specifically upregulated in fraction of lung adenocarcinomas and is associated with KRAS-mutant lung tumors and that KRAS-mutant cells are poor patient outcome. more sensitive to the glycolysis inhibitor 2-DG, are consistent with such changes in tumor metabolism. Moreover, a Discussion recent report showed that KRAS can also regulate glycolysis In this study, we characterized the KRAS amplicon at and glucose metabolism in a pancreatic cancer mouse 12p12 and found 18 genes that are coamplified with KRAS model (39). Our observations, however, show that in other in NSCLC tumors. Using a loss of function RNAi screen, we cancer types, such as colon cancer, KRAS-mutant tumors do subsequently knocked down all 18 genes and identified not display elevated dependence on glycolysis. Cumula- LDHB as an essential regulator of lung cancer. LDHB was tively, these studies suggest that examining the effect of upregulated and required for the growth of KRAS-mutant KRAS on cellular metabolism should be conducted in a and amplified lung cancers both in vitro and in vivo. Gene tumor-specific context. expression and functional analyses indicate that KRAS- Lactate dehydrogenase enzymes have important roles in mutant lung tumors, compared with KRAS wild-type aerobic glycolysis, as they are required to convert the surplus tumors, have a greater predilection for using glycolysis for of pyruvate generated by a high rate of glycolysis into lactate their energy demands. Consistent with such a role, we find (25). The LDHA isoform has received the most attention that in addition to LDHB, the lactate transporter MCT4 is because it is ubiquitously expressed in tumors and is a also upregulated in KRAS-mutant lung cancer, suggesting a downstream target of HIF1. While an essential function of

www.aacrjournals.org Clin Cancer Res; 19(4) February 15, 2013 OF9

McCleland et al.

Figure 6. Patients with lung tumors expressing high LDHB protein have a poor clinical outcome. A, representative IHC photomicrographs show LDHB protein expression in lung adenocarcinoma tumors. IHC score is indicated in parenthesis. B, IHC score breakdown for a panel of normal lung tissue (n ¼ 126) and lung adenocarcinoma tumors (n ¼ 383). C and D, Kaplan–Meier survival data for patients with lung adenocarcinoma, separated by LDHB IHC score. Patients with high LDHB expression (n ¼ 99; IHC score 2 or 3) had a worse overall survival compared with patients with low LDHB expression (n ¼ 71; IHC score 0 or 1) for a 5-year survival (C) or overall survival, as measured by death or being censored from the study (D).

LDHA in tumor metabolism has been described (40), the genic drivers. Importantly, LDHB is required to maintain role of LDHB is less clear. One report has shown that the cell growth in this context. While our data does not support mTOR pathway controls LDHB expression, whereas anoth- that LDHB transcription is downstream of KRAS signaling, er report has shown that LDHB transcription is shut off by we hypothesize that a pathway functioning in parallel with promoter methylation in bladder, colon, and prostate can- KRAS activity might drive LDHB expression. In normal cers (41–44). In the context of lung adenocarcinoma, we tissues, LDHB is expressed in liver, red blood cells, kidney, observed that LDHB is upregulated in KRAS aberrant and heart, raising concern that its inhibition may have tumors and in a subset of cell lines containing other onco- deleterious consequences (45, 46). Intriguingly, however,

