MAX Inactivation in Small Cell Lung Cancer Disrupts MYC–SWI/SNF Programs and Is Synthetic Lethal with BRG1

Published OnlineFirst December 20, 2013; DOI: 10.1158/2159-8290.CD-13-0799

RESEARCH ARTICLE

Octavio A. Romero 1 , Manuel Torres-Diz 1 , Eva Pros 1, Suvi Savola 6, Antonio Gomez 1 , Sebastian Moran 1 , Carmen Saez 3 , 4, Reika Iwakawa 7 , Alberto Villanueva 2 , Luis M. Montuenga 5 , Takashi Kohno 7 , Jun Yokota 7 , and Montse Sanchez-Cespedes 1

ABSTRACT Our knowledge of small cell lung cancer (SCLC) genetics is still very limited, amplifi cation of L-MYC , N-MYC , and C-MYC being some of the well-established gene alterations. Here, we report our discovery of tumor-specifi c inactivation of the MYC-associated factor X gene, MAX , in SCLC. MAX inactivation is mutually exclusive with alterations of MYC and BRG1 , the latter coding for an ATPase of the switch/sucrose nonfermentable (SWI/SNF) complex. We demonstrate that BRG1 regulates the expression of MAX through direct recruitment to the MAX promoter, and that depletion of BRG1 strongly hinders cell growth, specifi cally in MAX-defi cient cells, heralding a synthetic lethal interaction. Furthermore, MAX requires BRG1 to activate neuroendocrine transcriptional programs and to upregulate MYC targets, such as glycolysis-related genes. Finally, inactivation of the MAX dimerization protein, MGA, was also observed in both non–small cell lung cancer and SCLC. Our results provide evidence that an aberrant SWI/SNF–MYC network is essential for lung cancer development.

SIGNIFICANCE: We discovered that the MYC-associated factor X gene, MAX , is inactivated in SCLCs. Furthermore, we revealed a preferential toxicity of the inactivation of the chromatin remodeler BRG1 in MAX-deﬁ cient lung cancer cells, which opens novel therapeutic possibilities for the treatment of patients with SCLC with MAX-deﬁ cient tumors. Cancer Discov; 4(3); 292–303. ©2013 AACR.

See related commentary by Rudin and Poirier, p. 273.

INTRODUCTION pattern of gene alterations in SCLC is rather specifi c to this tumor type, probably refl ecting the different cell of origin Small cell lung cancer (SCLC) accounts for about 20% of of the distinct classes of lung cancers. For this reason, it lung cancer diagnoses and is a highly aggressive malignancy. has been suspected for some time that some SCLC subtype However, the genetics underlying its development are still arises from neuroendocrine cells in the lung, which are com- largely unknown. The genes most widely known to be fre- monly found in clusters known as neuroendocrine bodies quently altered in SCLC are TP53, RB1 , and those of the MYC ( 3 ). Recent observations, using mouse models for targeted family (1 ). PTEN , PIK3CA , and BRG1 (also called SMARCA4 ) Trp53 and Rb1 inactivation in distinct cell types of the adult are less frequently altered ( 1, 2 ). Novel high-throughput lung, support the explanation of the neuroendocrine origin sequencing screening approaches, such as exome sequenc- of at least some SCLCs (4 ). Either because of its possible ing, have been performed on SCLCs, revealing alterations at neuroendocrine origin or because of a specifi c neural tumor other genes, including the chromatin modifi ers CREBBP and differentiation, SCLCs are enriched in transcripts that are EP300 (2 ). related to neural tissues ( 5, 6 ). The neural origin of some Amplifi cations of L-MYC , N-MYC , and C-MYC are some of SCLCs may serve to explain why some of the genes mutated the best established gene alterations in lung cancer; L-MYC in this type of lung cancer are also altered in other neural- and N-MYC are more commonly amplifi ed in SCLC than related tumors. Such is the case of N-MYC and RB1 , which are in non–small cell lung cancer (NSCLC; ref. 1 ). In fact, the commonly altered in neuroblastomas and retinoblastomas, respectively ( 7, 8 ). Authors’ Affi liations: 1Genes and Cancer Group, Cancer Epigenetics Recently, germline-inactivating mutations at MAX , the and Biology Program (PEBC); 2Translational Research Laboratory, Cata- MYC-associated factor X gene, were found to be responsible lan Institute of Oncology (ICO), Bellvitge Biomedical Research Institute for hereditary pheochromocytoma, a tumor with neuroen- (IDIBELL), Hospitalet de Llobregat, Barcelona; 3Instituto de Biomedicina de Sevilla (IBiS), 4 Department of Pathology, Hospital Universitario Virgen docrine features ( 9 ). Homozygous inactivation of MAX in rat del Rocío, Consejo Superior de Investigaciones Cientifi cas (CSIC), Univer- adrenal pheochromocytoma PC12 cells had previously been sidad de Sevilla, Seville; 5 Division of Oncology, Centro para la Investigación reported ( 10 ). Inasmuch as the genes of the MYC family are Medica Aplicada (CIMA), University of Navarre, Pamplona, Spain; 6MRC- commonly activated in SCLC and that, similar to pheochro- 7 Holland, Amsterdam, the Netherlands; and Division of Genome Biology, mocytomas, SCLCs have neuroendocrinal features ( 11 ), we National Cancer Center Research Institute, Tokyo, Japan decided to test for MAX inactivation in lung cancer. Note: Supplementary data for this article are available at Cancer Discovery Online (http://cancerdiscovery.aacrjournals.org/). O.A. Romero and M. Torres-Diz contributed equally to this work. RESULTS Corresponding Author: Montse Sanchez-Cespedes, Genes and Cancer The MYC-Associated Factor X Gene, MAX , Group, Cancer Epigenetics and Biology Program (PEBC), Bellvitge Bio- Is Recurrently Inactivated in SCLC medical Research Institute (IDIBELL), 08908, Hospitalet de Llobregat, Barcelona 08907, Spain. Phone: 34-93-260-71-32; Fax: 34-93-260-72-19; We sequenced the entire coding region and the intron– E-mail: [email protected] exon boundaries of MAX in lung cancer cell lines (Supple- doi: 10.1158/2159-8290.CD-13-0799 mentary Table S1) and found MAX intragenic homozygous ©2013 American Association for Cancer Research. deletions, which caused the complete loss of MAX protein,

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AC E1 E2 E3 E4 E5 -PROBE-1 -PROBE-2 -PROBE-3 -PROBE-5a -PROBE-6 -PROBE-7 -PROBE-8 -Lu165 -H157 -H2170 -H1048 -Control (–) -COR-L47 -COR-L95 -E2 -E3 -E4 -E5 -E1 H1048- E1- 3.5 H1417 3 E2- Lu134- 2.5

E3- 2 NFKBIA-5 BMP4-2 OTX2-5 MAX-8 MAX-7 MAX-6 MAX-5a MAX-3 MAX-2 MAX-1 RDH12-8 NPC2-1 NRXN1* EDAR* PKHD1* SETX* ZNF25* MYBPC3* NOS1* NYO5B* RNASEH2A* KCNJ6* PPIL2* 1.5 H1417- Ratio E4- 1 0.5 E5- 0 3.5 Lu134 3 B 2.5 2 1.5 Ratio 1 -Lu165 -H1417 -LU134 -COR-L95 -HCC33 -H1963 -H69 -H446 -H82 0.5 0 20 kDa- -MAX PT2 3

2.5

-Tubulin 2

1.5 Ratio 1

0.5

D Tumor DNA Matched normal DNA 0 TTG CCC TTTAA TTC G TTTTTTTGC CCA GGC c.296-1G>A 1

