Merkel Cell Polyomavirus Small T Antigen Activates Noncanonical

Published OnlineFirst August 4, 2020; DOI: 10.1158/1541-7786.MCR-20-0587

MOLECULAR CANCER RESEARCH | CANCER GENES AND NETWORKS

Merkel Cell Polyomavirus Small T Antigen Activates Noncanonical NFkB Signaling to Promote Tumorigenesis Jiawei Zhao1, Yuemeng Jia2, Shunli Shen3, Jiwoong Kim4, Xun Wang2, Eunice Lee1, Isaac Brownell5, Jeong Hee Cho-Vega6, Cheryl Lewis7, Jade Homsi7,8, Rohit R. Sharma7,9, and Richard C. Wang1,7

ABSTRACT ◥ Multiple human polyomaviruses (HPyV) can infect the skin, NFkB signaling was required for SASP cytokine secretion, which but only Merkel cell polyomavirus (MCPyV) has been implicated promoted the proliferation of MCPyV sT–expressing cells in the development of a cancer, Merkel cell carcinoma (MCC). through autocrine signaling. Virus-positive MCC cell lines and While expression of HPyV6, HPyV7, and MCPyV small T tumors showed ncNFkB pathway activation and SASP gene antigens (sT), all induced a senescence-associated secretory expression, and the inhibition of ncNFkB signaling prevented phenotype (SASP), MCPyV sT uniquely activated noncanonical VP-MCC cell growth in vitro and in xenografts. We identify NFkB(ncNFkB), instead of canonical NFkBsignaling,toevade MCPyV sT–induced ncNFkB signaling as an essential tumori- p53-mediated cellular senescence. Through its large T stabiliza- genic pathway in MCC. tion domain, MCPyV sT activated ncNFkB signaling both by inducing H3K4 trimethylation-mediated increases of NFKB2 Implications: This work is the ﬁrst to identify the activation and RELB transcription and also by promoting NFKB2 stabili- of ncNFkB signaling by any polyomavirus and its critical role in zation and activation through FBXW7 inhibition. Noncanonical MCC tumorigenesis.

Introduction strongly linked to a malignancy in humans so far (5). The unique transforming properties of MCPyV appear to reside in the early region Human polyomaviruses (HPyV) are small, double-stranded DNA of the genome, which encodes for the small T antigen (sT) and through viruses most intensely studied for their transforming properties. alternative splicing, large T antigen (LT). This region is essential for HPyVs were first noted to be clinically relevant as causes of infectious viral DNA replication (6, 7) and also for the transforming properties of diseases in immunosuppressed patients. More recently, Merkel cell murine polyomavirus and the Rhesus macaque polyomavirus polyomavirus (MCV or MCPyV) has been identified as a critical factor SV40 (8). However, in contrast to other polyomaviruses, the cellular in the development of approximately 80% of Merkel cell carcinomas transforming activity of MCPyV appears to depend largely on its sT, (MCC; ref. 1), a rare but deadly skin cancer. Antibodies targeting the and not on its LT, as the knockdown of sT is sufficient to cause the programmed cell death-1 (PD-1) pathway can be effective and have arrest of MCC cells in vitro (9). A more detailed analysis of how become the standard of care for patients with advanced disease (2–4). MCPyV differs from other cutaneous HPyVs is essential for under- However, approximately 50% of patients with advanced MCC are standing conserved oncogenic pathways and may yield novel thera- resistant to checkpoint inhibitor therapy. Thus, a better understanding peutic targets for patients with virus-positive MCC (VP-MCC). of the role of MCPyV in MCC development may help to spur the One important difference between MCPyV and SV40 sTs is the development of novel therapies. presence of the large T stabilization domain (LSD domain) found in Up to 14 species of HPyVs have been identified, and as many as 5 MCPyV sT, but not in the sT of SV40 or other cutaneous polyoma- species have been detected on human skin, yet only MCPyV has been viruses, such as human polyomaviruses 6 and 7 (HPyV6/7). The LSD domain of MCPyV sT is essential for LT stabilization, LT-mediated 1 Department of Dermatology, UT Southwestern Medical Center, Dallas, Texas. viral replication, and its transforming properties. MCPyV sT can 2 ’ Children s Research Institute, UT Southwestern Medical Center, Dallas, Texas. inhibit the F-box/WD repeat-containing protein 7 (FBXW7) pro- 3Department of Hepatic Surgery, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong, China. 