The Transcriptional Regulator Sin3a Contributes to the Oncogenic

Published OnlineFirst January 28, 2019; DOI: 10.1158/0008-5472.CAN-18-0359 Cancer Tumor Biology and Immunology Research

The Transcriptional Regulator Sin3A Contributes to the Oncogenic Potential of STAT3 Giovanni Gambi1, Elisabetta Di Simone1, Veronica Basso1, Luisa Ricci1, Rui Wang2, Akanksha Verma3,4,5, Olivier Elemento3,4,5, Maurilio Ponzoni6, Giorgio Inghirami2,7,8, Laura Icardi1, and Anna Mondino1

Abstract

Epigenetic silencing of promoter and enhancer SIN3A “ ” SIN3A “deﬁcient” regions is a common phenomenon in malignant cells. The transcription factor STAT3 is aberrantly activated ALK ALK in several tumors, where its constitutive acetylation accounts for the transcriptional repression of a number STAT3 STAT3 Ac Ac of tumor suppressor genes (TSG) via molecular P P mechanisms that remain to be understood. Using STAT3 STAT3 nucleophosmin-anaplastic lymphoma kinase–posi- þ tive (NPM-ALK ) anaplastic large-cell lymphoma SIN3A (ALCL) as model system, we found in cells and Ac Ac SIN3A P P patient-derived tumor xenografts that STAT3 is con- STAT3 STAT3 stitutively acetylated as a result of ALK activity. STAT3 acetylation relied on intact ALK-induced PI3K- and mTORC1-dependent signaling and was sensitive to Ac SIN3A resveratrol. Resveratrol lowered STAT3 acetylation, P Ac SIN3A rescued TSG expression, and induced ALCL apoptotic STAT3 P STAT3 cell death. STAT3 constitutively bound the Sin3A PRDM1/TSG transcriptional repressor complex, and both STAT3 PRDM1/TSG and Sin3A bound the promoter region of silenced TSG silencing TSG expression TSG via a resveratrol-sensitive mechanism. Silencing SIN3A caused reexpression of TSG, induced ALCL apoptotic cell death in vitro, and hindered ALCL tumorigenic potential in vivo. A constitutive STAT3–Sin3A interaction was also found in breast adenocarcinoma cells and proved critical for TSG silencing and cell sur- Cell survival Cell death vival. Collectively, these results suggest that oncogene- driven STAT3 acetylation and its constitutive association with Sin3A represent novel and concomitant events contributing to STAT3 oncogenic potential.

Signiﬁcance: This study delineates the transcrip- Tumor development No tumor development tional regulatory complex Sin3A as a mediator of ALK-acetylated STAT3 associates with Sin3a to inhibit expression of tumor suppressor genes (TSG), which STAT3 transcriptional repressor activity and identi- promotes cell survival and tumor growth. Depletion of Sin3a promotes TSG expression and tumor cell death. © 2018 American Association for Cancer Research ﬁes the STAT3/Sin3A axis as a druggable target to antagonize STAT3-addicted tumors. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/00/0/0000/F1.large.jpg.

1Division of Immunology, Transplantation and Infectious Disease, IRCCS San Note: Supplementary data for this article are available at Cancer Research Raffaele Scientific Institute, Milan, Italy. 2Department of Pathology and Labo- Online (http://cancerres.aacrjournals.org/). ratory Medicine, Weill Cornell Medical College, New York, New York. 3Institute for Computational Biomedicine, Weill Cornell Medical College, New York, New L. Icardi and A. Mondino contributed equally to this article. York. 4Institute for Precision Medicine, Weill Cornell Medical College, New York, Corresponding Authors: Anna Mondino and Laura Icardi, San Raffaele Scientific New York. 5Department of Physiology and Biophysics, Weill Cornell Medical Institute, Via Olgettina 60, Milan 20132, Italy. Phone: 3902-2643-4801; Fax: 3902- College, New York, New York. 6Department of Pathology, IRCCS San Raffaele 2643-4844; E-mail: [email protected]; and Laura Icardi, [email protected] Scientific Institute, Milan, Italy. 7Department of Molecular Biotechnology and Health Science and Center for Experimental Research and Medical Studies doi: 10.1158/0008-5472.CAN-18-0359 (CeRMS), University of Turin, Turin, Italy. 8Department of Pathology and NYU Cancer Center, New York University School of Medicine, New York, New York. 2019 American Association for Cancer Research.

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Introduction Fragment Length Analysuis; Eurofins genomics), and confirmed The STAT3 is a transcription factor found in a constitutive for all, with the exception of JB6 and OCI-Ly12 cells, for which active form in a large number of malignances, including breast, there is no reference in the adopted database (https://www.dsmz. ovarian, pancreatic, gastric cancers, and leukemia, downstream de). ALCL samples were cultured in RPMI 1640 medium supple- to the aberrant activation of oncoproteins and soluble fac- mented with antibiotics, glutamine, and 10% FBS. MDA-MB-231, tors (1–4). It is also central to the oncogenic potential of the MCF7, and HEK-293 cells were cultured in DMEM supplemented nucleophosmin (NPM)-anaplastic lymphoma kinase (ALK) with antibiotics, glutamine, and 10% FBS. Cells were routinely chimeric protein (NPM-ALK), which is the result of a chromo- tested for Mycoplasma contamination and passaged no more than þ somal translocation found in a subset of CD30 non-Hodgkin 1 month prior to experiments. Where indicated, cells were treated T-cell anaplastic large-cell lymphomas (ALCL; refs. 5, 6). In with crizotinib (Sigma Aldrich, catalog no. PZ0191), LY294002 þ ALK ALCL, the NPM-ALK chimeric protein is constitutively (Promega, catalog no. V1201), rapamycin (Calbiochem, catalog activated via autophosphorylation and proved to be oncogenic no. 553210), resveratrol (Sigma Aldrich, catalog no. R5010), þ both in vitro and in vivo (7, 8). ALK cell lines and patient U0126 (Promega, catalog no. V1121). Cell growth and viability samples reveal constitutively activation of STAT3 (9–11), which were determined by Trypan Blue exclusion (Gibco, catalog contributes to NPM-ALK–dependent transformation by acting no. 15250-061). both as transcriptional activator and as transcriptional repressor. Indeed, while constitutive phosphorylation of STAT3 on Western blotting and immunoprecipitation Tyr705 sustains the canonical oncogenic role of STAT3 as Total lysates were recovered in 2% SDS Tris-HCl 65 mmol/L pH activator of genes involved in cell proliferation, survival, inva- 6.8 lysis buffer and sonicated. Cytosolic and nuclear extracts were sion, and immunosuppression (12, 13), deregulated STAT3 obtained by sequential hypotonic [HEPES 20 mmol/L pH 8, NaCl also leads to aberrant epigenetic silencing of several tumor 10 mmol/L, MgCl2 1.5 mmol/L, EDTA 0.2 mmol/L, Triton 0.1%, suppressor genes (TSG), including genes involved in T-cell glycerol 20%, NaV3O4, NaF, TSA 1 mmol/L, protease inhibitor identity and apoptosis (14–16). Interfering with STAT3 expres- cocktail set I (Calbiochem, catalog no. 539131)] and hypertonic sion, as well as the treatment with demethylating agents or (hypotonic buffer þ NaCl 400 mmol/L and DNAse I) extraction. deacetylases inhibitors, was shown to restore TSG expression Protein concentration was evaluated by a modified Lowry protein þ and induce apoptosis of ALK ALCL cells, underlying the assay (DC Protein Assay Reagents, Bio-Rad, catalog no. 500- important contribution of STAT3-mediated epigenetic silencing 0116). Equal amounts of lysate were separated by SDS-PAGE, to cell transformation (14, 15, 17, 18). transferred to nitrocellulose membrane, blocked with 5% milk in The interaction of STAT3 with epigenetic regulators is being TBS, and subjected to immunoblotting. investigated, in the effort of identifying novel therapeutic targets. For immunoprecipitation of endogenous proteins, nuclear Recent reports underscored a role for acetylation in STAT3- lysates were diluted to isotonic condition and immunoprecipi- mediated transcriptional repression. Indeed, besides being phos- tated using 1 mg of antibodies (anti-Sin3A sc-994/sc-767, normal phorylated in tyrosine and serine residues, STAT3 is found hyper- rabbit IgG sc-2027 were from Santa Cruz Biotechnology). Immu- acetylated in a number of malignancies and acetylated STAT3 was nocomplexes were recovered by adding Dynabeads protein A shown to mediate epigenetic TSG silencing (19–25). Of note, (Invitrogen, catalog no. 10002D) or Protein G Sepharose 4 Fast STAT3 acetylation was proven to promote the binding to Flow (GE Healthcare, catalog no. 71-7083-00) and analyzed by Sin3A (26), a member of the Sin3A transcriptional regulator immunoblotting. For immunoprecipitation of Sin3A and STAT3 complex that includes the histone deacetylases (HDAC) and DNA mutants, Hek293 cells were lysed in modified RIPA buffer 48 methyltransferases (DNMT). To date, whether Sin3A plays a role hours after transfection and immunoprecipitated with 1 mgof in STAT3-mediated tumor suppressor gene silencing in cancer anti-Sin3A antibodies (anti-Sin3A sc-994/sc-767 and D9D6). remains uninvestigated. Immunocomplexes were recovered by adding Protein G Sephar- þ Here, using NPM-ALK ALCL as model system, we show that ose 4 Fast Flow (GE Healthcare, catalog no. 71-7083-00) and STAT3 is hyperacetylated in ALCL cells and patient-derived tumor analyzed by immunoblotting. The following primary antibodies xenotransplant and that inducing STAT3 deacetylation with res- were used: anti-STAT3 (124H6), anti-Ac-Lys685-STAT3 (#2523), veratrol restores TSGs transcription. In addition, we identified the anti-P-Tyr705 STAT3 (#9131), anti-P-Ser727 STAT3 (#9134), anti- Sin3A complex as constitutive STAT3-binding partner and key S6 (#2317), anti-PS235/236S6 (#2211), anti-Sin3A (#D1B7), anti- mediator of STAT3-dependent TSGs transcriptional repression PY1606NPM-ALK (#3341), and anti-Blimp1 (#9115) from Cell þ and oncogenic potential in both NPM-ALK ALCL and breast Signaling Technology; anti-Sin3A (sc-994, sc-767) and anti-actin adenocarcinoma cells. Thus, we suggest that Sin3A contributes to (sc-1616) antibodies were from Santa Cruz Biotechnology; and STAT3-driven TSG silencing and oncogenic potential. anti-tubulin (T6074) antibody was from Sigma.