0.5 Ratio Normal 0

Figure 1. MAX is genetically inactivated in SCLC. A, PCR products of indicated exons show absence of ampliﬁ cation, indicating the presence of deletions at some exons (E) of the MAX gene in the indicated lung cancer cell lines (underlined). Appropriate positive controls from cells without MAX deletions are also included. B, Western blot analysis of endogenous MAX in the indicated lung cancer cell lines implying the lack of MAX protein in the MAX- mutant cells (underlined). C, top, schematic representation of the structure of MAX , with all its corresponding exons and with the different probes used in the multiplex ligation-dependent probe ampliﬁ cation (MLPA) assay. Probes 6 and 8 are located within alternative exons. Ratio charts of the MLPA depicting the intragenic deletions of various exons in the indicated lung cancer cell lines and in the lung primary tumor PT2. Appropriate controls from normal cells are also included. The black square indicates the location of the probes for the MAX gene. D, chromatogram depicting the indicated nucleotide substitution in a lung primary SCLC. The normal matched DNA is also included.

in H1417, Lu134, Lu165, and COR-L95 cells, all of which with MYC, and which binds to hexameric E-box motifs are of the SCLC type ( Fig. 1A and B ). We also sequenced (CACGTG) in the DNA to activate transcription. The MYC– MAX in primary SCLCs and performed multiplex ligation- MAX heterodimers also indirectly repress the expression dependent probe amplifi cation (MLPA; ref. 12 ) to test for of other genes (13, 14). In keeping with the current view intragenic deletions ( Fig. 1C and D ). We tested the tumor that proteins acting in a common biologic pathway are not xenograft directly derived from the same primary tumor as simultaneously altered in the same tumor specimen ( 1 ), the Lu134 cells and confi rmed the presence of an identical we found that the alterations in MAX and amplifi cation MAX alteration, which ruled out the possibility that the of the MYC genes were mutually exclusive. Moreover, none observation was a cell culture artifact (Supplementary Fig. of the MAX -mutant cells carried concomitant mutations of S1). Overall, we found homozygous and tumor-specifi c BRG1 (Supplementary Table S1), which are also known to MAX-inactivating alterations in about 6% of the 98 SCLCs be mutually exclusive with amplifi cation of the MYC genes tested (Table 1), a prevalence similar to that of the recently in lung cancer ( 15 ). identifi ed CREBBP and EP300 tumor-suppressor genes ( 2 ). All of the tumor specimens were surgically resected before BRG1, an ATPase of the SWI/SNF Chromatin treatment, so that the possibility can be ruled out that Remodeling Complex, Directly Regulates the alterations of MAX are secondary alterations due to chemo- Expression Levels of MAX therapy or radiotherapy. The functional relationship of MYC with both MAX and The MAX protein contains a basic helix–loop–helix BRG1 is well established ( 13, 14 , 16 ). Here, we aimed to elu- zipper domain, which is required to form heterodimers cidate the mechanistic interaction between MAX and BRG1

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MAX Inactivation in Small Cell Lung Cancer RESEARCH ARTICLE

Table 1. List of the MAX alterations found among the 98 SCLCs (53 cell lines and 45 primary tumors) tested

Sample Nucleotide change Exon/intron Protein change LOH Tumor speciﬁ c Other gene mutations H1417 c.(?_-170)_171+?del E1-3 No protein Yes NACDKN2A , RB1 , TP53 Lu134 c.172-?_(*1349_?)del E4-5 No protein Yes NAPTEN , RB1 , TP53 Lu165 c.(?_-170)_(*1349_?)del E1-5 No protein Yes NARB1 , TP53 COR-L95 c.(?_-170)_(*1349_?)del E1-5 No protein Yes NARB1 , TP53 PT-1 c.296-1G>A I4 Unknown Yes YesTP53 a PT-2 c.64-?_295+?del E3-4 No protein Yes YesTP53 a

NOTE: The presence of LOH indicates the homozygous nature of the alterations at MAX . The presence of alterations at other genes is also indicated ( 39 ). Abbreviations: NA, not analyzable; PT, primary tumor. aOnly TP53 , MYC , MYCN , and MYCL have been tested in these samples.

and to explore the role of MAX inactivation in cancer. To this expression of ectopic MAX from the 5′-UTR-MAX was very end, we took advantage of three lung cancer cell lines lack- low in the BRG1-mutant cells, in a hormone-free environ- ing MAX and studied the effects of restoring MAX activity ment, and increased slightly upon the addition of gluco- and depleting BRG1 in these cells ( Fig. 2A ). Reconstitution corticoids. In contrast, neither the status of BRG1 nor the of MAX signifi cantly reduced cell growth in the three cancer presence of glucocorticoids impinged on the levels of MAX cell lines (Fig. 2B and Supplementary Fig. S2A), which is from the construct lacking the 5′-UTR (Supplementary consistent with a previous observation in PC12 cells (10 ) and Fig. S2C). provides evidence of the tumor-suppressor function of MAX. We then examined whether BRG1 is recruited to the Likewise, depletion of BRG1 gave rise to a highly signifi cant MAX promoter. Chromatin from the H1299tr-BRG1wt reduction in cell viability. and H1299tr-BRG1mut cells was precipitated [chromatin We observed that the 5′-untranslated region (5′-UTR) of immunoprecipitation (ChIP)] using a BRG1 antibody. DNA MAX contains putative glucocorticoid response elements. enrichment was measured by quantitative PCR (qPCR) using Glucocorticoids are critical in normal lung differentiation, primer sets fl anking a region of about 3,000 bp. We observed and BRG1 is required to mediate the transcriptional activi- enrichment of BRG1 in a region of about 700 bp within the ties of the glucocorticoid receptor (3 , 16 ). Therefore, we 5′-UTR of MAX (Fig. 2G). These fi ndings demonstrate, for examined whether MAX was responsive to, or involved in, the fi rst time, a direct functional connection between these mediating the response to treatment with glucocorticoids. two tumor suppressors, in virtue of which BRG1, as part of For this purpose, we cloned MAX with the 5′-UTR (hence- the switch/sucrose nonfermentable (SWI/SNF) complex, forth 5′-UTR–MAX; Fig. 2C). Ectopic levels of MAX were facilitates the access of the glucocorticoid receptor to the low in cells infected with 5′-UTR–MAX, in a hormone-free MAX promoter, thereby activating its expression. environment, and became upregulated after treatment with glucocorticoids. In contrast, cells carrying MAX devoid of Depletion of BRG1 Is Preferentially Toxic in the 5′-UTR exhibited high basal levels of MAX, which MAX-Defi cient Cells increased moderately upon treatment with glucocorticoids Depletion of BRG1 dramatically impaired (by >95%) (Fig. 2D). This indicates that the 5′-UTR contains regula- cell viability in the MAX -deficient cells (Fig. 2B and Sup- tory elements that modulate the expression of MAX in plementary Fig. S2A). To test whether this behavior also response to glucocorticoids. took place in cancer cells with wild-type MAX , we depleted Unexpectedly, we found a severe reduction in the levels of BRG1 in a panel of six lung cancer cell lines with amplifi- ectopic MAX after depleting BRG1 in the glucocorticoid- cation of either MYC , MYCL , or MYCN . Only a moderate treated 5′-UTR–MAX cells, implying that the regulation of decrease (<25%) in cell growth was found in some cells, MAX expression through its 5′-UTR is strongly dependent implying that the depletion of BRG1 was preferentially on BRG1 (Fig. 2E). The requirement of BRG1 for transac- toxic in MAX-deficient cells (Fig. 3A). It is of particular tivation of endogenous MAX was verified in several lung note that the MAX–shBRG1 cells, from parental Lu134 cancer cell lines and in neuroblastoma-derived SHSY-5Y and Lu165 cells, were more viable than either the MAX- cells. These exhibited a strong reduction of MAX follow- reconstituted or shBRG1 cells ( Fig. 3B and Fig. 2B ). This ing depletion of BRG1 (Fig. 2F and Supplementary Fig. suggests that MAX restores, to some extent, the cell viabil- S2B). In addition, the findings were reproduced in cells ity that is undermined by shBRG1 , or vice versa . Taken derived from the H1299 lung cancer cells, which are BRG1 together, these results imply the existence of a synthetic deficient. These were the isogenic cells H1299tr-BRG1wt lethal type of interaction between MAX and BRG1, and and H1299tr-BRG1mut, which express the wild-type and raise the possibility of developing a therapeutic strategy a mutant version of BRG1, respectively ( 16 ). The level of for patients with MAX -deficient tumors.