4Department of Clinical Science, UT tein (6), a tumor suppressor protein that promotes the degradation of Southwestern Medical Center, Dallas, Texas. 5Cutaneous Development and many proto-oncogenes including c-Myc and Cyclin E (10). However, Carcinogenesis Section, NIAMS, Bethesda, Maryland. 6Department of Pathology as FBXW7 knockdown alone does not rescue transformation by an and Laboratory Medicine, Sylvester Comprehensive Cancer Center and Miller LSD domain mutant MCPyV sT (6), it is likely that the MCPyV sT 7 School of Medicine, University of Miami, Miami, Florida. Harold C. Simmons possesses additional transforming properties. 8 Cancer Center, UT Southwestern Medical Center, Dallas, Texas. Department of There have been conflicting reports on the impact of MCPyV sT on Internal Medicine, UT Southwestern Medical Center, Dallas, Texas. 9Department of Surgery, UT Southwestern Medical Center, Dallas, Texas. NFkB signaling, with some reports suggesting an inhibition of canonical NFkB pathways through an indirect interaction with the NEMO/ Note: Supplementary data for this article are available at Molecular Cancer IKKg protein (11) while others have shown an activation of the same Research Online (http://mcr.aacrjournals.org/). pathway (12). Because of NFkB’s central role in coordinating immune Corresponding Author: Richard C. Wang: UT Southwestern Medical Center, and inflammatory responses, its inhibition has been proposed as a Harry Hines Blvd Dallas, TX 75390-9069. Phone: 214-648-3430; Fax: 214-648- 5554; E-mail: [email protected]; and Jiawei Zhao, E-mail: mechanism to allow MCPyV to subvert the immune responses against [email protected] viral infection. In addition to its role in immune signaling, NFkB activation has been implicated in pleiotropic, and sometimes contra- Mol Cancer Res 2020;XX:XX–XX dictory activities with respect to transformation. The activation doi: 10.1158/1541-7786.MCR-20-0587 of canonical NFkB signaling has been linked to the development of 2020 American Association for Cancer Research. senescence and to the upregulated secretion of a diverse set of

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cytokines, growth factors, and proteases (e.g., IL1b, IL6, IL8, and doxycycline-inducible expression system, small T antigens were GM-CSF), also known as the senescence-associated secretory pheno- cloned into pLVX-TRE3G vector through InFusion cloning type (SASP; refs. 13–15). In contrast, signaling from the noncanonical (Clontech) with single digestion by BamHI. The annealed oligos were NFkB (ncNFkB) pathway, in which NFkB2/p52 and RELB are then ligated into predigested pLKO.1-hygro vector (AgeI þ EcoRI). activated, bypasses cellular senescence, at least in part through the For lentiviral packaging and transduction, transfer plasmid, pMD2. induction of the EZH2 histone methyltransferase (16, 17). Notably, G and psPAX2 plasmids were cotransfected into Lenti-X 293T cells while MCPyV sT signaling has been studied in the context of canonical by Lipofectamine 3000 at molar ratio of 1:1:1. Viral supernatant was NFkB activation, its impact on ncNFkB signaling has not been directly harvested at 48 and 72 hours posttransfection. A total of 8 104 BJ examined. cells/well were plated into 6-well plate the day before transduction. Here, we express MCPyV sT and the sTs of two