ChIP, ChIP-re-ChIP, and ChIP-seq Materials and Methods For chromatin immunoprecipitation (ChIP) experiments, Cell lines and drug treatments cells were fixed with methanol-free formaldehyde (Thermo Sci- Human ALCL cells SU-DHL-1, Sup-M2, JB6, and OCI-Ly12 entific, catalog no. 28906) and lysed in Lysis Buffer 1 (LB1, cells were kindly provided by Roberto Chiarle and Roberto Piva 50 mmol/L HEPES-KOH pH7.5, 140 mmol/L NaCl, 1 mmol/L (University of Torino, Torino, Italy). Human breast cancer cells EDTA, 10% glycerol, 0.5% NP40, 0.25% TritonX100), LB2 MDA-MB-231, MCF7, and HEK-293 cells were kindly provided by (10 mmol/L TrisHCl pH 8, 200 mmol/L NaCl, 1 mmol/L EDTA, Rosa Bernardi (San Raffaele Scientific Institute, Milan, Italy). Cell 0.5 mmol/L EGTA), and LB3 (10 mmol/L TrisHCl pH 8, 100 identity was finally analyzed in June and July 2018 by short mmol/L NaCl, 1 mmol/L EGTA, 0.1% Na-deoxycholate, 0.5% tandem repeat analysis (GenePrint 10 System, Promega and N-lauroylsarcosine) supplemented with protease inhibitor

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cocktail. Samples were sonicated using the Diagenode Bioruptor a single "A" base and subsequent ligation of the adapter. The sonication device. Sonicated lysates were incubated with antibo- products are purified and enriched with PCR to create the final dies (anti-STAT3 #124H6, anti-Sin3A #sc-994/sc-767 or normal cDNA library. Paired-end RNA-sequencing at read lengths of rabbit IgG #sc-2027, Cell Signaling Technology) at 4C on rota- 100 bp was performed with the HiSeq2500 (Illumina). Raw tion overnight. Immunocomplexes were recovered by adding sequenced reads were aligned to the Human reference genome protein A magnetic beads (Invitrogen, catalog no. 10002D), (Version hg19 from UCSC) using STAR (Version 2.4.2) aligner. resuspended in 1% SDS NaHCO3 and treated with proteinase Aligned reads were quantified against the reference annotation K (Promega, catalog no. V3021) and then with RNase A. DNA was (hg19 from UCSC) to obtain FPKM (Fragments per Kilobase purified using the MiniElute Reaction Cleanup Kit (Qiagen, per million) and raw counts using CuffLinks (v 2.2.1) and catalog no. 28204). One percent of sonicated chromatin was HTSeq,respectively. used as input. Immunoprecipitated DNA and input were assayed by qRT-PCR (a list of primers is available in Supplementary Table Plasmid and siRNA transfection, shRNAmir lentivirus S1). Data were expressed according to the Adjusted input formula production, and transduction [100 2(Adjusted input Ct(IP)]. In ChIP-re-ChIP, 3 107 cells were Hek293 and MDA-231 cells were transiently transfected sonicated (Covaris) in LB3 omitting NaCl. The soluble chromatic using the PolyFect Transfection Reagent (Qiagen) following fraction was supplemented with NaCl and SDS and incubated manufacturer's instruction. The pMet7-etag-STAT3WT, pMet7- with anti-SIN3A [SIN3A (D9D6) rabbit mAb, Cell Signaling etag-STAT3K685Q, and pMet7-etag-STAT3K685R were kindly Technology; or STAT3)] antibody. Mouse IgG1 (M5284, Sigma) provided by Dr. Jan Tavernier (VIB, Ghent, Belgium). The and rabbit E-Tag (13419s, Cell Signaling Technology) were used lentiviral vectors pGIPZ-GFP-SIN3A-shRNAmir (V2LHS_96677 as isotype controls. Immunocomplexes were recovered on Protein and V2LHS_96678) and pGIPZ-GFP-control-shRNAmir A-beads, sequentially washed in WB1 (20 mmol/L Tris-HCl pH 8, (RHS4346) were purchased from Open Biosystems. Lentiviral 150 mmol/L NaCl, 2 mmol/L EDTA, 1% Triton, 0,1% SDS) and packaging pCMV-dR8.74 and pseudotyping VSV-G/pMD2.G WB2 (20 mmol/L Tris-HCl pH 8, 150 mmol/L NaCl, 2 mmol/L (kindly provided by Dr. Vincenzo Calautti, Dulbecco Telethon EDTA, 1% Triton, 0,1% SDS), and eluted in TE buffer (10 mmol/L Institute, c/o Molecular Biotechnology Center, University of Tris-HCl pH 8, 0.25 mmol/L EDTA pH 8). Eluted fraction was then Torino, Torino, Italy) were used. The siGENOME control non- diluted in 20 mmol/L Tris-HCl pH 8, 2 mmol/L EDTA pH 8, 1% targeting siRNA and the STAT3 30-UTR siRNA was purchased TRITON X-100, 150 mmol/L NaCl, and incubated with anti- from Thermo Fisher Scientific (D-001210-03 and D-003544- STAT-3 (or anti-Sin3A) Ag or control IgG, followed by Protein 19), and the SIN3A siRNA silencer was purchased from Ambion A-beads, and processed as described above. For the ChIP-seq (s24800). experiment, a total of 107 cells were fixed with 1% formaldehyde, For individual silencing experiments, 50 nmol/L siGENOME lysed, and sonicated (Branson Sonicator; Branson), leading to a nontargeting siRNA Control or STAT3 siRNA (Thermo Fisher DNA average size of 200 bp. Anti-STAT3 (Cell Signaling Tech- Scientific) or SIN3A siRNA Silencer (Ambion) were transfected nology, 9139; 5 mg) or control IgG (Millipore 12-370, 12-371) in MDA-MB-231 cells using Dharmafect 1 transfection reagent antibodies were added to the precleared sample and incubated (Thermo Fisher Scientific) following the manufacturer's instruc- overnight at 4C. The complexes were purified using protein-A tions. Where indicated, DNA plasmid transfection was performed beads (Roche), followed by elution from the beads and decross- after 24 hours. Western blot analysis and qRT-PCR analysis were linking. DNA was purified using PCR purification columns performed 5 days after siRNA transfection. Lentiviral stocks were (QIAGEN) and was amplified by qRT-PCR using SYBR Green produced in Hek293 cells transfected with pGIPZ-shSIN3A, (Applied Biosystems) on 7900HT Fast Real-Time PCR System pGIPZ shSIN3A#2, or pGIPZshControl (Open Biosystems) (Applied Biosystems). Raw ChIP-seq samples were aligned using together with the pCMV-dR8.74 and VSV-G/pMD2.G vectors. þ bwa aligner. Peak calling was performed using MACS2 (Version Virus was titrated on Hek293 cell line. NPM-ALK cells were 2.1.1) to identify transcription factor–binding sites. Only peaks infected in the presence of polybrene, harvested 1 day after called with FDR < 0.05 were called as significantly enriched. To transduction, and cultured in fresh medium supplemented with create signal tracks, macs2 bdgcmp command was used, to cal- puromycin. After 48 hours of selection, viable cells were recovered culate the fold change in bedgraph format and then converted to by density centrifugation on a Ficoll gradient (GE Healthcare, bigwig using UCSC toolkit. catalog no.17-1440-02).