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∅ A Parental B MAX LU134 shBRG1 10 5 Scramble

LU134 LU165 ∅ Scramble MAX GFP 5 *** *** Scramble PURO ∅ GFP shBRG1 PURO MAX sh BRG1

Relative no. of viable cells of viable no. Relative 0 0 0 5 10 15 0510 15 Days Days ∅ MAX shBRG1 C CMV 5′-UTR MAX 3′-UTR IRES ZsGreen -H1417 -LU134 -LU165 -H1417 -LU134 -LU165 -H1417 -LU134 -LU165 ATG TAA MAX-

BRG1-

Tubulin- –81 –26 –8 +1

D FGScramble shBRG1 ∅ 5′-UTR–MAX MAX H1299tr-BRG1mut GC (μmol/L) HF 0.5 1 2 HF 0.5 1 2 10 GC (μmol/L) HF 0.5 2 HF 0.5 2 HF 0.5 2 H1299tr-BRG1wt MAX- MAX- Tubulin- BRG1- 5

Lu134 Tubulin- % Input E H460 HF GC 5′-UTR–MAX/ MAX– 0 ∅ 5′-UTR–MAX shBRG1 MAX shBRG1 TSS –1,500 +1,200 –372 +562 -H1417 -LU134 -LU165 -H1417 -LU134 -LU165 -H1417 -LU134 -LU165 -H1417 -LU134 -LU165 -H1417 -LU134 -LU165 -Scramble -sh BRG1 #1 -sh BRG1 #4 -Parental -Scramble -sh BRG1 #1 -sh BRG1 #4 MAX- MAX- MAX- BRG1- BRG1- Input- Tubulin- Tubulin- SH-SY5Y

Figure 2. Effects of MAX reconstitution and of BRG1 depletion in lung cancer cells. A, top, schematic depiction of the experimental design using different lentiviral constructs expressing human MAX , and the shRNA targeting BRG1 (shBRG1 ). Control (∅) and scramble RNAs are also shown. The shBRG1 used were validated in our previous study (16 ). Bottom, Western blot analyses, from total lysates, depicting MAX and BRG1 in the indicated cells. Tubulin is shown as a loading control. B, left, cell proliferation measured using MTT assays. Lines, the number of viable cells relative to the total number of cells at 0 hours. Error bars, SD. ***, P < 0.0005. Right, images of the MTT assays. C, schematic representation of the 5′-UTR–MAX construct. The putative glucocorticoid receptor–binding region is highlighted in green. D, Western blot analysis, from total lysates, depicting the ectopic expression of MAX from the 5′-UTR–MAX construct and from that lacking the 5′-UTR (MAX) in cells cultured in hormone-free (HF) medium or at the indicated glucocorticoid (GC) concentrations. E, Western blot analysis, from total lysates, showing the levels of ectopic expression of MAX and coexpression of MAX–shBRG1 and 5′-UTR–MAX/shBRG1 in the indicated cell lines. F, reduction of levels of endogenous MAX, after depletion of BRG1, in one MYC -ampliﬁ ed lung cancer cell line (H460) and in the neuroblastoma-derived SHSY-5Y cells, treated with 2 μmol/L of glucocorticoid. In the latter, two sh BRG1s (#1 and #4) have been used. G, ChIP of BRG1 in the indicated cells after inducing BRG1 expression with doxycycline, followed by qPCR to determine DNA enrichment in the MAX promoter, relative to the input. The bars represent the data for the BRG1 ChIP in H1299tr-BRG1wt and the H1299tr-BRG1mt cells, as indicated. Error bars, SDs of three replicates. Under the graph there is a schematic representation of the region screened and the position (in bp) of each amplicon (vertical lines) relative to the ATG (+1). TSS, transcription start site. The bottom corresponds to the 2% agarose gel of the qPCR of the top, shown for comparison.

MAX Requires BRG1 to Activate Neuroendocrine refl ects the activation of prodifferentiation programs. Recon- Transcriptional Programs and to Upregulate MYC stitution of MAX also upregulated genes encoding glycolysis Targets Such as Glycolysis-Related Genes enzymes (e.g., LDHA , HK2 , PDK1 , PKM2 , and PGK1 ), which To elucidate the functional relationship between MAX are targets of MYC. Conversely, the levels of these glycolysis- and BRG1, we performed global gene expression analysis in related genes were lower in the shBRG1 cells than in the con- the ectopic MAX and shBRG1 cell models. On the basis of trol cells (Fig. 4A), whereas the MAX–shBRG1 cells showed a their Gene Ontology (GO) function, reconstitution of MAX reversion in the expression of glycolysis- and neural-related showed a signifi cant enrichment of transcripts related to genes toward the profi le of the control cells ( Fig. 4B ). neural differentiation and to glycolysis/carbohydrate metab- Most of the MYC targets that were upregulated by MAX olism (Supplementary Fig. S3). Some of these transcripts are were inversely associated with the gene expression profi le well-established targets of MYC (Fig. 4A; refs. 17, 18). Because of sh BRG1 cells (Fig. 4C). The MAX signature was also SCLCs have neuroendocrine features ( 6 , 11 ), the upregulation negatively correlated with that of embryonic lungs from mice of neural-related genes upon MAX reconstitution possibly overexpressing Nmyc and Cmyc , whereas there was a direct

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A -Scramble -sh BRG1 -Parental -Scramble -sh BRG1 -Parental -Parental -Scramble -sh BRG1 BRG1- BRG1- BRG1- Tubulin- Tubulin- 12 Tubulin- 12 15

NS NS 8 8 10 NS

Relative no. Relative 4 4 5 of viable cells of viable H446-scr H460-scr BRG1 H82-scr H446-sh H460-shBRG1 H82-shBRG1 0 0 051015 0 05100 51015 -Parental -Scramble -sh BRG1 -Parental -Scramble -sh BRG1 -Parental -Scramble -sh BRG1 BRG1- BRG1- BRG1-

Tubulin- Tubulin- Tubulin- 12 12 6 * * * 8 8 3

Relative no. Relative 4 4 H69-scr of viable cells of viable H1963-scr HCC33-scr H69-shBRG1 H1963-shBRG1 HCC33-shBRG1 0 0 0 0 5 10 15 05100 5 10 15 Days Days Days

B ∅ 5′-UTR– 5′-UTR–MAX/ 5′-UTR–MAX ∅ MAX shBRG1 9 5′-UTR–MAX/shBRG1 5

4 LU134 LU165 6 ** 3 **

2 Relative no. Relative

of viable cells of viable 3 ** ** 1

00 051015 0 5 10 15 Days Days

Figure 3. Effects of BRG1 depletion in the proliferation of lung cancer cells with amplifi cation at the MYC family of genes and in MAX -defi cient cells, after reconstitution of MAX. A, MTT assay to determine viability, after depleting BRG1 and in the scramble (scr) control, of cells carrying amplifi cation of MYC (H446, H460, and H82), MYCN (H69), or MYCL (HCC33 and H1963). Lines, the number of viable cells relative to the total number of cells at 0 hours. Error bars, SD. NS, not signifi cant; *, P < 0.05; **, P < 0.005. B, left, MTT assays of MAX-defi cient cells carrying the indicated constructs. Right, images of the MTT assay of Lu134 cells.