cutaneous poly- The cells were then spin-transduced at 35C, 2,500 rpm for 90 omaviruses (HPyV6/7) currently not thought to be transforming. minutes. The cells were selected 48 hours posttransduction While HPyV6/7 sTs induce senescence, MCPyV sT bypasses cellular with 2 mg/mL puromycin (for constitutive expression plasmid), senescence and transforms primary human fibroblasts. MCPyV sT 15 mg/mL blasticidin, and 0.5 mg/mL G418 (for doxycycline- upregulated ncNFkB signaling by increasing the levels of NFKB2 and inducible expression system). RELB through transcriptional and posttranslational mechanisms. Using both chemical and genetic inhibition of the pathway, we find Soft agarose colony formation assay that the activation of ncNFkB signaling was essential for the induction HPyV6, HPyV7, and MCPyV small T–expressing vectors were of SASP gene transcription and the serum independent growth of transduced into Rat2 fibroblasts and selected with puromycin at MCC. Using a specific inhibitor of ncNFkB, we find that VP-MCC cell 4 mg/mL. The stable cell lines were then resuspended and plated in lines require ncNFkB signaling for their growth in vitro and in vivo. 0.35% agarose DMEM on top of 0.5% agarose DMEM base in 6-well plate at 25,000 cells/well of 6-well plate. The cells were allowed to grow for 3 weeks and stained with 0.005% crystal violet dye in 6.25% Materials and Methods ethanol for more than 1 hour at room temperature avoiding light. Cell culture and tumor tissue Thecolonieswerethenidentified and counted under dissecting BJ dermal fibroblasts were cultured in complete BJ medium (4:1 microscope. ratio of high glucose DMEM and Medium 199, 10% FBS, 2 mmol/L glutamine, and 1 mmol/L sodium pyruvate; Sigma-Aldrich). Lenti-X MTT/XTT–mediated cell growth assay HEK-293T cells (Clontech) and Rat2 (ATCC) were cultured in DMEM Doxycycline-inducible HPyV6, HPyV7, and MCPyV small T anti- supplemented with 10% FBS and used at early (<5 passages). All media gen–expressing cells were plated at 1,000/well of 96-well plate for a were tested for Mycoplasma positivity by PCR-based method monthly. total of 8 plates without addition of doxycycline. The cells were assayed VP-MCC cancer cell lines (MKL-1, MKL-2, MS-1, and WaGa) were for MTT per plate for every 24 hours. Doxycycline (1 mg/mL) was cultured in RPMI1640 supplemented with 20% FBS. VN-MCC cancer added to doxycycline-induced plates after 24 hours (after the first plate cell lines (MCC13, MCC26, UISO) were cultured in RPMI1640 was assayed). MTT assay was performed according to manufacturer’s supplemented with 10%FBS. MCC lines were confirmed through instructions. Briefly, 10 mL of 12 mmol/L MTT was added to 100 mL verifying the expression of sT (Supplementary Fig. S13A) and/or medium and incubated for 8 hours at 37C. MTT-containing medium assessment of cell morphology. For chemical inhibition of RELB, was then removed and dissolved in 50 mL DMSO and absorbance read DMSO vehicle control or 50 nmol/L calcitriol were added into virally at 540 nm. All the later time points were normalized by the first plate infected cell culture medium. For experiments utilizing conditioned readings. For XTT assays (Sigma Aldrich) of VP-MCC cells—MKL-1, media, BJ fibroblasts pretransduced with either vector, HPyV6-sT, MKL-2 and MS-1—were trypsinized into single-cell suspension and HPyV7-sT, or MCPyV-sT and were cultured in 2% FBS for 48 hours to cells were plated at 5,000/well in 96-well plate. The XTT reaction was produce cytokine-rich, conditioned medium. For MTT-mediated done according to manufacturer’s instructions with absorbance read at growth curve quantification, vector or LSD mutant–transduced 450 nm. cells were plated in 96-well plate in triplicate. Cells cultured in 2% FBS medium or MCPyV-sT WT cells cultured with protein Senescence-associated b-galactosidase