RNA-seq library preparation qRT-PCR RNA-seq was performed as described previously (27). All RNA was purified with the RNeasy Mini Kit (QIAGEN, catalog TruSeq Stranded Total RNA kits follow the same workflow. no. 74104) following manufacturer's instructions. One micro- Briefly, the removal of ribosomal RNA (rRNA) was achieved gram of total RNA was retrotranscribed for 1 hour at 42C with using biotinylated, target-specific oligos combined with Ribo- retrotranscriptase M-MLV (Invitrogen, catalog no. 28025-013) in Zero rRNA removal beads. The Ribo-Zero Human/Mouse/Rat the presence of oligo(dT)15 Primer (Promega, catalog no. C1101), kit depletes samples of cytoplasmic rRNA and the Ribo-Zero dNTPs (Euroclone), and RNAsin Ribonuclease Inhibitor (Pro- Gold kit depletes samples of both cytoplasmic and mitochon- mega, catalog no. N2111). cDNA was diluted 10 times to perform drial rRNA. After purification, the RNA was fragmented into qRT-PCR with SYBR Green Master Mix (Applied Biosystems, small pieces using divalent cations under elevated temperature. catalog no. 4309155) with gene-specific primers (summarized The cleaved RNA fragments were then copied into first-strand in Supplementary Table S1). Ct values were normalized to house- cDNA using reverse transcriptase and random primers, fol- keeping genes expression using the DDCt method. The list of lowed by second-strand cDNA synthesis using DNA polymerase primer pairs used in this work is mentioned in Supplementary I and RNase H. These cDNA fragments then had the addition of Table S1.

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Flow cytometry analysis diameter. Tumors were excised, dissected into 5-mm cubes, and Apoptotic cell death was measured by Annexin V-APC and frozen for additional transplants or digested with Collagenase I 7AAD staining (BD Biosciences, catalog nos. 550474 and and DNAse I for 1 hour at 37C. Viable cells were recovered by 559925), according to the manufacturer's instructions, where density centrifugation on a Ficoll gradient (GE Healthcare, catalog indicated cells were stained for surface IL2Rg expression with no. 17-1440-02) and directly used for RNA and total protein anti-IL2Rg-APC (catalog no. 563682, Clone TUGh4) or a corre- extraction or cultured for 48 hours in the presence of resveratrol or spondent isotype control. Samples were acquired with a BD Accuri vehicle. To exclude contamination from mouse stromal and C6 analyzer. Singlets were defined by plotting FSC area versus FSC erythroid cells, samples were surface-stained with anti-mouse height and analyzed using FlowJo software. H-2K[d] (clone SF1-1.1, catalog no. 562004), anti-mouse TER- 119 (clone TER-119, catalog no. 561032), and anti-human-CD45 Immunofluorescence (catalog no. 555485, Clone HI30) or correspondent isotype þ NPM-ALK cells were seeded by cytospin centrifugation on controls and acquired with a BD Accuri C6 analyzer. þ Thermo Fisher Scientific Superfrost Plus microscope slides, fixed In selected experiments, NPM-ALK cells were used. A total of in 4% formaldehyde, washed with PBS, and permeabilized in ice- 105 cells were transduced with the pGIPZ shSIN3A or pGIPZ- cold 100% methanol at 20C for 10 minutes. Slides were shControl lentivirus, suspended in BD Matrigel Matrix Growth blocked in PBSþ5% normal goat serumþ0.3% TritonX100 for Factor Reduced (catalog no. 356230) and injected subcutaneous- 1 hour and stained with primary antibodies (anti-STAT3 #9139 ly into the rear flank of NSG mice. Matrigel was used to increase from Cell Signaling Technology; anti-Sin3A #sc-767 from Santa the efficiency of engraftment and growth of malignant cells. Cruz Biotechnology) in PBS þ BSA 1% þ 0.3% TritonX100) Tumor growth was monitored by caliper and mice were sacrificed overnight at 4C. Slides were then stained with secondary anti- when tumors reached approximately 1.5 cm in diameter. Tumor bodies and with DAPI. Images were acquired with a confocal masses were collected, weighted, and reduced to single-cell sus- (Leica DMI6000) microscope with a 40 objective and analyzed pension for further analysis. with ImageJ (NIH).

In vivo animal studies Results þ Studies were performed in accordance with European Union STAT3 is constitutively acetylated in ALK ALCL guidelines and with the approval of the San Raffaele Institute STAT3 sustains NPM-ALK–driven transformation by promot- (Milan, Italy; IACUC-814) and the Weill Cornell Medical College ing the transcriptional silencing of a subset of TSGs via molecular (New York, NY; IACUC 2014-0024) Institutional Ethical Com- mechanisms that remain largely unknown (14–16, 18, 28, 29). As mittees. Animals were housed and bred in specific pathogen-free STAT3 acetylation was associated with transcriptional repression animal facilities. Patient-derived tumor xenografts (PDTX) were in several cancer models (23, 24), we investigated STAT3 post- þ established as follows: from the surgical theater, fresh tissue translational modifications in NPM-ALK cells and PDTX. As fragments (pregraft tissues) were stored in PBS and/or in complete previously reported (10), we found STAT3 to be constitutively media (RPMI 1640 supplemented with 10% FCS and antibiotics), phosphorylated on both Tyr705 and Ser727 residues in NPM- and rapidly delivered into the surgical pathology triage room. ALK–expressing cells (Sup-M2, JB6, and SU-DHL-1), and not in Dedicated tissue fragments were selected for tumor graft implants NPM-ALK ALCL control cells (OCI-Ly12; Fig. 1A; Supplementary þ only when sufficient material was available for routine diagnostic Fig. S1A). STAT3 was also constitutively acetylated in NPM-ALK þ and molecular analyses. All samples were processed in sterile cells (Fig. 1A; Supplementary Fig. S1A and S1B) and ALK PDTXs conditions. Tumor graft samples were cut into multiple 3 3 1 (Fig. 1B), and not in ALK controls. Of note, while STAT3 Tyr705 mm pieces (multiple pieces/specimen) in complete media and phosphorylation was uniquely dependent on ALK, STAT3 Ser727 implanted fresh and/or cryopreserved in 10% DMSO-RPMI 1640 phosphorylation and acetylation also relied on the Ser/Thr frozen media supplemented with 20% FCS. Six- to 8-week-old kinases mTOR, PI3K, and MAPK, previously described targets of NSG were first anesthetized (Rompun 0.05 mL/g and Zoletil 1.6 ALK (30, 31). Indeed, treatment with crizotinib, a potent and mg/g intramuscularly). Then, with animals lying on their ventral selective ALK inhibitor, inhibited both Tyr and Ser phosphoryla- site, the dorsal region was sterilized (70% ethanol). A skin tion and Lys acetylation (Fig. 1C; Supplementary Fig. S1C). In incision of 0.3 cm was subsequently made along the dorsal contrast, rapamycin (targeting mTOR), LY294009 (targeting midline retro-nuchal region and a small pocket was created by PI3K), and U0126 (targeting MAPK) inhibited STAT3 Ser727 blunt dissection. Multiple tumor graft tissue fragments (2–4) were phosphorylation and Lys685 acetylation, without affecting transferred into the subcutaneous in each pocket using blunt- Tyr705 phosphorylation (Supplementary Fig. S1D). Thus, STAT3 þ ended forceps with or without Matrigel. The cut edges were sealed is constitutively phosphorylated and acetylated in ALK cells. with a single metal clip. After the procedure, mice were regularly Next, we compared the expression of a number of TSGs, þ checked until they became vigilant. Implanted animals were reported to be repressed by STAT3 in ALK cells (IL2Rg, LAT, housed in same-sex groups. Similar frequencies were seen in both and LEF1; refs. 14, 15, 32) or in other cancer models (DDIT3, male and female mice. Implant growth was assessed by palpation ATF3, PHLDA1, and DUSP4; ref. 33). We observed TSG expression þ and or by MRI scanning and harvested when tumor masses were levels to be lower in ALK cells compared with ALK controls þ required. Recipient animals were checked regularly and sacrificed (Fig. 1D). In contrast, ALK cells displayed higher SOCS3 expres- at early sign of distress. At harvesting, mice were sacrificed in a CO2 sion, a canonical STAT3-transcribed gene, when compared with chamber and grafts were collected for histologic evaluation, ALK controls (Supplementary Fig. S1E), supporting the notion þ regrafting, or snap-freezing in liquid nitrogen. When regrafting, that, in NPM-ALK ALCL, STAT3 is transcriptionally active. By lymphoma fragments were transplanted in NSG mice, and mice ChIP-seq analysis, we found STAT3 to be bound to the promoter were sacrificed when tumors reached approximately 1.5 cm in of a number of TSGs, among which PRDM1, coding for the