OF10 Clin Cancer Res; 19(4) February 15, 2013 Clinical Cancer Research

LDHB Is a Regulator of KRAS-Dependent Lung Cancer

patients with complete loss of LDHB expression due to a lung cancers develop a dependence on glycolysis for their hereditary recessive trait have no significant phenotypic metabolic demands. impairment (47, 48). This suggests that an LDHB small- molecule inhibitor may have minimal off-target affects in Disclosure of Potential Conflicts of Interest normal tissue. Future studies are necessary to identify sig- All authors are employed by Genentech, Inc. naling pathways that act upstream of LDHB. Authors' Contributions Our data showing that LDHB inhibition reduced cell Conception and design: M.L. McCleland, L. Deming, E.M. Blackwood, R. growth, even in the presence of LDHA, raises an important Firestein question about the biochemical activity of the LDHB iso- Development of methodology: M.L. McCleland, L. Deming, W.F. Forrest Acquisition of data (provided animals, acquired and managed patients, form. Because the LDH enzymes function as both homo- provided facilities, etc.): M.L. McCleland, A.S. Adler, L. Deming, E. Cosino, and heterotetramers (46), it is possible that reducing LDHB L. Lee, E.M. Blackwood, M. Solon, J. Tao, E. Jackson somehow affects LDHA activity and the forward reaction. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): M.L. McCleland, A.S. Adler, L. Deming, E. However, an alternative explanation is that LDHB has an Cosino, L. Lee, E.M. Blackwood, L. Li, D. Shames, E. Jackson, W.F. Forrest exclusive function from LDHA. While biochemical data Writing, review, and/or revision of the manuscript: M.L. McCleland, A.S. Adler, L. Deming, E.M. Blackwood, L. Li, D. Shames, W.F. Forrest, R. Firestein indicates that LDHA and LDHB can run in both the forward Study supervision: E.M. Blackwood, R. Firestein and reverse direction, LDHB has a biochemical predilection for the reverse direction (45). Perhaps LDHB functions in Acknowledgments certain contexts to oppose LDHA or act as a governor to The authors thank Laura Corson, Tom Hunsaker, Anna Hitz, David Shames, Marie Evangelista, and Georgia Hatzivassiliou for helpful comments LDHA function. In this scenario, LDHB would at the very and discussion, David Davis for reagents, and Pete Haverty for assistance in least slow down the conversion of pyruvate to lactate. Future creating gene signatures. work is necessary to test these hypotheses, but is beyond the The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked scope of this work. advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate In conclusion, our work identifies LDHB as a promising this fact. new therapy target for lung adenocarcinomas that overexpress LDHB and supports the notion that KRAS-driven Received August 13, 2012; revised October 22, 2012; accepted November 12, 2012; published OnlineFirst December 6, 2012.