staining transport inhibitor (PTI) cocktail (proprietary mixture of Brefeldin Senescence-associated b-galactosidase (SA-b-gal) staining was A and Monensin; Thermo Fisher Scientific) were supplemented performed according to manufacturer’s instruction (Thermo Fisher with conditioned medium harvested from either HPyV6, 7, or Scientific). Briefly, cells were plated at 70% confluence and then fixed MCPyV sT–expressing cell culture. Cell growth was then monitored by formaldehyde and glutaraldehyde fixative agents for 15 minutes at for 6 days by MTT. Frozen MCC tumors were obtained from the room temperature. Cells were then washed and stained by X-gal at final UTSW Tissue Management Shared Resource. Tissue collection was concentration (1 mg/mL) in staining buffer at pH ¼ 6.0 (exact) in performed with written informed consent from the patients and presence of potassium ferricyanide and potassium ferrocyanide in the studies were approved by the UT Southwestern institutional 37 C bacteria culture incubator (free of CO2) for 16 hours. review board. Immunofluorescence staining Cloning and lentiviral packaging and transduction For immunofluorescence staining, cells were fixed in 4% parafor- Primers used for cloning and qRT-pCR are listed in Supplementary maldehyde at room temperature avoiding light for 10 minutes. Fixed Table S1. For constitutive expression system, HPyV6, HPyV7, and cells were then permeabilized with 0.1% Triton X-100 at room MCPyV sT antigens were amplified from the viral genome plasmid temperature for 10 minutes. Cells were then blocked with 5% normal and MCPyV sT antigen was tagged with 3XFLAG at the N terminus. goat serum at room temperature for 1 hour. Primary antibody was then Amplified cDNAs were then cloned into pLEX vector with SpeIþNotI incubated at 4C overnight, followed by 1 hour room temperature (for HPyV6 and MCPyV) and BamHIþNotI (for MCPyV). For incubation of secondary antibody.

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BrdU incorporation assay N-lauroylsarcosine) using a Covaris M220 Focused-ultrasonicator BrdU incorporation assay was done as previously reported until the chromatin was sonicated to 200–700 bp fragments. The with slight modifications (18). BrdU was added to cell culture medium fragmented chromatin was then precleared by mouse IgG and incu- at a final concentration of 10 mmol/L and incubated for 4 hours at bated with M2 FLAG antibody bound to protein A/G dynabeads 37C. Then BrdU-containing medium was removed and cells were overnight at 4C. The magnetic beads were then washed by the same washed with PBS before proceeding to immunofluorescence staining wash buffers in native ChIP. Input and ChIP DNA were reverse protocol. Fixed cells were treated with 1.5 N HCl at room temperature crosslinked by the same SDS elution buffer at 65C for at least 6 hours. for 20 minutes for antigen retrieval before permeabilization. The eluted DNA was then treated with RNase and protease K and recovered by phenol–chloroform extraction. Immunoblot analysis and subcellular fractionation Cells were harvested and lysed in RIPA buffer (50 mmol/L Tris qRT-PCR analysis pH 8.0, 150 mmol/L NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium Total RNAs were isolated by Qiagen RNeasy RNA Miniprep Kit (BJ deoxycholate) in the presence of 1 protease/phosphatase inhibitor cells) or TRIzol (MCC xenografts and patient samples). RNA con- cocktail (Thermo Fisher Scientific) on ice for 15 minutes. Patient centration and purity were measured and checked by Nanodrop. samples and xenografted tumors were homogenized in liquid cDNA was synthesized using 1 mg of total RNA by iScript cDNA nitrogen and protein was extracted by T-PER protein extraction Synthesis Kit. The reverse transcribed cDNA was then used for real- buffer. Chromatin and cell debris were then