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Figure 1. Constitutive STAT3 phosphorylation and acetylation in NPM-ALKþ ALCL correlates with the silencing of oncosuppressor genes. A–C, NPM- ALKþ (Sup-M2, JB6, SU-DHL-1), ALK (OCI-Ly12) ALCL cells, and NPM-ALKþ PDTXs (PDTX#1, PDTX#2) were lysed in SDS sample buffer and analyzed by Western blot with the indicated antibodies. C, Cells were treated with 100 nmol/L crizotinib for 24 hours. Data in A–C are representative of at least three independent experiments. D, Gene expression was assessed by qRT-PCR. Results indicate relative mRNA levels calculated over the OCI-Ly12 reference cells. Error bars, SD (n ¼ 2).

Blimp1 protein, frequently found downregulated in both B- and same promoter regions (Fig. 2D). To more directly demonstrate T-cell lymphomas (Supplementary Fig. S2A; ref. 34). Crizotinib that STAT3 and Sin3A simultaneously associate with the same inhibited STAT3 binding to PRDM1 promoter regions (Supple- genomic sites, we performed sequential ChIP-re-ChIP experi- mentary Fig. S2A) and, while it lowered SOCS3 expression, it ments. In two of three independent determination, we found increased the expression of some of the STAT3-repressed TSGs STAT3 and Sin3A to be specifically enriched for the promoter (DDIT3, PRDM1, and LAT; Supplementary Fig. S2B) supporting region of PRDM1 (Fig. 2E). This was the case in both STAT3/Sin3A their ALK-dependent transcriptional repression. and Sin3A/STAT3 sequential ChIPs. While similar trends were þ Thus, in NPM-ALK ALCL, STAT3 is constitutively phosphor- found when interrogating the binding to the IL2Rg, results were ylated and acetylated, and this correlates with repression of a not indicative for LAT. We believe that some variability might be number of TSGs. attributed to the nature of the STAT3–Sin3A complex, likely including several proteins, and dynamically regulated in asyn- þ STAT3 and Sin3A constitutively associate in ALK cells and bind chronously dividing transformed cells. Thus, given the ability of TSG promoter regions Sin3A to bind HDACs and DNMTs and to regulate transcription As cytokine-driven STAT3 acetylation induces transient STAT3 by epigenetic means, these data suggest a role for Sin3A in STAT3- association with the transcriptional regulator Sin3A complex (26), mediated transcriptional repression of TSGs. we asked whether the constitutive acetylation of STAT3 in NPM- þ ALK cells could promote its binding to Sin3A. We first assessed Resveratrol inhibits STAT3 acetylation, Sin3A binding to TSG Sin3A and acetylated STAT3 subcellular localization and Western promoters, and rescues TSGs expression blot analysis and found both to be represented almost exclusively To investigate whether STAT3 acetylation was required for in the nucleus (Fig. 2A). Immunofluorescence confocal analysis Sin3A recruitment to the TSG promoters and for their tran- þ identified STAT3 and Sin3A to colocalize in the nucleus of NPM- scriptional repression, we treated NPM-ALK cells with resver- þ ALK cells (Fig. 2B). Furthermore, by coimmunoprecipitation, we atrol, a polyphenol previously shown to inhibit STAT3 acety- found endogenous STAT3 to interact with endogenous Sin3A in a lation (35, 36). We found that resveratrol significantly reduced constitutive, stimulus-independent manner (Fig. 2C). STAT3 acetylation, as evidenced in Western blot analysis with To investigate putative effects of the STAT3–Sin3A complex, we an antibody specific for acetyl-Lys685. Resveratrol also reduced analyzed the ability of these factors to bind the promoters of Ser727 phosphorylation, without significantly affecting STAT3 STAT3-repressed genes in ChIP assays. We selected three repre- Tyr705 phosphorylation (Fig. 3A; Supplementary Fig. S3A), sentative ALK-repressed genes (PRDM1, IL2Rg, LAT) and found further underling the cross-regulation between acetylation and that both STAT3 and Sin3A were constitutively associated to the Ser727 phosphorylation. Concomitantly, resveratrol reduced

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Figure 2. The transcriptional repressor Sin3A binds to STAT3 and to promoter regions of STAT3-repressed oncosuppressor genes in NPM-ALKþ ALCLs. A and B, Sin3A, ac-STAT3 (Lys685), and STAT3 subcellular localization was evaluated in cytosolic and nuclear fractions by Western blot analysis (A) and by immunofluorescence (B) in NPM-ALKþ cells. B, DNA was stained with DAPI and images were acquired with a confocal microscope (magnification, 40). C, Nuclear lysates were immunoprecipitated with anti-Sin3A antibodies or normal rabbit IgG and analyzed with the indicated antibodies. Results are representative of three independent experiments. D, Cross-linked chromatin extracts were immunoprecipitated with anti-Sin3A, anti-STAT3 antibodies, or with polyclonal immunoglobulin (IgG). Purified DNA was analyzed by qRT-PCR. Results indicate the percentage of immunoprecipitated DNA over input; error bars, SD (n ¼ 4). One-tailed paired t test, , P < 0.05. E, Cross-linked chromatin extracts were subjected to sequential ChIP-re-ChIP with anti-Sin3A and anti-STAT3 antibodies (left) or vice versa (right). Matched isotype control antibodies were used to control specific enrichments. One experiment representative of two independent determinations is shown.

the recruitment of both Sin3A and STAT3 to promoter regions Fig. S4, resveratrol interfered with LIF-induced STAT3 acetylation of PRDM1 and IL2Rg (Fig. 3B) and caused the reexpression of (Supplementary Fig. S4A), only partially reducing STAT3 nuclear þ these and several other STAT3-repressed TSGs in NPM-ALK localization (Supplementary Fig. S4B), and completely abrogated cells (Fig. 3C; Supplementary Fig. S3B and S3C). Gained IL2Rg, STAT3 binding to Sin3A (Supplementary Fig. S4C). Importantly, PRDM1/Blimp-1 and DDIT3/CHOP expression was conﬁrmed although resveratrol hindered STAT3 Lys685 acetylation, the at the protein level either by FACS (Fig. 3D) or Western blot unique mutation of STAT3 Lys685 in either acetyl-mimicking analyses (Fig. 3E and F). (STAT3K685Q) or acetyl-deﬁcient (STAT3K685R) did not affect Treating cells with resveratrol improved TSG expression also in STAT3 binding to Sin3A (Supplementary Fig. S4D and S4E), þ ex vivo treated ALK PDTXs (Supplementary Fig. S3D). Among strongly suggesting that other STAT3 lysine residues beside Lys685 other genes, the expression of STAT3 proved insensitive to the regulate STAT3–Sin3A interaction. þ drug in both ALK and ALK cells (Supplementary Fig. S3E). Together, these results indicate that resveratrol causes decreased Concomitantly to the induction of TSG reexpression, resveratrol STAT3 acetylation and S727 phosphorylation, the release of Sin3A þ induced NPM-ALK cells to undergo apoptotic cell death (Sup- and of STAT3 from TSG promoters, the upregulation of TSGs þ plementary Fig. S3F and S3G). In contrast, ALK OCI-Ly12 cells expression, and NPM-ALK apoptotic cell death. proved insensitive to the drug (Supplementary Fig. S3F and S3G). We next asked whether resveratrol inhibits the association of SIN3A silencing unleashes TSG expression, induces apoptotic þ STAT3 with Sin3A. We investigated this issue in Hek293 cells cell death, and hinders tumorigenic potential of ALK ALCL stimulated with LIF, previously shown to acutely induce STAT3 To more directly test whether the TSG transcriptional brake þ acetylation and Sin3A binding (26). As shown in Supplementary relied on Sin3A, we transduced NPM-ALK ALCL cells with a