association with the expression proﬁ le after BRG1 reconstitu- Genetic Inactivation of the MAX Dimerization tion in lung cancer cells (Fig. 4C; ref. 16 ). These observations Protein, MGA, in Lung Cancers with Wild-Type imply that BRG1, MAX, and MYC orchestrate the transcrip- Components of the SWI/SNF or MYC Pathways tional regulation of a common set of genes, and suggest that MAX interacts not only with MYC, but also with other MYC represses cell differentiation–related transcripts in a BHLHZ-containing proteins (i.e., MXD1, MXI1, MXD3, MAX-independent manner. MXD4, MNT, and MGA), which antagonize the transcriptional

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A CARD10 B

PARP14

NDRG1

GDF15

VEGFA TRIB3 SMARCA2

HK2 2

A N

CXCR4 O

P LDH RT S AATK CXXC4 TE MXD DLX6

NOTCH1 1 –5 5

EGR1 -Lu134- ∅ -Lu134 MAX -Lu134 sh BRG1 -Lu134-MAX/sh BRG1 HK2 hexokinase 2 (muscle) PFKFB3 PARP12 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 PDK1 pyruvate dehydrogenase kinase, isozyme 1 PFKFB4 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 LDHA lactate dehydrogenase A PGK1 phosphoglycerate kinase 1 FO S ENO2 enolase 2 (gamma, neuronal) ADCY4 ALDOA aldolase A, fructose-bisphosphate ENO2 PGM1 phosphoglucomutase 1 EGR4 Glycolisis-related PKM2 pyruvate kinase, muscle NEUR OD1 –4 logFC +11 APOE hypothetical LOC100129500; apolipoprotein E HSPG2 heparan sulfate proteoglycan 2 PCP4 Purkinje cell protein 4 Neural related SPON2 spondin 2, extracellular matrix protein MAX BRG1 MYC/MAX and NMYC sh ADM2 targets adrenomedullin 2 MAPT microtubule-associated protein tau Glycolis and metabolism MAL of carbohydrates CSPG5 chondroitin sulfate proteoglycan 5 (neuroglycan C) Apoptosis and cell MAL mal, T-cell differentiation protein death FGF11 fibroblast growth factor 11 CXCR4 chemokine (C-X-C motif) receptor 4 SMAD6 AHNAK AHNAK nucleoprotein PKM2 LAMB2 laminin, beta 2 (laminin S) ODZ1 odz, odd Oz/ten-m homolog 1 (Drosophila) MYC NES nestin MYC N ISL1 ISL LIM homeobox 1 Neural-related N HEY1 OTC hairy/enhancer-of-split related with YRPW motif 1 H SLITRK6 SLIT and NTRK-like family, member 6 2 LHX2 LIM homeobox 2 GAL3ST1 galactose-3-O-sulfotransferase 1 VIM SEMA3B semaphorin 3B SMAD4 EMX1 empty spiracles homeobox 1 DLX6 distal-less homeobox 6 SMAD9 DLG4 disce, large homolog 4 (Drosophila) A CLDN1 claudin 1 LDOA BMF EPOR GK1 erythropoietin receptor GM1 P P UCN urocortin

BRG1 C MAX upregulated vs. Lu134sh shBRG1 downregulated vs. Lu134MAX MAX upregulated vs. dataset GSE10954 expression profile as reference dataset expression profile as reference dataset from lungs of mice overexpressing cMyc

FDR < 0.01 FDR < 0.0001 FDR < 0.0001 NES = –1.455 NES = 2.593 NES = –1.583

‘na_pos’ (positively correlated) ‘na_pos’ (positively correlated) ‘na_pos’ (positively correlated)

Zero cross at 18796 Zero cross at 24430 Zero cross at 8278

‘na_neg’ (negatively correlated) ‘na_neg’ (negatively correlated) ‘na_neg’ (negatively correlated)

MAX upregulated vs. dataset GSE6077 MAX upregulated vs. dataset shBRG1 downregulated vs. dataset from lungs of mice overexpressing Nmyc GSE35168 from H1299tr-BRG1wt cells GSE35168 from H1299tr-BRG1wt cells

FDR < 0.0001 FDR < 0.0001 FDR < 0.0476 NES = –2.008 NES = 1.22 NES = 1.09

‘na_pos’ (positively correlated) ‘na_pos’ (positively correlated) ‘na_pos’ (positively correlated)

Zero cross at 12985 Zero cross at 12895 Zero cross at 5383

‘na_neg’ (negatively correlated) ‘na_neg’ (negatively correlated) ‘na_neg’ (negatively correlated)

Figure 4. Gene expression proﬁ les of 5′-UTR–MAX and of shBRG1 -expressing cells. A, circos plot of the heatmap of the approximately 2,030 transcripts that constitute the MAX- and shBRG1 -gene expression signatures (Supplementary Tables S2 and S3). The GO categories for those genes associated with neural development, glucose metabolism, targets of MYC, and apoptosis are indicated in orange, light blue, pink, and gray, respectively. Selected genes from these GO categories are highlighted in blue in the outer part of the circle. B, expression heatmap for genes in the indicated GO categories (from Supplementary Table S2). Gene expression of Lu134-derived cells. Controls (∅), 5′-UTR–MAX, shBRG1 , and 5′-UTR– MAX/sh BRG1 . C, graph of the ranked gene lists derived from the comparison (using gene set enrichment analysis) of the indicated datasets and gene lists. Probabilities and FDRs are indicated. NES, normalized enrichment score.

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ABc.6870delG c.2188+G>A Cell line Type Protein change Resequenced (p.R2291QfsX44) (p.V730_splice) T CCCTTA CCC TTTTAACC TTT H2291 NSCLC R2291QfsX44 Yes H650 NSCLC p.G809_splice No

LOUNH91 NSCLC p.V730_splice Ye s H2291 LOU-NH91

CORL279 SCLC p.R1053* No TTCCCC A CCCT TTTAA TTTCCC DMS153 SCLC Hom. deletion No Control Control

C MYC BRG1 MYCL MGA MYCN ARID1A MAX SCLC SMARCB1 NSCLC PBRM1 0102030

Figure 5. Mutation proﬁ le of MGA and of MYC - and BRG1-related genes in lung cancer. A, inactivating MGA alterations in lung cancer cell lines (from http://www.broadinstitute.org/ccle/ ). The last column indicates whether MGA has been resequenced in our laboratory. B, chromatogram depicting mutations at MGA in the indicated lung cancer cell lines. Normal controls are also included. Arrows indicate the nucleotide changes. C, schematic representation of the co-occurrence analysis of alterations at the indicated genes in a panel of 180 lung cancer cell lines. Information has been gathered from different sources, including our current and previous ( 1 , 15 ) results, and publicly available databases (i.e., http://cancer.sanger.ac.uk/ and http://www .broadinstitute.org/ccle/). Red and white squares indicate the presence and absence of alterations, respectively. Gray squares indicate that no data were available. Bar graph, left, indicates the frequency of alterations and the distribution between the two main lung cancer types. A detailed list including the identity of each cell line and exhaustive information on speciﬁ c mutations is provided in Supplementary Table S4.