pelleted down by time PCR by CFX96 Real Time Cycler (Bio-Rad) as per manufacturer’s centrifuging at 12,000 rpm at 4C. The supernatant was collected, instruction using 2X SYBR green PCR Master Mix (Applied and BCA assay was used to quantify protein concentration. Protein Biosystems). Target gene expression was normalized by housekeeping lysates were fully denatured by 1 Laemmli sample buffer and gene ACTB (b-actin). boiled at 95C for 10 minutes. Protein lysates were then separated in SDS-PAGE gel and blotted onto polyvinylidene difluoride mem- c-Myc turnover assay brane. Membrane was then blocked by 5% nonfat milk in TBS/T (20 293T cells were cotransfected with pLEX vector, HPyV6-sT, mmol/L Tris pH 7.4, 150 mmol/L NaCl, 0.1% (w/v) Tween-20) for HPyV7-sT, wild-type MCPyV-sT, or LSD-mutant MCPyV-sT and at least 1 hour before incubation with primary antibody at 4Cfor pcDNA3.1-cMyc expression vector. After 48 hours, cells were treated overnight. The blot was then probed with horseradish peroxidase with cycloheximide (0.1 mg/mL) to halt translation. The cells were (HRP)-conjugated secondary antibody and developed using ECL then harvested at 0 (immediately), 1, 2, and 4-hour intervals. c-Myc reagent. Subcellular fractionation was performed according to protein expression were then assessed by Western blot analysis. manufacturer’s instructions. The protein concentration of each fraction was quantified by BCA assay. Mice and xenograft of MCC lines NOD-scid/IL2Rgamma immunodeficient mice were housed in a Chromatin immunoprecipitation sterile facility and all mouse experiments were approved by UT Native ChIP Southwestern Medical Center’s institutional Animal Care and Use Total chromatin was purified by lysing cells with hypotonic buffer Committee. For xenografts, MKL-1 and WaGa cells were cultured in þ 7 (10 mmol/L HEPES pH7.9, 10 mmol/L KCl, 1.5 mmol/L MgCl2, RPMI 20% FBS and allowed to grow until at least 1 10 viable cells 0.34 mol/L sucrose, 10% glycerol, and 1 mmol/L DTT) followed by were obtained. A total of 1 107 cells in PBS were subcutaneously No-salt buffer (3 mmol/L EDTA, 0.2 mmol/L EGTA, 1 mmol/L DTT) injected into NSG immunodeficient mice and allowed to grow for in the presence of protease inhibitor cocktail and phenylmethylsulfo- 1 month. Five days after injection of the cancer cells, mice were treated nylfluoride. Purified chromatin was washed by hypotonic buffer for at with either SN52-mutant peptide or SN52 functional peptide twice least twice to fully remove residual EDTA and EGTA before proceed- weekly at 40 mg/injection in situ by subcutaneous injection. Tumors ing to micrococcal nuclease (MNase) digestion. 5% of the digested were then harvested for downstream analysis. Fraction of each tumor chromatin was reserved as input DNA. The rest of the digested was harvested and flash frozen for protein/RNA extraction. genomic DNA was precleared by rabbit IgG and then immunopre- cipiated by H3K4me3 antibody bound to protein A/G dynabeads in ELISA RIPA buffer for at 4C. The complex was then washed by RIPA buffer Cells were typically harvested 7 days after transduction with a fresh twice, high salt RIPA buffer (RIPA buffer þ 300 mmol/L NaCl) twice, medium changed 2 days before harvesting to allow for cytokine LiCl wash buffer (250 mmol/L LiCl, 0.5% NP-40, 0.5% sodium accumulation. Uncoated human IL8 ELISA kit was purchased from deoxycholate) twice and 1XTE buffer þ 50 mmol/L NaCl. The DNA PeproTech and the experiment was performed according to manu- was then eluted in SDS elution buffer (50 mmol/L Tris pH 8.0, facturer’s instructions. Briefly, 1 mg/mL in PBS capture antibody was 10 mmol/L EDTA, 1% SDS) at 65C for at least 6 hours and treated plated onto ELISA microplate for overnight incubation (at least with RNase and protease K. The DNA was finally recovered by phenol 12 