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Figure 3. Resveratrol inhibits STAT3 acetylation and Ser 727 phosphorylation and Sin3A binding to oncosuppressor gene promoters, promoting their expression and apoptotic cell death. NPM-ALKþ cells were cultured in the absence (NT) or the presence of resveratrol (20 mmol/L) for 24 hours and then analyzed by Western blot (A, E,and F), ChIP (B), or qPCR (C). Results in B indicate fold increase (% of input DNA) versus the untreated sample; error bars are SD from at least two independent experiments. Results in C indicate relative mRNA levels over the untreated sample (dashed line). Error bars, SD (n > 3). D, IL2Rg expression was measured by FACS. Untreated cells and isotype control– stained, treated cells are shown as controls. E and F, CHOP and BLIMP expression was investigated by Western blot analysis. E, A representative analysis is depicted, while in F, expression relative to actin levels over independent investigations (n ¼ 3) is shown. Two-tailed paired t test, , P < 0.05; , P < 0.01; , P < 0.005.

lentiviral vector encoding for a SIN3A-specific short hairpin We next asked whether Sin3A silencing would also impact on þ RNA (shSIN3A). A nonsilencing control shRNA was used NPM-ALK cells' tumorigenic potential in vivo. To this aim, (shControl), while GFP and puromycin resistance were shControl and shSIN3A cells were injected subcutaneously in adopted to enable transduced cells' tracing and selection. By NSG-3GS immunocompromised mice and tumor development five days of selection, shSIN3A cells expressed significantly was followed over time. We found that mice challenged with þ lower Sin3A levels compared with shControl-transduced cells, ALK tumor cells lacking SIN3A developed tumors at later times while retaining comparable STAT3 expression (Fig. 4A). In (Fig. 5A) and displayed significantly increased survival (Fig. 5B) addition, knocking down SIN3A induced the reexpression of when compared with mice challenged with shControl cells. Of resveratrol-sensitive STAT3-controlled TSGs both at the RNA note, at the time of sacrifice, cells retrieved from shSIN3A tumors (Fig. 4B) and protein level (Fig. 4C and D). When comparing showed lower GFP expression (Fig. 5C) and higher SIN3A levels cell behaviors in culture, we found that shSIN3A cells showed a compared with those measured at the time of injection (Fig. 5D), slower growth rate compared with shControl ones, and after while GFP levels remained comparable in cells from shControl day 5, concomitantly to TSG reexpression, they no longer tumors at the time of injection and sacrifice. This suggested the increased in numbers (Fig. 4E) and were enriched for apoptotic in vivo outgrowth of cells with suboptimal SIN3A silencing. cells (Fig. 4F). Similar results were obtained with a second Together, these data indicate that Sin3A participates in the repres- þ unrelated shSIN3A (shSIN3A#2), thus overruling possible sion of TSGs in NPM-ALK cells and that its inhibition promotes off-target effects. Also, in this case, SIN3A silencing (Supple- TSG expression and apoptosis, hindering tumor cell growth in vitro mentary Fig. S5A) unleashed the expression of PRDM1, LAT and in vivo. These data thus highlight a role for Sin3A in ALK (Supplementary Fig. S5B) and IL2Rg (Supplementary Fig. tumorigenic potential, and suggest the silencing/inhibition of Sin3A S5C), and favored cell apoptosis (Supplementary Fig. S5D). as a novel strategy to inhibit STAT3-driven transformation.

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Figure 4. SIN3A silencing unleashes oncosuppressor gene expression and induces NPM-ALKþ cell apoptosis. NPM-ALKþ cells were transduced with shControl or shSIN3A and analyzed by Western blot (A and D) or by qRT-PCR (B)5 and 8 days after transduction, respectively. B, Results indicate relative mRNA levels calculated over the shControl sample (dashed line). Error bars, SD (n > 4). Two- tailed paired t test, , P < 0.05; , P < 0.01; , P < 0.005. C, The dot plots depict FACS analysis 8 days upon transduction. D, Relative expression is depicted. Data are representative of two independent experiments. E, Growth of shControl or shSIN3A-transduced NPM-ALKþ cells at the indicated time points was evaluated by Trypan blue. Results indicate cell counts. One day after silencing, cells were plated at 105 cells/mL. At day 3 and 5, cells were counted and replated (105 cells/mL) to avoid confluence. Data represent cell/mL. Error bars, SD of independent experiments (n ¼ 2–4). F, Apoptosis of shControl or shSIN3A NPM-ALKþ cells was evaluated 8 days upon transduction by flow cytometry via Annexin V-FITC and 7AAD staining. Percentages SD of Annexin Vþ cells of a representative experiment are shown (n ¼ 3). Statistical significance was analyzed by two-tailed paired t test. , P < 0.05; , P < 0.01.

þ Sin3A contributes to STAT3 oncogenic potential also in breast in MCF7 control cells. In addition, as observed in ALK cells, cancer cells STAT3 and Sin3A coprecipitated in MDA-MB-231 nuclear lysates STAT3 is constitutively activated in numerous cancer types and (Fig. 6B), while in MCF7 cells, STAT3 and Sin3A formed a complex this correlates with a more severe prognosis (37). In breast cancer, only upon stimulation with the STAT3-activating cytokine LIF aberrant STAT3 signaling is found in more than 40% of cases, in (Supplementary Fig. S6A). which it promotes tumor progression through deregulation of the To assess the contribution of Sin3A to STAT3-mediated tran- expression of a number of downstream target genes, involved in scriptional repression, MDA-MB-231 cells where transfected with proliferation, angiogenesis, epithelial-to-mesenchymal transition a control siRNA (siControl) or with SIN3A siRNA (siSIN3A). and invasion (38). To investigate whether the contribution of the Interestingly, we found that SIN3A silencing increased the expres- Sin3A complex to STAT3-driven TSG transcriptional repression sion of a number of genes, among which the DDIT3/CHOP gene þ could be observed in other than ALK ALCL tumors, we selected (Fig. 6C), via a resveratrol-sensitive mechanism (Supplementary MCF7 and MDA-MB-231 breast tumor cells, of which only the Fig. S6B). In addition, Sin3A silencing also increased CHOP latter carries constitutive STAT3 activation. As depicted in Fig. 6A, protein levels (Fig. 6D), reduced MDA-MB-231 cell growth STAT3 was found to be acetylated in MDA-MB-231 cells, and not (Fig. 6E), and promoted cell apoptosis (Fig. 6F). Also, in this

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Figure 5. SIN3A silencing hinders ALKþ ALCL in vivo tumorigenic potential. A and B, shControl or shSIN3A- transduced NPM-ALKþ cells were subcutaneously injected in the flank of NSG mice (n ¼ 6). Tumor volumes ( SD; two-tailed unpaired t test, , P < 0.05; A) and overall survival (log rank, Mantel–Cox test, , P < 0.01; n ¼ 6; B) are shown. At the time of sacrifice, tumors were dissociated into single cells and analyzed by flow cytometry for GFP expression (C) or by qRT-PCR (D) compared with cells at the time of injection or to untransduced cells. C, Results are representative of 6 mice for group. D, SIN3A expression ( SD) in individual mice is depicted.