References 1. Herbst RS, Heymach JV, Lippman SM. Lung cancer. N Engl J Med 13. Barbie DA, Tamayo P, Boehm JS, Kim SY, Moody SE, Dunn IF, et al. 2008;359:1367–80. Systematic RNA interference reveals that oncogenic KRAS-driven 2. Singh M, Lima A, Molina R, Hamilton P, Clermont AC, Devasthali V, cancers require TBK1. Nature 2009;462:108–12. et al. Assessing therapeutic responses in Kras mutant cancers using 14. Dolma S, Lessnick SL, Hahn WC, Stockwell BR. Identification of genetically engineered mouse models. Nat Biotechnol 2010;28: genotype-selective antitumor agents using synthetic lethal chemical 585–93. screening in engineered human tumor cells. Cancer Cell 2003;3: 3. Camidge DR, Doebele RC. Treating ALK-positive lung cancer-early 285–96. successes and future challenges. Nat Rev Clin Oncol 2012;9:268–277. 15. Luo J, Emanuele MJ, Li D, Creighton CJ, Schlabach MR, Westbrook 4. Carter CA, Giaccone G. Treatment of nonsmall cell lung cancer: TF, et al. A genome-wide RNAi screen identifies multiple synthetic overcoming the resistance to epidermal growth factor receptor inhi- lethal interactions with the Ras oncogene. Cell 2009;137:835–48. bitors. Curr Opin Oncol 2012;24:123–9. 16. Torrance CJ, Agrawal V, Vogelstein B, Kinzler KW. Use of isogenic 5. Lynch TJ, Bell DW, Sordella R, Gurubhagavatula S, Okimoto RA, human cancer cells for high-throughput screening and drug discovery. Brannigan BW, et al. Activating mutations in the epidermal growth Nat Biotechnol 2001;19:940–5. factor receptor underlying responsiveness of non-small-cell lung can- 17. Gray DC, Hoeflich KP, Peng L, Gu Z, Gogineni A, Murray LJ, et al. cer to gefitinib. N Engl J Med 2004;350:2129–39. pHUSH: a single vector system for conditional gene expression. BMC 6. Paez JG, Janne PA, Lee JC, Tracy S, Greulich H, Gabriel S, et al. EGFR Biotechnol 2007;7:61. mutations in lung cancer: correlation with clinical response to gefitinib 18. Adler AS, McCleland ML, Truong T, Lau S, Modrusan Z, Soukup TM, therapy. Science 2004;304:1497–500. et al. CDK8 maintains tumor de-differentiation and embryonic stem cell 7. Kwak EL, Bang YJ, Camidge DR, Shaw AT, Solomon B, Maki RG, et al. pluripotency. Cancer Res 2012;72:2129–39. Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer. N 19. Okayama H, Kohno T, Ishii Y, Shimada Y, Shiraishi K, Iwakawa R, Engl J Med 2010;363:1693–703. et al. Identification of genes upregulated in ALK-positive and EGFR/ 8. Riely GJ, Kris MG, Rosenbaum D, Marks J, Li A, Chitale DA, et al. KRAS/ALK-negative lung adenocarcinomas. Cancer Res 2012;72: Frequency and distinctive spectrum of KRAS mutations in never 100–11. smokers with lung adenocarcinoma. Clin Cancer Res 2008;14: 20. Hoeflich KP, O'Brien C, Boyd Z, Cavet G, Guerrero S, Jung K, et al. In 5731–4. vivo antitumor activity of MEK and phosphatidylinositol 3-kinase 9. Sasaki H, Hikosaka Y, Kawano O, Moriyama S, Yano M, Fujii Y. inhibitors in basal-like breast cancer models. Clin Cancer Res Evaluation of Kras gene mutation and copy number gain in non-small 2009;15:4649–64. cell lung cancer. J Thorac Oncol 2011;6:15–20. 21. Mootha VK, Lindgren CM, Eriksson KF, Subramanian A, Sihag S, Lehar 10. Weir BA, Woo MS, Getz G, Perner S, Ding L, Beroukhim R, et al. J, et al. PGC-1alpha-responsive genes involved in oxidative phos- Characterizing the cancer genome in lung adenocarcinoma. Nature phorylation are coordinately downregulated in human diabetes. Nat 2007;450:893–8. Genet 2003;34:267–73. 11. Karnoub AE, Weinberg RA. Ras oncogenes: split personalities. Nat 22. Takeuchi T, Tomida S, Yatabe Y, Kosaka T, Osada H, Yanagisawa K, Rev Mol Cell Biol 2008;9:517–31. et al. Expression profile-defined classification of lung adenocarcinoma 12. Downward J. Targeting RAS signalling pathways in cancer therapy. shows close relationship with underlying major genetic changes and Nat Rev Cancer 2003;3:11–22. clinicopathologic behaviors. J Clin Oncol 2006;24:1679–88.