hours). Then excess capture antibody was then removed with wash chloroform extraction. buffer (0.05% Tween-20 in PBS). The plate was then blocked with 1% BSA in PBS for 1 hour at room temperature. After washing three times Crosslinked ChIP with wash buffer, 100 mL of standard and samples (1:10–1:10,000 The cells were crosslinked by paraformaldehyde at final con- dilution for all samples except for LSD mutant to fit into detection centration of 1% at room temperature for 10 minutes and range) were added and incubated for 2 hours at room temperature. quenched by 0.125 mol/L glycine. The crosslinked cells were Detection antibody was then added at 0.25 mg/mL and incubated for collected with cell scraper. The chromatin was purified as native 2 hours at room temperature. Avidin–HRP conjugate was then added ChIPN-ChIP. The purified chromatin was then resuspended and incubated for 30 minutes at room temperature. The color was in sonication buffer (10 mmol/L Tris, pH 8, 100 mmol/L NaCl, 1 developed by ABTS liquid substrate for <5 minutes and plate was read mmol/L EDTA, 0.5 mmol/L EGTA, 0.1% sodium deoxycholate, 0.5% at absorbance 405 nm.

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Lentiviral RNAi Statistical analysis The shRNAs (TP53, RELB, GFP) were cloned into pLKO.1-hygro Student t test (for two group comparison) and one-way ANOVA backbone by AgeI þ EcoRI. BJ cells were then lentivirally infected (for three group comparison) were used in this study. Levene’s mean and selected with hygromycin B at 200 mg/mL or puromycin at test was used to test violation of homogeneity of variance of 2 mg/mL for more than 7 days. The shRNA constructs (NIK, each dataset before one-way ANOVA was used. If tested positive NFKB2, MAP3K14, IKKA) in pLKO.1-puro backbone were (assumption of homogeneity of variance was violated), the dataset was obtained from Sigma-Aldrich. Sequences of shRNAs can be found transformed to log10 fold change to correct for violation of homoge- in Supplementary Table S1. neity of variance and one-way ANOVA was then used with a Tukey post hoc analysis for comparison among each pair of groups. RNA sequencing and data analysis Total RNAs were extracted by RNeasy Miniprep Kit according to manufacturer’s instructions. The total RNA samples were then Results sent to Genewiz for RNA quality control, library preparation, HPyV6 and HPyV7 sT induce p53-dependent cellular and sequencing. RNA samples were quantified using Qubit 2.0 senescence Fluorometer RNA assay (Invitrogen) and RNA integrity was MCPyV sT transforms rodent fibroblasts in vitro (24), but the checked with Agilent 4200 TapeStation (Agilent Technologies). transforming properties of other cutaneous polyomaviruses, such as RNA library preparations, sequencing reactions were conducted at HPyV6 and HPyV7, have not been explored in detail. HPyV6 and 7 GENEWIZ, LLC. Ribosomal RNA depletion was performed using have a high degree of similarity, while MCPyV is less closely related Ribo-Zero Gold Kit (Human/Mouse/Rat probe; Illumina). RNA- (Supplementary Fig. S1). Rat fibroblasts (Rat2)-expressing lentivirally sequencing library preparation used NEBNext Ultra RNA Library transduced HPyV6 and 7 sTs could not generate colonies in soft agar in Prep Kit from Illumina by following the manufacturer’sinstruc- contrast to MCPyV sT (Fig. 1A and B). However, their expression did tions (NEB). Briefly, enriched RNAs were fragmented for 15 not impair cell proliferation (Supplementary Fig. S2A and S2B). minutes at 94C. First strand and second strand cDNA were Because dermal fibroblasts have been reported as a host cell for subsequently synthesized. cDNA fragments were end repaired MCPyV (25), we next expressed cutaneous sTs in primary human andadenylatedat30ends, and universal adapters were ligated foreskin fibroblasts (BJ fibroblasts). In