case, the mutation of STAT3 Lys685 did not affect DDIT3/CHOP Previous studies described the interaction between STAT3 and expression (Supplementary Fig. S6C and S6D), further supporting Sin3A as a transient and cytokine-dependent event, by which the that other lysines, in addition to Lys685, play a role in Sin3A- acetylation of STAT3 would promote the binding to Sin3A, which dependent STAT3-driven transcriptional repression. in turn mediates its deacetylation, downregulating its transcrip- þ Taken together, these results suggest the STAT3/Sin3A complex tional activity (26). In NPM-ALK ALCLs, we found that STAT3 also acts in breast cancer cells as a transcriptional repressor, was constitutively phosphorylated and acetylated and also con- supporting the idea that Sin3A contributing to the oncogenic stitutively bound to Sin3A in a stimulus-independent manner. We potential of STAT3 might represent a shared characteristic of speculate that STAT3 hyperphosphorylation and acetylation is cancers with constitutive STAT3 activation. responsible for prolonged binding to Sin3A and the silencing of critical TSGs by epigenetic means, and that this contributes to the transformed phenotype. Accordingly resveratrol, previously Discussion shown to promote STAT3 deacetylation and to hinder its activity Here, we show that STAT3 is constitutively phosphorylated and as transcriptional repressor (23, 24), also inhibited STAT3 Ser727 þ þ acetylated in ALK cells and ALCL PDTXs and it is bound to the phosphorylation and acetylation in NPM-ALK cells, STAT3/ transcriptional repressor complex Sin3A, and that these events are Sin3A complex formation and Sin3A binding to the STAT3- critical for the repression of a number of TSGs previously reported controlled PRDM1 and IL2Rg genes, caused reexpression of a þ to be repressed by STAT3 and critical for NPM-ALK tumorigenic subset of STAT3-repressed genes, and promoted NPM-ALK potential. ALCL apoptotic cell death. Although resveratrol might control a þ The role of STAT3 in the transformed phenotype of ALK ALCLs plethora of substrates, it had no effect on TSGs expression in and in controlling tumor growth in vitro and in vivo has long been ALK cells. recognized (9). Although the oncogenic potential of STAT3 was The mechanism by which resveratrol controls STAT3 acetyla- originally linked to its function as transcriptional activator, it was tion downstream to NPM-ALK remains largely to be defined, and later reported that, following STAT3 silencing, the number of may involve activation of deacetylases or inhibition of acetyl- þ upregulated genes outnumbered the downregulated ones in ALK transferases. While resveratrol is a known activator of the deace- ALCLs (16). In these cells, STAT3-dependent TSG silencing was tylase Sirtuin 1 (39, 40), this did not appear to be solely respon- shown to involve the methylation of TSG-regulatory regions (14), sible for STAT3 deacetylation. Indeed, Sirtuin1 knockdown via the recruitment of epigenetic modifiers such DNMT1, HDAC1, caused an increase of Lys685 acetylation [Supplementary Fig. and MeCP2 (15, 28, 29). As these are well-known interactors of S7A, and yet it did not abrogate the ability of resveratrol to the Sin3A repressor complex, our data showing that STAT3 upregulate TSG expression (DDIT3 is depicted in the Supplemen- constitutively binds to Sin3A profile a more detailed landscape tary Fig. S7B]. Thus, other deacetylases, such as HDACs, other of the oncogenic potential of STAT3 via its ability to act as both Sirtuins, or the Lysyl Oxidase 3, all previously described to transcriptional activator and repressor (Fig. 7). deacetylate STAT3 (41–43), might also play a role.

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TSGs expression. In line with this notion, although Sin3A levels were reduced as soon as four days after silencing, TSG reexpression and apoptotic cell death were observed only after an additional 24–72 hours. Whether active or passive (via cell division) demethylation plays a role remains to be determined. The cooperation between STAT3 and Sin3A in repressing TSG transcription did not appear peculiar of NPM-ALK–transformed ALCL cells, but was also observed in breast adenocarcinoma cells, suggesting the STAT3/Sin3A axis might operate whenever STAT3 is constitutively activate and, in particular, when it is acetylated. As several events can lead to overt activation of STAT3, including aberrant or chronic stimulation via cytokines and growth factors, constitutive engagement of wild-type and mutated RTK receptors, and deregulated activation of G protein–coupled receptors (51), it is likely that the STAT3–Sin3A axis described here is active in a large number of STAT3-addicted tumors. We speculate that tumor- or oncogene-specific effects might be in place, and lead to diverse and possibly cell-type–specific epigenetic remodeling. Indeed, we found that only a subset of the STAT3 and Sin3A þ coregulated genes identified in ALK cells were also sensitive to SIN3A silencing in MDA-MB-231 cells. Furthermore, we found that the STAT3-repressed genes were not equally sensitive to PI3K inhibition or to the epigenetic drugs 50-aza-20-deoxycitidine and Trichostatin A (Supplementary Fig. S7C). Thus, it is possible that cell-type–specific signatures evolve, secondary to STAT3 posttranslational modifications and also coregulation by other Figure 6. pathways. Sin3A interacts with STAT3 and controls gene expression in breast cancer cells. A, Western blot analyses of nuclear lysates of human breast adenocarcinoma MDA-MB-231 and MCF7 cells. Data are representative of three independent determinations. B, Nuclear lysates of MDA-MB-231 cells were immunoprecipitated with anti-Sin3A antibodies or normal rabbit IgG and analyzed with the indicated antibodies. One representative experiment out of three is depicted. MDA-MB-231 cells were transfected with siControl or siSIN3A and analyzed by qRT-PCR (C) or Western blot (D). C, DDIT3 mRNA levels calculated over the siControl sample are shown. Error bars, SD (n ¼ 4). Two-tailed paired t test, P < 0.005. E and F, Cell growth and viability of siControl and siSIN3A-transfected MDA-MB-231 cells were evaluated 5 days after transfection by Trypan Blue counts (E) and by FACS analysis of Annexin V expression (F; n > 3).

STAT3 acetylation is normally catalyzed by the acetyltrans- ferases p300/CBP (19, 41). Although p300/CBP was not previously associated with NPM-ALK, the Ser/Thr kinases MAPK, PI3K, and mTOR, activated downstream to NPM-ALK (30, 31), could represent putative missing links. Indeed, phosphorylation of Ser727 acts as a docking site for p300/CBP (44). We found that inhibition of MAPK, PI3K, and mTOR reduced Ser727 phosphorylation and also Lys685 acetylation. As resveratrol also impacts on mTOR activity, this could represent an additional mechanism of controlling STAT3 acetylation (45, 46). In this view, overt Ser/Thr kinases activity, a shared event in a number of cancers (41, 47– 50), might be responsible for constitutive STAT3 Ser727 phosphorylation and Lys acetylation and ultimately for its activity as a Figure 7. transcriptional repressor. Schematic model of the cooperation between Sin3A and STAT3 in TSGs Interfering with Sin3A expression unleashed the transcriptional transcriptional repression. The aberrant expression of the ALK chimeric break imposed on TSG expression. This correlated with the kinase leads to constitutive STAT3 Y705 phosphorylation and transcriptional induction of tumor cell death in vitro and with a reduction of activity, promoting oncogene expression. In addition, ALK activates several Ser/Thr kinases that, in turn, lead to STAT3 Ser727 phosphorylation and ALCL tumorigenic potential in vivo. The mechanism by which lysine acetylation. Acetylated STAT3 binds to the Sin3A complex, likely Sin3A contributes to TSGs repression remains to be established. It promoting its recruitment to the regulatory elements of several tumor is reasonable to suggest that via STAT3, Sin3A docks epigenetic suppressor genes (PRDM1 in the scheme), jointly leading to epigenetic modiﬁers to critical promoters and/or enhancer regions, silencing silencing.