control of MYC, at the same E-box target DNA sequences pheochromocytoma, a tumor with neuroendocrine features ( 14 ). Some of these MAX-binding proteins promote differ- (9 ). This is interesting because MAX inactivation was found entiation in vivo , block cellular growth and MYC-induced only in SCLC and not in the NSCLC type, the latter com- transformation, and suppress the development of cancer prising about 80% of the lung cancers. Taking into account (19 ). Furthermore, apart from BRG1 , genes coding for other that, unlike NSCLCs, SCLCs have neuroendocrine features, components of the SWI/SNF complex are altered in cancers, it can be hypothesized that MAX is preferentially mutated in including lung cancer (20, 21). We have exhaustively searched neuroendocrine-related malignancies. public databases for gene alterations in partners of MAX in None of the MAX-mutant cells carried concomitant ampli- all types of lung cancer and found inactivation of the MAX fi cation at the MYC family of genes or mutations at BRG1. dimerization protein, MGA , in lung cancer cell lines ( Fig. 5A ) In lung cancer, genetic inactivation of BRG1 is mutually and in lung primary tumors ( http://www.cbioportal.org/ ) exclusive with amplifi cation of MYC genes, which is consist- of both the NSCLC and SCLC type. We have tested a panel ent with the biologic connection between these two cancer of these cell lines and confi rmed the alterations, most of proteins (15 ). BRG1 encodes one ATPase of the SWI/SNF which are homozygous and predictive of truncated proteins chromatin remodeling complex and is involved in the tran- (Fig. 5B). Moreover, inactivation of MGA and alterations at scriptional control of various cell processes, such as embry- different members of the SWI/SNF complex at MAX and MYC onic development and cell differentiation (23, 24). Some were mutually exclusive in lung cancer (Fig. 5C). of these activities also require the complex to interact with nuclear receptors ( 25 ). The functional relationship of MYC with BRG1 and with the SWI/SNF complex is well estab- DISCUSSION lished. For example, MYC physically interacts with the SWI/ We report the discovery of the recurrent inactivation SNF component, SMARCB1 (26 ), and BRG1 is required to of MAX in SCLC. The alterations were tumor-specifi c and regulate the expression of MYC and MYC target genes ( 16 , homozygous, leaving little doubt that MAX constitutes a 27 ). In tumors carrying BRG1 mutations, this regulation is bona fi de tumor-suppressor gene. MAX -inactivating altera- abolished, thereby preventing cell differentiation and pro- tions occurred in about 6% of the SCLCs, a prevalence similar moting cell growth ( 16 ). Here, we provide evidence that BRG1 to that of the recently identifi ed CREBBP and EP300 tumor- also regulates the levels of MAX , stimulated by the presence of suppressor genes ( 2 ). Most alterations of MAX were intra- glucocorticoids. In virtue of this regulation, and as part of the genic deletions, which may explain why inactivation of MAX SWI/SNF complex, BRG1 would be directly recruited to the has not been picked up in recent exome or whole-genome 5′-UTR of MAX to facilitate the access of the glucocorticoid sequencing of SCLCs ( 2 , 22 ). Germline-inactivating muta- receptor to the MAX promoter, thereby activating its expres- tions at MAX were found to be responsible for hereditary sion. This constitutes the fi rst demonstration of a direct

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functional connection between these two tumor suppressors. intriguing that the reconstitution of a tumor suppressor, In addition to glucocorticoid receptor, the SWI/SNF complex MAX, activates the expression of genes that are typically interacts with other nuclear receptors, such as estrogens, upregulated in cancer cells. Although additional research is retinoic acid, and vitamin D3 receptors ( 25 , 28 , 29 ). There- likely to provide an explanation for these observations, we fore, we cannot rule out the possibility that the activation of can speculate in the meantime that a substantial increase these other receptors also infl uences the expression levels of (i.e., after reconstitution of MAX) or decrease (i.e., after MAX in an SWI/SNF–dependent manner. depletion of BRG1) of glycolysis enzymes, in a predomi- We also noted that depletion of BRG1 triggered a strong nantly tumor-genetic context, triggers an energetic imbal- inhibition of lung cancer cell growth. The effect was very ance, with dire consequences for cell viability. In support of specifi c to the MAX -defi cient cells, as growth was not affected this concept, the rescue of cell viability observed in MAX– after depleting BRG1 in a panel of lung cancer cells with sh BRG1 cells but not MAX-reconstituted or BRG1-depleted amplifi ed MYC genes. Regardless of the mechanism underly- cells is accompanied by a reversion in the expression of ing this specifi c and dramatic impairment in the growth of glycolysis- and neural-related genes toward the profi le of MAX-defi cient cells after BRG1 depletion, it is important the control cells. to highlight its potential clinical relevance. This informa- MAX was believed to be essential to the oncogenic function suggests therapeutic possibilities for the treatment of tion of MYC ( 30 ). However, in Drosophila , an Myc mutant patients with SCLC, and possibly for patients with pheochro- lacking the Max-interaction domain still retained partial mocytoma, with MAX-defi cient tumors. activity ( 31 ). These observations, coupled with the existence The reconstitution of MAX in SCLC cells triggered the of MAX inactivation in pheochromocytomas (9 ) and now in expression of neural-related and glycolysis/carbohydrate SCLC, imply that some MYC activities are independent of its metabolism-related transcripts, some of which are well- heterodimerization with MAX. Here, we found that most of established targets of MYC ( 17, 18 ). Because it has been the MYC targets that were upregulated by MAX were inversely speculated that some SCLCs arise from neuroendocrine associated with the gene expression signatures of shBRG1 cells ( 4 ), the upregulation of neural-related genes upon cells and of embryonic lungs from mice overexpressing Nmyc MAX reconstitution may refl ect the activation of prodif- and Cmyc . In contrast, there was a direct association with the ferentiation programs. Genes encoding glycolysis enzymes expression profi le after BRG1 reconstitution in lung cancer (e.g., LDHA , HK2 , PDK1 , PKM2 , and PGK1 ) are targets of cells (16 ). These fi ndings imply that BRG1, MAX, and MYC MYC and are commonly overexpressed in cancer cells ( 18 ). orchestrate the transcriptional regulation of a common set Conversely, the levels of these glycolysis-related genes were of genes and suggest that MYC represses cell differentiation– lower in the shBRG1 cells than in the control cells. It is related transcripts in a MAX-independent manner. Figure 6

SWI/SNF X MYC -Low levels of any of the MYC genes. Normal differentiated MAX MAX -Chromatin accessibility in prodifferentiation genes. cell Closed chromatin -MAX-mediated transactivation of prodifferentiation genes. -No MYC-mediated transactivation of stemness-related genes

SWI/SNF MYC MYC X MYC -Moderate levels of any of the MYC genes. MAX-deficient MYC -Chromatin accessibility. -No MAX-mediated transactivation of prodifferentiation genes -MYC-mediated transactivation of stemness-related genes.

MYC MYC MAX MAX MYC MYC -Moderately high levels of any of the MYC genes. X MYC -No chromatin accessibility in prodifferentiation genes. BRG1 (or SWI/SNF) MYC -No MAX-mediated transactivation of prodifferentiation genes. -MYC-mediated transactivation of stemness-related genes. -deficient Closed chromatin

MYC MYC MYC MYC MYC MYC MAX MYC MYC MYC MAX -Very high levels of any of the genes. MYC MYC MYC -Chromatin accessibility. SWI/SNF -MYC-mediated repression of prodifferentiation genes. X MYC -MYC-mediated transactivation of stemness-related genes. MYC-amplified MYC MYC MYC

Figure 6. Schematic model for depicting some possible scenarios of the interplay among BRG1 (or the SWI/SNF complex), MYC, and MAX to regulate the transcriptional programs of normal differentiated cells and for cancer cells with alterations at the indicated genes. White boxes, genes involved in cell differentiation; black boxes, genes involved in cell stemness; blue circles, MAX-interacting proteins.