www.aacrjournals.org Clin Cancer Res; 19(4) February 15, 2013 OF11

McCleland et al.

23. Taatjes DJ. The human mediator complex: a versatile, genome-wide 37. Gaglio D, Metallo CM, Gameiro PA, Hiller K, Danna LS, Balestrieri C, regulator of transcription. Trends Biochem Sci 2010;35:315–22. et al. Oncogenic K-Ras decouples glucose and glutamine metabolism 24. Bayley JP, Devilee P. The Warburg effect in 2012. Curr Opin Oncol to support cancer cell growth. Mol Syst Biol 2011;7:523. 2012;24:62–7. 38. Vizan P, Boros LG, Figueras A, Capella G, Mangues R, Bassilian S, 25. Porporato PE, Dhup S, Dadhich RK, Copetti T, Sonveaux P. Anticancer et al. K-ras codon-specific mutations produce distinctive metabolic targets in the glycolytic metabolism of tumors: a comprehensive phenotypes in NIH3T3 mice [corrected] fibroblasts. Cancer Res review. Front Pharmacol 2011;2:49. 2005;65:5512–5. 26. Vogelstein B, Kinzler KW. Cancer genes and the pathways they 39. Ying H, Kimmelman AC, Lyssiotis CA, Hua S, Chu GC, Fletcher-Sana- control. Nat Med 2004;10:789–99. nikone E, et al. Oncogenic Kras maintains pancreatic tumors through 27. Singh A, Greninger P, Rhodes D, Koopman L, Violette S, Bardeesy N, regulation of anabolic glucose metabolism. Cell 2012;149:656–70. et al. A gene expression signature associated with "K-Ras addiction" 40. Fantin VR, St-Pierre J, Leder P. Attenuation of LDH-A expression reveals regulators of EMT and tumor cell survival. Cancer Cell uncovers a link between glycolysis, mitochondrial physiology, and 2009;15:489–500. tumor maintenance. Cancer Cell 2006;9:425–34. 28. Warburg O. On the origin of cancer cells. Science 1956;123:309–14. 41. Leiblich A, Cross SS, Catto JW, Phillips JT, Leung HY, Hamdy FC, et al. 29. Halestrap AP. The monocarboxylate transporter family—structure and Lactate dehydrogenase-B is silenced by promoter hypermethylation in functional characterization. IUBMB Life 2012;64:1–9. human prostate cancer. Oncogene 2006;25:2953–60. 30. Pinheiro C, Longatto-Filho A, Azevedo-Silva J, Casal M, Schmitt FC, 42. Liao AC, Li CF, Shen KH, Chien LH, Huang HY, Wu TF. Loss of lactate Baltazar F. Role of monocarboxylate transporters in human cancers: dehydrogenase B subunit expression is correlated with tumour prostate of the art. J Bioenerg Biomembr 2012;44:127–39. gression and independently predicts inferior disease-specific survival 31. Levine AJ, Puzio-Kuter AM. The control of the metabolic switch in in urinary bladder urothelial carcinoma. Pathology 2011;43:707–12. cancers by oncogenes and tumor suppressor genes. Science 43. Thangaraju M, Carswell KN, Prasad PD, Ganapathy V. Colon cancer 2010;330:1340–4. cells maintain low levels of pyruvate to avoid cell death caused by 32. Deberardinis RJ, Sayed N, Ditsworth D, Thompson CB. Brick by brick: inhibition of HDAC1/HDAC3. Biochem J 2009;417:379–89. metabolism and tumor cell growth. Curr Opin Genet Dev 2008;18: 44. Zha X, Wang F, Wang Y, He S, Jing Y, Wu X, et al. Lactate dehydro- 54–61. genase B is critical for hyperactive mTOR-mediated tumorigenesis. 33. Gao P, Tchernyshyov I, Chang TC, Lee YS, Kita K, Ochi T, et al. c-Myc Cancer Res 2011;71:13–8. suppression of miR-23a/b enhances mitochondrial glutaminase 45. Dawson DM, Goodfriend TL, Kaplan NO. Lactic dehydrogenases: expression and glutamine metabolism. Nature 2009;458:762–5. functions of the two types rates of synthesis of the two major forms 34. Wise DR, DeBerardinis RJ, Mancuso A, Sayed N, Zhang XY, Pfeiffer can be correlated with metabolic differentiation. Science 1964;143: HK, et al. Myc regulates a transcriptional program that stimulates 929–33. mitochondrial glutaminolysis and leads to glutamine addiction. Proc 46. Markert CL, Shaklee JB, Whitt GS. Evolution of a gene. Multiple genes Natl Acad Sci U S A 2008;105:18782–7. for LDH isozymes provide a model of the evolution of gene structure, 35. Chiaradonna F, Gaglio D, Vanoni M, Alberghina L. Expression of function and regulation. Science 1975;189:102–14. transforming K-Ras oncogene affects mitochondrial function and 47. Kanno T, Sudo K, Takeuchi I, Kanda S, Honda N, Nishimura Y, et al. morphology in mouse fibroblasts. Biochim Biophys Acta 2006;1757: Hereditary deficiency of lactate dehydrogenase M-subunit. Clin Chim 1338–56. Acta 1980;108:267–76. 36. Chiaradonna F, Sacco E, Manzoni R, Giorgio M, Vanoni M, Alberghina 48. Joukyuu R, Mizuno S, Amakawa T, Tsukada T, Nishina T, Kitamura M. L. Ras-dependent carbon metabolism and transformation in mouse Hereditary complete deficiency of lactate dehydrogenase H-subunit. fibroblasts. Oncogene 2006;25:5391–404. Clin Chem 1989;35:687–90.

OF12 Clin Cancer Res; 19(4) February 15, 2013 Clinical Cancer Research

Lactate Dehydrogenase B Is Required for the Growth of KRAS-Dependent Lung Adenocarcinomas

Mark L. McCleland, Adam S. Adler, Laura Deming, et al.

Clin Cancer Res Published OnlineFirst December 6, 2012.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-12-2638

Supplementary Access the most recent supplemental material at: Material http://clincancerres.aacrjournals.org/content/suppl/2012/12/06/1078-0432.CCR-12-2638.DC1

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

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

Permissions To request permission to re-use all or part of this article, use this link http://clincancerres.aacrjournals.org/content/early/2013/01/29/1078-0432.CCR-12-2638. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.