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Resveratrol increased Blimp-1 and Chop levels. Although MAD1 (54) and Sin3A-Foxo1 (55), while sparing additional changes were very small, they reached statistical significance over specific functions should prove more selective, and less toxic than independent determinations. Whether such small changes per se the ones tested to date. would be biologically relevant could be debatable and remains to be determined. Single overexpression of some of the reexpressed fl genes (i.e., DDIT3/CHOP, Blimp1, FOS) was sufficient to induce Disclosure of Potential Con icts of Interest fl cancer cells to apoptose in previous studies (32, 33). Yet, knocking No potential con icts of interest were disclosed. down only one protein at a time did not rescue cells from apoptosis induced by STAT3 inhibition, suggesting that several Authors' Contributions factors contribute to tumor cell death consequent to STAT3 Conception and design: G. Gambi, G. Inghirami, L. Icardi, A. Mondino blocking. We suggest that a concomitant upregulation of several Development of methodology: G. Gambi, L. Ricci, R. Wang, L. Icardi of the STAT3-repressed TSG contributes to the delay in tumor cell Acquisition of data (provided animals, acquired and managed patients, growth and the observed apoptotic phenotype. provided facilities, etc.): G. Gambi, M. Ponzoni, G. Inghirami The identity of STAT3 acetylated lysines and their influence on Analysis and interpretation of data (e.g., statistical analysis, biostatistics, other posttranslational modifications might also shape the STAT3 computational analysis): G. Gambi, L. Ricci, A. Verma, O. Elemento, G. Inghirami, L. Icardi, A. Mondino interactome. We found Lys685 to be constitutively acetylated. Writing, review, and/or revision of the manuscript: G. Gambi, M. Ponzoni, Nevertheless, our results together with previous studies (26) do G. Inghirami, L. Icardi, A. Mondino not support Lys685 to be uniquely responsible for STAT3–Sin3A Study supervision: L. Icardi, A. Mondino interaction. Indeed, mutation of STAT3 Lys685 in either acetyl- Other (performed experiments): E.Di Simone mimicking (STAT3K685Q) or acetyl-deficient (STAT3K685R) did Other (performed experiments and acquired samples): V. Basso not affect STAT3 binding to Sin3A. Whether additional Lys residues could be acetylated and control the interaction with Sin3A allow- Acknowledgments ing for cell-specific events remains to be defined. As the human The authors are grateful to Dr. Chiarle and Dr. Piva (University of Torino, STAT3 homolog contains several, extremely conserved lysine resi- Torino, Italy), Dr. Bernardi and Dr. Bondanza (San Raffaele Scientific dues ( 47), it is likely that different STAT3 acetylation patterns Institute, Milan, Italy), Dr. Tavernier (VIB, Ghent, Belgium), and Dr. Calautti might exist and account for different biological functions. The (Dulbecco Telethon Institute, c/o Molecular Biotechnology Center, Univer- fi sity of Torino) for useful reagents. This work was supported by the generous identi cation of the critical residues will be needed to address this gift of an anonymous donor and by grants from the Associazione Italiana issue. Given the notions that (i) the N-terminal domains of STAT3 Ricerca sul Cancro (AIRC IG15883; to A. Mondino) and from AIRC 5 1000 and STAT5 are required for TSG transcriptional repression (33, 52), (no. 10007) and the Leukemia Lymphoma Society (SCOR 2015; to G. and (ii) the acetylation of two lysine residues located in the Inghirami). L. Icardi was supported by fellowships funded by the San N-terminal domain of STAT3 (i.e., on Lys49 and Lys87) promotes Raffaele International Postdoctoral Programme (INVEST) and the Associa- fi STAT3–Sin3A interaction (26), it is tempting to speculate a role for zione Italiana per la Ricerca sul Cancro (AIRC-iCare) with partial nancial support by the European Commission (FP7–Marie Curie Actions–People– the N-terminal domain of STAT3 in Sin3A-mediated transcription- COFUND). L. Ricci was supported by an intramural competitive "Seed al repression. In support to this possibility, cell-permeable analo- Grant" financed with FY2014 5 1000 funds from the Italian Ministry of gues of this region were found to specifically bind to STAT3 and Health. inhibit STAT3-dependent cancer cell survival and growth (53). In conclusion, together, our data support a previously unrec- The costs of publication of this article were defrayed in part by the ognized role for Sin3A in STAT3 oncogenic potential. Because the payment of page charges. This article must therefore be hereby marked binding of STAT3 to Sin3A is controlled by extracellular signals in advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate physiologic conditions (26), their constitutive interaction in this fact. cancer cells might represent a promising druggable target. The generation of molecules able to outcompete or disrupt the bind- Received February 2, 2018; revised June 14, 2018; accepted January 23, 2019; ing with selected interactors, as described previously for Sin3A- published first January 28, 2019.

References 1. Garcia R, Bowman TL, Niu G, Yu H, Minton S, Muro-Cacho CA, et al. 6. Tabbo F, Barreca A, Piva R, Inghirami G. ALK signaling and target therapy in Constitutive activation of Stat3 by the Src and JAK tyrosine kinases anaplastic large cell lymphoma. Front Oncol 2012;2:41. participates in growth regulation of human breast carcinoma cells. Onco- 7. Chiarle R, Gong JZ, Guasparri I, Pesci A, Cai J, Liu J, et al. NPM-ALK gene 2001;20:2499–513. transgenic mice spontaneously develop T-cell lymphomas and plasma cell 2. Huang M, Page C, Reynolds RK, Lin J. Constitutive activation of stat 3 tumors. Blood 2003;101:1919–27. oncogene product in human ovarian carcinoma cells. Gynecol Oncol 2000; 8. Zhang Q, Wei F, Wang HY, Liu X, Roy D, Xiong QB, et al. The potent 79:67–73. oncogene NPM-ALK mediates malignant transformation of normal human 3. Scholz A, Heinze S, Detjen KM, Peters M, Welzel M, Hauff P, et al. Activated CD4(þ) T lymphocytes. Am J Pathol 2013;183:1971–80. signal transducer and activator of transcription 3 (STAT3) supports the 9. Chiarle R, Simmons WJ, Cai H, Dhall G, Zamo A, Raz R, et al. Stat3 is malignant phenotype of human pancreatic cancer. Gastroenterology 2003; required for ALK-mediated lymphomagenesis and provides a possible 125:891–905. therapeutic target. Nat Med 2005;11:623–9. 4. Kanda N, Seno H, Konda Y, Marusawa H, Kanai M, Nakajima T, et al. 10. Zamo A, Chiarle R, Piva R, Howes J, Fan Y, Chilosi M, et al. Anaplastic STAT3 is constitutively activated and supports cell survival in associa- lymphoma kinase (ALK) activates Stat3 and protects hematopoietic cells tion with survivin expression in gastric cancer cells. Oncogene 2004;23: from cell death. Oncogene 2002;21:1038–47. 4921–9. 11. Zhang Q, Raghunath PN, Xue L, Majewski M, Carpentieri DF, Odum N, 5. Inghirami G, Pileri SA. Anaplastic large-cell lymphoma. Semin Diagn et al. Multilevel dysregulation of STAT3 activation in anaplastic lymphoma Pathol 2011;28:190–201. kinase-positive T/null-cell lymphoma. J Immunol 2002;168:466–74.

www.aacrjournals.org Cancer Res; 2019 OF11

Gambi et al.