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depicts a model that speculates about the different scenarios homozygous deletions at MAX in the lung primary tumors. The that could take place in the context of MAX , MYC , or BRG1 MLPA protocol used here has been described elsewhere (12 ). In genetic alterations in a cancer cell ( Fig. 6 ). short, 100 ng of genomic DNA was denatured for 5 minutes at 98°C, μ Finally, through a search of public cancer databases, we after which 3 L of the probe mixture was added. The sample DNA ° discovered recurrent inactivation at the MAX dimerization and P429-A1 MLPA probe mixture was heated at 95 C for 1 minute and then incubated at 60°C for 16 hours, followed by a ligation protein, MGA, which was mutually exclusive with alterations step (Ligase-65 enzyme; MRC-Holland). Subsequently, a 10-μL mix- of members of the SWI/SNF complex and with MAX and ture was added containing deoxynucleotide triphosphates (dNTP), MYC. Thus, MGA is a tumor-suppressor gene in lung cancer Taq polymerase, and one unlabeled and one 6-carboxyfl uorescein and is probably another cog on the SWI/SNF–MYC func- amidite–labeled (FAM) universal PCR primer in each reaction. PCR tional axis. In this regard, it is important to highlight that was carried out for 35 cycles, and the fragments were analyzed with in recent RNA interference screening, Brg1 , Max , and Mga an ABI model 3130XL capillary sequencer (Applied Biosystems) were identifi ed as repressors of germ-cell gene expression in using GeneScan 500 LIZ size standard (Applied Biosystems). MLPA murine embryonic stem cells. This is additional evidence of fragment analysis and comparative analysis were performed using a functional connection of these proteins with the control of CoffalyserNET software (34 ). cell differentiation ( 32 ). Expression Vectors and Lentiviral Production In conclusion, our current genetic and molecular fi ndings provide powerful evidence that MAX is a tumor-suppressor Two main forms of MAX have been described: the short and gene involved in SCLC development. We propose that the long forms, containing 151 and 160 amino acids, respectively (35 ). These arise by alternate mRNA splicing that inserts nine amino abnormal function of the SWI/SNF–MYC axis, arising from acids between residues 12 and 13 of the short form of MAX. Here, genetic alterations of any of its components, prevents cell dif- we cloned the short form (NM_145112.2) because it was the more ferentiation and constitutes an acquired ability of the cancer abundant transcript in the tumors and lung cancer cell lines tested cells that should be added to the Hanahan and Weinberg and was functionally equivalent to the long form. Human wild-type model (33 ). MAX was cloned into the Eco RI and Not I restriction sites of the vector pLVX–IRES–ZsGreen1 (Clontech Laboratories, Inc.). All constructs were verifi ed by automatic sequencing. Short hairpin RNAs (shRNA) METHODS were purchased from SIGMA-MISSION (LentiExpress Technology; Lung Tumor Specimens and Cancer Cell Lines Sigma-Aldrich) as a glycerol stock of 5 pLKO plasmids carrying Lung cancer cell lines representative of the most common lung BRG1-specifi c shRNA sequences. Two of these shRNAs had previ- histopathologies were included (Supplementary Table S1). Cell ously been shown to deplete BRG1 expression effi ciently and specifi - lines were obtained from the American Type Culture Collection, cally (depleted BRG1 but not BRM expression; ref. 16 ). A scramble grown under recommended conditions, and maintained at 37°C in shRNA (Sigma MISSION shRNA non-mammalian control SHC002) was used as a control. The lentiviruses were generated within the a humidifi ed atmosphere of 5% CO 2 /95% air. All cells tested negative for Mycoplasma infection. The cells lines were authenticated by geno- 293T packaging cells. Lentiviruses carrying MAX constructs were typing for TP53 and other known mutations (e.g., BRG1 /SMARCA4 generated using Lenti-X (LentiExpress Technology; Clontech Labora- and STK11). The genotyping of the mutations was done before tories), following the manufacturer’s recommendations. The lentivi- starting the experiments or, in some particular cases, also during the rus carrying the shBRG1 and scrambled shRNAs, were cotransfected experimental part of the study. The genotyping was done using direct with each construct and the packaging plasmids psPAX and pMD2.6 Sanger sequencing of PCR products, as indicated previously (1 ). The (Sigma-Aldrich). After 48 hours, 293T fi ltered supernatants were col- mutations found were in agreement with those provided in public lected and subconfl uent cells were infected with harvested virus and databases, as indicated in Supplementary Tables. Most samples were selected with puromycin for 72/96 hours. from the National Cancer Center Biobank at the National Cancer Center Hospital (Tokyo, Japan). Four samples were obtained from ChIP Assays the Biobanco del SSPA (Sistema Sanitario Público de Andalucía, ChIP assays were performed as previously described ( 16 ). Briefl y, Spain). Genomic DNA and total RNA were extracted by standard preliminary fi xation experiments were performed over a predeter- protocols. The study was approved by the relevant Institutional mined period. Cells were then fi xed in 1% formaldehyde for 10 Review Boards and ethics committees. minutes and fi nal conditions were chosen that yielded the best combi- nation of in vivo fi xed chromatin, high DNA recovery, and small aver- Screening for MAX Gene Alterations: Sanger Direct age size of chromatin fragments (an average length of 0.25–1.00 kb). Sequencing and MLPA Three independent ChIP experiments were performed. qPCRs were To extract genomic DNA, freshly frozen tissue from tumors was performed using SYBR Green Master Mix (Applied Biosystems). meticulously dissected to ensure enriched material containing at least Relative enrichment was determined from a standard curve of serial 40% tumor cells. Approximately 10- to 20-μm sections were collected dilutions of input samples. For semi-qPCR, amplifi cations were μ and placed in 1% SDS/proteinase K (10 mg/mL) at 58°C overnight. performed with 30 cycles in a total volume of 25 L and run on 2% Digested tissue was then subjected to phenol–chloroform extrac- agarose gels. qPCR was performed using Power SYBR Green Master tion and ethanol precipitation following standard protocols. For Mix (Applied Biosystems). The sequences of primer sets used in each mutation screening of the MAX gene, exons 1 to 5 (NM_002382.3) case are available upon request. and MGA (exons 1–23; NM_001164273.1) were amplifi ed from 30 ng of genomic DNA extracted from all tumors. Cycle sequencing Antibodies and Western Blot Analyses of PCR products was carried out using Big Dye Terminator chem- The following primary antibodies were used for Western blot istry (Applied Biosystems) with an ABI PRISM 3700 DNA Analyzer analyses: polyclonal anti-MAX, sc-197 antibody (Santa Cruz Bio- (PerkinElmer Life Sciences, Inc.). All of the variants identifi ed in the technology), polyclonal anti-BRG1, H88 (1:1,000; Santa Cruz Bio- study were confi rmed by resequencing of independent PCR products. technology), anti–C-MYC 9E10 (1:500; Santa Cruz Biotechnology), MLPA technique was used to determine the presence of intragenic anti-tubulin (T6199 mouse; Sigma-Aldrich), and anti–β-actin (13854;