contrast to Rat2 fibroblasts, BJ to cDNA fragments, followed by index addition and library cells demonstrated a pronounced flattened and enlarged cell mor- enrichment with limited cycle PCR. Sequencing libraries were phology within 2 days of lentiviral delivery of HPyV6 and 7 sT, while validated using Agilent 4200 TapeStation (Agilent Technologies), MCPyV sT did not induce similar changes (Fig. 1C). Similar and quantified by using Qubit 2.0 Fluorometer (Invitrogen) as morphologic changes occurred in BJ fibroblasts immortalized by well as by using KAPA qPCR assay (KAPA Biosystems). The hTERT expression (Supplementary Fig. S2B). To control the expres- sequencing libraries were multiplexed and clustered on two lanes sion dynamics more precisely, we also generated lentiviral, doxycy- of a flowcell. After clustering, the flowcell was loaded on the cline-inducible expression systems for each of the sT antigens. We Illumina HiSeq instrument according to manufacturer’sinstruc- detected expression for all PyV sTs with doxycycline induction, tions. The samples were sequenced using a 2 150 Paired End although the expression level varied compared with the constitutive (PE) configuration. Image analysis and base calling were con- (CMV) promoter (Supplementary Fig. S2C). Consistent with the cell ducted by the HiSeq Control Software (HCS) on the HiSeq morphology, both HPyV6 and 7 sT–expressing fibroblasts showed instrument. Raw sequence data (.bcl files) generated from Illumina significantly slower growth rate compared with the uninduced (no HiSeq was converted into fastq files and demultiplexed using doxycycline) controls as early as 2 days after induction (Fig. 1D and E; Illumina bsl2fastq v. 2.17 program. Supplementary Fig. S3A and S3B). In contrast, MCPyV sT expression For data analysis, the data files of the reference genome, hg19, were did not have a strong impact on cell proliferation (Fig. 1F; Supple- downloaded from UCSC Genome Browser (https://genome.ucsc.edu/) mentary Fig. S3C and S3D). and Illumina iGenomes (https://support.illumina.com/sequencing/ Because the pronounced morphologic change of the HPyV6/7 sequencing_software/igenome.html). The qualities of sequencing sT–expressing cells were suggestive of cellular senescence, we reads were evaluated using NGS QC Toolkit (19) and high-quality performed SA-b-gal staining on the cells. HPyV6 and 7 sT, but not reads were extracted. STAR (20) was employed to map the reads onto MCPyV sT, cells showed a significantly higher percentage of the reference genome and HTSeq Python package (21) was employed SA-b-gal–positive cells compared with the vector control, with more to count reads for genes. DESeq R Bioconductor package (22, 23) was than 80% of cells staining (Fig. 1G and H) regardless of hTERT used to normalized read counts and identify differentially-expressed expression (Supplementary Fig. S2B). The expression of CDK inhi- (DE) genes. Gene Set Enrichment Analysis (GSEA) was performed bitors involved in cellular senescence, including p53, p21, and p16, was using Broad Institute GSEA software. A custom gene set for SASP strongly induced only in HPyV6/7 sT–expressing cells (Fig. 1I). The signaling was generated using genes previously identified as part of the methyltransferase EZH2 represses p21 and p16 expression and is SASP program (15). downregulated after passage or oncogene-induced senescence (26–28). Consistent with its reported functions in inhibiting senescence, EZH2 Protein sequence alignment and protein modeling was modestly decreased by HPyV6/7 sT, but strongly induced by Protein sequences were aligned in Protein BLAST program MCPyV sT expression (Fig. 1I). Finally, we directly assessed cell-cycle (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE¼Proteins). MCPyV progression through the BrdU incorporation assay. Consistent small T (wild-type and LSD mutant) structures were modeled with the cell proliferation assays, we found that HPyV6 and 7 sT, but by Expasy SWISS-MODEL program using SV40 small T antigen not MCPyV sT, significantly inhibited BrdU incorporation after as template. Protein structures were then annotated in Pymol doxycycline induction (Fig. 1J and K). HPyV6 and 7 are constitutively software. shed from the skin, and keratinocytes, rather than fibroblasts, have