12. Piva R, Pellegrino E, Mattioli M, Agnelli L, Lombardi L, Boccalatte F, et al. 32. Boi M, Rinaldi A, Kwee I, Bonetti P, Todaro M, Tabbo F, et al. PRDM1/ Functional validation of the anaplastic lymphoma kinase signature identifies BLIMP1 is commonly inactivated in anaplastic large T-cell lymphoma. CEBPB and BCL2A1 as critical target genes. J Clin Invest 2006;116:3171–82. Blood 2013;122:2683–93. 13. Marzec M, Liu X, Wong W, Yang Y, Pasha T, Kantekure K, et al. Oncogenic 33. Timofeeva OA, Tarasova NI, Zhang X, Chasovskikh S, Cheema AK, Wang H, kinase NPM/ALK induces expression of HIF1alpha mRNA. Oncogene et al. STAT3 suppresses transcription of proapoptotic genes in cancer cells 2011;30:1372–8. with the involvement of its N-terminal domain. Proc Natl Acad Sci U S A 14. Ambrogio C, Martinengo C, Voena C, Tondat F, Riera L, di Celle PF, et al. 2013;110:1267–72. NPM-ALK oncogenic tyrosine kinase controls T-cell identity by transcrip- 34. Boi M, Zucca E, Inghirami G, Bertoni F. PRDM1/BLIMP1: a tumor sup- tional regulation and epigenetic silencing in lymphoma cells. Cancer Res pressor gene in B and T cell lymphomas. Leuk Lymphoma 2015;56: 2009;69:8611–9. 1223–8. 15. Zhang Q, Wang HY, Liu X, Bhutani G, Kantekure K, Wasik M. IL-2R 35. Nie Y, Erion DM, Yuan Z, Dietrich M, Shulman GI, Horvath TL, et al. STAT3 common gamma-chain is epigenetically silenced by nucleophosphin- inhibition of gluconeogenesis is downregulated by SirT1. Nat Cell Biol anaplastic lymphoma kinase (NPM-ALK) and acts as a tumor suppressor 2009;11:492–500. by targeting NPM-ALK. Proc Natl Acad Sci U S A 2011;108:11977–82. 36. Kulkarni SS, Canto C. The molecular targets of resveratrol. Biochim 16. Piva R, Agnelli L, Pellegrino E, Todoerti K, Grosso V, Tamagno I, et al. Gene Biophys Acta 2015;1852:1114–23. expression profiling uncovers molecular classifiers for the recognition of 37. Wu P, Wu D, Zhao L, Huang L, Shen G, Huang J, et al. Prognostic role of anaplastic large-cell lymphoma within peripheral T-cell neoplasms. J Clin STAT3 in solid tumors: a systematic review and meta-analysis. Oncotarget Oncol 2010;28:1583–90. 2016;7:19863–83. 17. Hassler MR, Klisaroska A, Kollmann K, Steiner I, Bilban M, Schiefer AI, et al. 38. Banerjee K, Resat H. Constitutive activation of STAT3 in breast cancer cells: Antineoplastic activity of the DNA methyltransferase inhibitor 5-aza-2'- A review. Int J Cancer 2016;138:2570–8. deoxycytidine in anaplastic large cell lymphoma. Biochimie 2012;94: 39. Borra MT, Smith BC, Denu JM. Mechanism of human SIRT1 activation by 2297–307. resveratrol. J Biol Chem 2005;280:17187–95. 18. Piazza R, Magistroni V, Mogavero A, Andreoni F, Ambrogio C, Chiarle R, 40. Hou X, Rooklin D, Fang H, Zhang Y. Resveratrol serves as a protein- et al. Epigenetic silencing of the proapoptotic gene BIM in anaplastic large substrate interaction stabilizer in human SIRT1 activation. Sci Rep 2016; cell lymphoma through an MeCP2/SIN3a deacetylating complex. Neo- 6:38186. plasia 2013;15:511–22. 41. Zhuang S. Regulation of STAT signaling by acetylation. Cell Signal 2013;25: 19. Icardi L, De Bosscher K, Tavernier J. The HAT/HDAC interplay: multilevel 1924–31. control of STAT signaling. Cytokine Growth Factor Rev 2012;23:283–91. 42. Ma L, Huang C, Wang XJ, Xin DE, Wang LS, Zou QC, et al. Lysyl oxidase 3 is 20. Yuan ZL, Guan YJ, Chatterjee D, Chin YE. Stat3 dimerization regulated by a dual-specificity enzyme involved in STAT3 deacetylation and deacetyli- reversible acetylation of a single lysine residue. Science 2005;307:269–73. mination modulation. Mol Cell 2017;65:296–309. 21. Ray S, Boldogh I, Brasier AR. STAT3 NH2-terminal acetylation is activated 43. Lei Y, Liu L, Zhang S, Guo S, Li X, Wang J, et al. Hdac7 promotes lung by the hepatic acute-phase response and required for IL-6 induction of tumorigenesis by inhibiting Stat3 activation. Mol Cancer 2017;16:170. angiotensinogen. Gastroenterology 2005;129:1616–32. 44. Schuringa JJ, Schepers H, Vellenga E, Kruijer W. Ser727-dependent tran- 22. Wang R, Cherukuri P, Luo J. Activation of Stat3 sequence-specific DNA scriptional activation by association of p300 with STAT3 upon IL-6 binding and transcription by p300/CREB-binding protein-mediated acet- stimulation. FEBS Lett 2001;495:71–6. ylation. J Biol Chem 2005;280:11528–34. 45. Frojdo S, Cozzone D, Vidal H, Pirola L. Resveratrol is a class IA phosphoi- 23. Lee H, Zhang P, Herrmann A, Yang C, Xin H, Wang Z, et al. Acetylated nositide 3-kinase inhibitor. Biochem J 2007;406:511–8. STAT3 is crucial for methylation of tumor-suppressor gene promoters and 46. Cao Z, Fang J, Xia C, Shi X, Jiang BH. trans-3,4,5'-Trihydroxystibene inhibits inhibition by resveratrol results in demethylation. Proc Natl Acad Sci U S A hypoxia-inducible factor 1alpha and vascular endothelial growth factor 2012;109:7765–9. expression in human ovarian cancer cells. Clin Cancer Res 2004;10: 24. Li J, Cui G, Sun L, Wang SJ, Li YL, Meng YG, et al. STAT3 acetylation- 5253–63. induced promoter methylation is associated with downregulation of the 47. Dhillon AS, Hagan S, Rath O, Kolch W. MAP kinase signalling pathways in ARHI tumor-suppressor gene in ovarian cancer. Oncol Rep 2013;30: cancer. Oncogene 2007;26:3279–90. 165–70. 48. Yuan TL, Cantley LC. PI3K pathway alterations in cancer: variations on a 25. Albrengues J, Bertero T, Grasset E, Bonan S, Maiel M, Bourget I, et al. theme. Oncogene 2008;27:5497–510. Epigenetic switch drives the conversion of fibroblasts into proinvasive 49. Yi KH, Lauring J. Recurrent AKT mutations in human cancers: functional cancer-associated fibroblasts. Nat Commun 2015;6:10204. consequences and effects on drug sensitivity. Oncotarget 2016;7:4241–51. 26. Icardi L, Mori R, Gesellchen V, Eyckerman S, De Cauwer L, Verhelst J, et al. 50. Kim LC, Cook RS, Chen J. mTORC1 and mTORC2 in cancer and the tumor The Sin3a repressor complex is a master regulator of STAT transcriptional microenvironment. Oncogene 2017;36:2191–201. activity. Proc Natl Acad Sci U S A 2012;109:12058–63. 51. Ram PT, Iyengar R. G protein coupled receptor signaling through the Src 27. Crescenzo R, Abate F, Lasorsa E, Tabbo F, Gaudiano M, Chiesa N, et al. and Stat3 pathway: role in proliferation and transformation. Oncogene Convergent mutations and kinase fusions lead to oncogenic STAT3 acti- 2001;20:1601–6. vation in anaplastic large cell lymphoma. Cancer Cell 2015;27:516–32. 52. Moriggl R, Sexl V, Kenner L, Duntsch C, Stangl K, Gingras S, et al. Stat5 28. Zhang Q, Wang HY, Marzec M, Raghunath PN, Nagasawa T, Wasik MA. tetramer formation is associated with leukemogenesis. Cancer Cell 2005;7: STAT3- and DNA methyltransferase 1-mediated epigenetic silencing of 87–99. SHP-1 tyrosine phosphatase tumor suppressor gene in malignant T lym- 53. Timofeeva OA, Gaponenko V, Lockett SJ, Tarasov SG, Jiang S, phocytes. Proc Natl Acad Sci U S A 2005;102:6948–53. Michejda CJ, et al. Rationally designed inhibitors identify STAT3 29. Zhang Q, Wang HY, Liu X, Wasik MA. STAT5A is epigenetically silenced by N-domain as a promising anticancer drug target. ACS Chem Biol the tyrosine kinase NPM1-ALK and acts as a tumor suppressor by recip- 2007;2:799–809. rocally inhibiting NPM1-ALK expression. Nat Med 2007;13:1341–8. 54. Farias EF, Petrie K, Leibovitch B, Murtagh J, Chornet MB, Schenk T, et al. 30. Werner MT, Zhao C, Zhang Q, Wasik MA. Nucleophosmin-anaplastic Interference with Sin3 function induces epigenetic reprogramming and lymphoma kinase: the ultimate oncogene and therapeutic target. Blood differentiation in breast cancer cells. Proc Natl Acad Sci U S A 2010;107: 2017;129:823–31. 11811–6. 31. Palmer RH, Vernersson E, Grabbe C, Hallberg B. Anaplastic lymphoma 55. Langlet F, Haeusler RA, Linden D, Ericson E, Norris T, Johansson A, et al. kinase: signalling in development and disease. Biochem J 2009;420: Selective Inhibition of FOXO1 activator/repressor balance modulates 345–61. hepatic glucose handling. Cell 2017;171:824–35.

OF12 Cancer Res; 2019 Cancer Research

The Transcriptional Regulator Sin3A Contributes to the Oncogenic Potential of STAT3

Giovanni Gambi, Elisabetta Di Simone, Veronica Basso, et al.

Cancer Res Published OnlineFirst January 28, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-18-0359

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2019/01/26/0008-5472.CAN-18-0359.DC1

Visual A diagrammatic summary of the major findings and biological implications: Overview http://cancerres.aacrjournals.org/content/early/2019/04/16/0008-5472.CAN-18-0359/ F1.large.jpg

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