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Sigma-Aldrich). For Western blot analyses, whole-cell lysates were gene set enrichment analysis (GSEA; ref. 37 ) using the indicated gene collected in a buffer containing 2% SDS, 50 mmol/L Tris–HCl (pH expression signatures as the gene set. After Kolmogorov–Smirnov 7.4), 10% glycerol, and protease inhibitor cocktail (Roche Applied testing, our gene sets were considered signifi cantly enriched between Science). Protein concentrations were determined using a Bio-Rad comparison classes for values of FDR < 0.25, a widely accepted cutoff DC Protein Assay kit (Life Science Research). Equal amounts of for the identifi cation of biologically relevant gene sets ( 38 ). lysates (20 μg) were separated by SDS-PAGE and transferred to a polyvinylidene difl uoride (PVDF) membrane that was blocked with Disclosure of Potential Confl icts of Interest 5% nonfat dry milk. Membranes were incubated with the primary S. Savola is employed by MRC-Holland, manufacturer of commer- antibody overnight at 4°C, and then washed before incubation with cially available MLPA probemixes. No potential confl icts of interest species-appropriate horseradish peroxidase (HRP)–conjugated sec- were disclosed by the other authors. ondary antibodies for 1 hour at room temperature. Authors’ Contributions Treatments Conception and design: O.A. Romero, M. Sanchez-Cespedes Dexamethasone was used for glucocorticoid treatment. Cells were Development of methodology: O.A. Romero, M. Torres-Diz, fi rst depleted of FBS and subjected to a hormone-free medium by E. Pros, S. Savola, A. Villanueva transfer to 10% charcoal-dextran–treated, heat-inactivated FBS for Acquisition of data (provided animals, acquired and managed 24 hours before hormone treatment (16 ). Cells were then treated for patients, provided facilities, etc.): O.A. Romero, M. Torres-Diz, 24 to 72 hours with the indicated concentrations of dexamethasone C. Saez, L.M. Montuenga, T. Kohno before harvesting. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): O.A. Romero, M. Torres-Diz, MTT Assay E. Pros, A. Gomez, S. Moran, A. Villanueva, M. Sanchez-Cespedes Writing, review, and/or revision of the manuscript: O.A. Romero, μ For cell viability assays, 10 L of a solution of 5 mg/mL MTT M. Torres-Diz, S. Savola, C. Saez, L.M. Montuenga, M. Sanchez- (Sigma Chemical Co.) was added. After incubation for 3 hours at Cespedes ° 37 C, the medium was discarded, the formazan crystals that had Administrative, technical, or material support (i.e., reporting or μ formed were dissolved in 100 L dimethyl sulfoxide (DMSO), and organizing data, constructing databases): O.A. Romero, M. Torres-Diz, absorbance was measured at 596 nm. Results are presented as the E. Pros, S. Savola, R. Iwakawa, J. Yokota, M. Sanchez-Cespedes median of at least two independent experiments performed in tripli- Study supervision: O.A. Romero, M. Sanchez-Cespedes cate for each cell line and for each condition. Acknowledgments Microarray Global Gene Expression Analysis The authors thank Patricia Cabral and Sara Verdura (Genes and RNA (100 ng) was used for the gene expression microarray analy- Cancer Group) at IDIBELL for technical assistance and HaciAli Yigit- sis. RNA integrity values were in the range of 9.0 to 10.0 when top for performing the MLPA experiments. assayed by Lab-chip technology in an Agilent 2100 Bioanalyzer. For labeling, we used a commercial “One-Color Microarray-Based Gene Grant Support Expression Analysis” version 5.5 kit and followed the manufacturer’s This work has been supported by the Spanish Ministry of Econ- instructions (Agilent manual G4140-90050, February 2007). Hybridi- omy and Competitiveness (grant SAF2011-22897 to M. Sanchez- zation was performed on the Human Gene Expression v2 microarray Cespedes), the Red Temática de Investigación del Cáncer-RTICCs × 8 60K (Agilent microarray design ID 014850, P/N G4112F). For (RD12/0036/0045 to M. Sanchez-Cespedes and RD12/0036/0040 scanning, we used a G2505B DNA microarray scanner. Images were to L.M. Montuenga), Red de Biobancos (RD09/0076/00085) and the quantifi ed using Agilent Feature Extraction Software (v. 9.5). The European Community’s Seventh Framework Programme (FP7/2007- cells used for microarray gene expression analysis were as follows: 13 to M. Sanchez-Cespedes, L.M. Montuenga, and S. Savola), under (i) control cells: H1417, Lu134, and Lu165 containing the control grant agreement no. HEALTH-F2-2010-258677–CURELUNG. Addi- ′ vector; (ii) H1417, Lu134, and Lu165, each containing 5 -UTR– tional support came from a Grant-in-Aid from the Ministry of MAX; (iii) H1417, Lu134, and Lu165, each expressing shBRG1 ; and Health, Labor and Welfare for the Third-term Comprehensive 10-year ′ (iv) Lu134, expressing both 5 -UTR–MAX and sh BRG1. To generate Strategy for Cancer Control, Japan, to T. Kohno and J. Yokota. M. the lists of upregulated and downregulated transcripts for each Torres-Diz is the recipient of a Fellowship (FPI) from the Spanish condition, we chose the following criteria: (i) transcripts induced Ministry of Economy and Competitiveness. or repressed by a factor of at least 1.5 under each MAX or shBRG1 condition with respect to their matched cell line carrying the empty Received October 28, 2013; revised December 13, 2013; accepted vector, and (ii) statistical signifi cance (see below). The genes are listed December 16, 2013; published OnlineFirst December 20, 2013. in Supplementary Tables S2 and S3.

Statistical and Bioinformatic Analysis REFERENCES Data were analyzed using a χ 2 test or a two-tailed Student unpaired- 1. Blanco R , Iwakawa R , Tang M , Kohno T , Angulo B , Pio R , et al. A samples t test. Group differences were presented as mean and SDs. gene-alteration profi le of human lung cancer cell lines . Hum Mut Differences were considered statistically signifi cant for any value of P 2009 ; 30 : 1199 – 206 . < 0.05. Expression data were analyzed using the R program. Raw data 2. Peifer M , Fernández-Cuesta L , Sos ML , George J , Seidel D , Kasper LH , were normalized by the robust multiarray average (RMA) algorithm et al. Integrative genome analyses identify key somatic driver muta- available in Bioconductor’s affy package (36 ). We used limma package tions of small-cell lung cancer. Nat Genet 2012 ; 44 : 1104 – 10 . to obtain limma-moderated t statistics to derive the signatures of dif- 3. Maeda Y , Davé V , Whitsett JA . Transcriptional control of lung mor- ferentially expressed genes. Test P values were adjusted with respect phogenesis. Physiol Rev 2007 ; 87 : 219 – 44 . to the false discovery rate (FDR). Test statistics with an associated 4. Sutherland KD , Proost N , Brouns I , Adriaensen D , Song JY , Berns A . value of P < 0.05 were considered to be statistically signifi cant. The Cell of origin of small cell lung cancer: inactivation of Trp53 and Rb1 lists of genes (ranked by the n -fold values of change) were subjected to in distinct cell types of adult mouse lung. Cancer Cell 2011 ; 19 : 754 – 64 .

302 | CANCER DISCOVERYMARCH 2014 www.aacrjournals.org

MAX Inactivation in Small Cell Lung Cancer RESEARCH ARTICLE

5. Bangur CS , Switzer A , Fan L , Marton MJ , Meyer MR , Wang T . Iden- 23. Seo S , Richardson GA , Kroll KL . The SWI/SNF chromatin remodeling tifi cation of genes over-expressed in small cell lung carcinoma using protein Brg1 is required for vertebrate neurogenesis and mediates suppression subtractive hybridization and cDNA microarray expres- transactivation of Ngn and NeuroD. Development 2005 ; 132 : 105 – 51 . sion analysis. Oncogene 2002 ; 21 : 3814 – 25 . 24. De la Serna IL , Carlson KA , Imbalzano AN . Mammalian SWI/SNF 6. Castillo SD , Matheu A , Mariani N , Carretero J , Lopez-Rios F , L ovell- complexes promote MyoD-mediated muscle differentiation . Nat Badge R , et al. Novel transcriptional targets of the SRY-HMG box Genet 2001 ; 27 : 187 – 90 . transcription factor SOX4 link its expression to the development of 25. Chiba H , Muramatsu M , Nomoto A , Kato H . Two human homo- small cell lung cancer. Cancer Res 2012 ; 72 : 176 – 86 . logues of Saccharomyces cerevisiae SWI2/SNF2 and Drosophila brahma are 7. Lee WH , Murphree AL , Benedict WF . Expression and amplifi cation transcriptional coactivators cooperating with the estrogen receptor of the N-myc gene in primary retinoblastoma. Nature 1984 ; 309 : and the retinoic acid receptor. Nucleic Acids Res 1994 ; 22 : 1815 – 20 . 458 – 60 . 26. Cheng SW , Davies KP , Yung E , Beltran RJ , Yu J , Kalpana GV . c-MYC 8. Small MB , Hay N , Schwab M , Bishop JM . Neoplastic transformation interacts with INI1/hSNF5 and requires the SWI/SNF complex for by the human gene N-myc. Mol Cell Biol 1987 ; 7 : 1638 – 45 . transactivation function. Nat Genet 1999 ; 22 : 102 – 5 . 9. Comino-Méndez I , Gracia-Aznárez FJ , Schiavi F , Landa I , Leandro- 27. Pal S , Yun R , Datta A , Lacomis L , Erdjument-Bromage H , Kumar J , García LJ , Letón R , et al. Exome sequencing identifi es MAX mutations et al. mSin3A/histone deacetylase 2- and PRMT5-containing Brg1 as a cause of hereditary pheochromocytoma . Nat Genet 2011 ; 4 : 663 – 7 . complex is involved in transcriptional repression of the Myc target 10. Ribon V , Leff T , Saltiel AR . c-Myc does not require max for transcrip- gene cad. Mol Cell Biol 2003 ; 23 : 7475 – 87 . tional activity in PC-12 cells . Mol Cell Neurosci 1994 ; 5 : 277 – 82 . 28. Hsiao PW , Fryer CJ , Trotter KW , Wang W , Archer TK . BAF60a medi- 11. Brambilla E , Lantuejoul S , Sturm N . Divergent differentiation in ates critical interactions between nuclear receptors and the BRG1 neuroendocrine lung tumors . Semin Diagn Pathol 2000 ; 17 : 138 – 48 . chromatin-remodeling complex for transactivation . Mol Cell Biol 12. Schouten JP , McElgunn CJ , Waaijer R , Zwijnenburg D , Diepvens 2003 ; 23 : 6210 – 20 . F , Pals G . Relative quantifi cation of 40 nucleic acid sequences by 29. Johnson TA , Elbi C , Parekh BS , Hager GL , John S . Chromatin remod- multiplex ligation-dependent probe amplifi cation. Nucleic Acids Res eling complexes interact dynamically with a glucocorticoid receptor- 2002 ; 30 : e57 . regulated promoter. Mol Biol Cell 2008 ; 19 : 3308 – 22 . 13. Dang CV , O’Donnell KA , Zeller KI , Nguyen T , Osthus RC , Li F. The 30. Amati B , Brooks MW , Levy N , Littlewood TD , Evan GI , Land H. c-Myc target gene network. Semin Cancer Biol 2006 ; 16 : 253 – 64 . Oncogenic activity of the c-Myc protein requires dimerization with 14. Hurlin PJ , Huang J . The MAX-interacting transcription factor net- Max. Cell 1993 ; 72 : 233 – 45 . work . Semin Cancer Biol 2006 ; 16 : 265 – 74 . 31. Steiger D , Furrer M , Schwinkendorf D , Gallant P . Max-independent 15. Medina PP , Romero OA , Kohno T , Montuenga LM , Pio R , Yokota J , functions of Myc in Drosophila melanogaster. Nat Genet 2008 ; 40 : et al. Frequent BRG1/SMARCA4-inactivating mutations in human 1084– 91 . lung cancer cell lines . Hum Mut 2008 ; 29 : 617 – 22 . 32. Maeda I , Okamura D , Tokitake Y , Ikeda M , Kawaguchi H , Mise N , 16. Romero OA , Setien F , John S , Gimenez-Xavier P , Gómez-López G , et al. Max is a repressor of germ cell-related gene expression in mouse Pisano D , et al. The tumour suppressor and chromatin-remodelling embryonic stem cells. Nat Commun 2013 ; 4 : 1754 . factor BRG1 antagonizes Myc activity and promotes cell differentia- 33. Hanahan D , Weinberg RA . Hallmarks of cancer: the next generation . tion in human cancer. EMBO Mol Med 2012 ; 4 : 603 – 16 . Cell 2011 ; 144 : 646 – 74 . 17. Kim JW , Gao P , Liu YC , Semenza GL , Dang CV . Hypoxia-inducible 34. Coffa J , van de Wiel MA , Diosdado B , Carvalho B , Schouten J , Meijer factor 1 and dysregulated c-Myc cooperatively induce vascular GA . MLPAnalyzer: data analysis tool for reliable automated normali- endothelial growth factor and metabolic switches hexokinase 2 and zation of MLPA fragment data. Cell Oncol 2008 ; 30 : 323 – 35 . pyruvate dehydrogenase kinase 1. Mol Cell Biol 2007 ; 27 : 7381 – 93 . 35. Prochownik EV , Van Antwerp ME . Differential patterns of DNA bind- 18. Cantor JR , Sabatini DM . Cancer cell metabolism: one hallmark, many ing by myc and max proteins . Proc Natl Acad Sci U S A 1993 ; 90 : 960 – 4 . faces. Cancer Discov 2012 ; 2 : 881 – 98 . 36. Gautier L , Cope L , Bolstad BM , Irizarry RA . affy—Analysis of Affyme- 19. Luscher B . Function and regulation of the transcriptional factors of trix GeneChip data at the probe level . Bioinformatics 2004 ; 20 : 307 – 15 . the Myc/Max/mad network. Gene 2001 ; 277 : 1 – 14 . 37. Subramanian A , Tamayo P , Mootha VK , Mukherjee S , Ebert BL , 20. Imielinski M , Berger AH , Hammerman PS , Hernandez B , Pugh TJ , Gillette MA , et al. Gene set enrichment analysis: a knowledge-based Hodis E , et al. Mapping the hallmarks of lung adenocarcinoma with approach for interpreting genome-wide expression profi les. Proc Natl massively parallel sequencing. Cell 2012 ; 150 : 1107 – 20 . Acad Sci U S A 2005 ; 102 : 15545 – 50 . 21. Romero OA , Sanchez-Cespedes M . The SWI/SNF genetic blockade: 38. Edgar R , Domrachev M , Lash AE . Gene Expression Omnibus: NCBI effects in cell differentiation, cancer and developmental diseases. gene expression and hybridization array data repository. Nucleic Oncogene. 2013 Jun 10. [Epub ahead of print]. Acids Res 2002 ; 30 : 207 – 10 . 22. Rudin CM , Durinck S , Stawiski EW , Poirier JT , Modrusan Z , Shames DS , 39. Iwakawa R , Takenaka M , Kohno T , Shimada Y , Totoki Y , Shibata T , et al. Comprehensive genomic analysis identifi es SOX2 as a frequently et al. Genome-wide identifi cation of genes with amplifi cation and/or amplifi ed gene in small-cell lung cancer. Nat Genet 2012 ; 44 : 1111 – 6 . fusion in small cell lung cancer . Genes Chrom Cancer 2013 ; 52 : 802 – 16 .

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MAX Inactivation in Small Cell Lung Cancer Disrupts MYC−SWI/SNF Programs and Is Synthetic Lethal with BRG1

Octavio A. Romero, Manuel Torres-Diz, Eva Pros, et al.

Cancer Discovery 2014;4:292-303. Published OnlineFirst December 20, 2013.

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