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Figure 1. HPyV6 and HPyV7, but not MCPyV, sT induce senescence. A, A total of 2.5 104 Rat2 fibroblasts expressing vector control, HPyV6, HPyV7, and MCPyV small T (sT) antigens were seeded in soft agar (21 days). Representative images of soft agar colonies formed by indicated cell type. Scale bar, 1,000 mm. Only MCPyV sT–expressing cells promote anchorage-independent growth. B, Quantification of soft agar colonies in three biological replicates from each group reveals that only MCPyV sT transforms Rat2 fibroblasts. C, Representative light microscopy of BJ fibroblasts transduced with HPyV6, HPyV7, and MCPyV sT (2 days posttransduction). HPyV6/7 sT cause cells to adopt a flattened and senescent morphology. Scale bar, 200 mm. D–F, Relative growth rates of BJ fibroblasts stably transduced with doxycycline-inducible HPyV6, HPyV7, and MCPyV sT. w –dox (no expression) control. HPyV6/7, but not MCPyV, sTs induce growth arrest. G, Representative light microscopy of SA-b-gal staining of HPyV6, HPyV7, and MCPyV sT–transduced BJ cells (4 days) highlights the induction of SA-b-gal staining by HPyV6/7 sT. Scale bar, 200 mm. H, Quantification of SA-b-gal–positive cells of three biological replicates of each group confirms the significant induction of senescence by HPyV6/7 sT. I, Western blot analysis of p53, p21, p16, and EZH2 expression in HPyV6, HPyV7, and MCPyV sT–transduced BJ fibroblasts (7 days postinfection). HPyV6/7 sT–expressing cells show increased expression of p53, p21, and p16 consistent with the induction of senescence. MCPyV sT shows increased expression of H3K27 methytransferase, EZH2. 1t1.13 confirms HPyV6/7 sT expression; Flag confirms MCPyV sT expression; and HSP90 is the loading control. J, Doxycycline induction (3 days) of HPyV6/7 sT, but not MCPyV sT, results in a marked reduction of BrdU-positive BJ cells. Images show representative IF images of BrdU staining of cells without or with doxycycline induction (1 mg/mL, 24 hours before the assay). Scale bar, 100 mm. K, Expression of HPyV6/7 sT results in significantly less BrdU incorporation. Quantification of BrdU-positive cells of three biological replicates of each group. L, BJ cells were transduced with the indicated shRNA (7 days), then HPyV sT. The knockdown of p53 prevents the morphologic induction of senescence by HPyV6/7 sT (3 days). Representative light microscopy of HPyV6/7 sT–transduced shTP53-3 KD and shGFP (shRNA control) cells. See Supplementary Fig. S2. M, BJ cells were transduced with the indicated shRNA (7 days), then HPyV sT. The knockdown of p53 prevents the induction of SA-b-gal staining by HPyV6/7 sT (3 days). Representative light microscopy of transduced shGFP and shTP53-3 cells. Scale bar, 200 mm. N, Quantification of SA-b-gal–positive cells of three biological replicates of each shRNA group confirms the rescue of senescence induction by p53 knockdown. In B, D, E, F, H, K, L,andO, data represents mean SD. , P < 0.05; , P < 0.01; , P < 0.001; , P < 0.0001. Significance assessed by one-way ANOVA followed by Tukey post hoc analysis for B and H and Student t test for J.

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been suggested as a possible host cell for these PyVs (5, 29). expressing cells all induce the expression and secretion of inflamma- Therefore, we also expressed HPyV6/7 sT in keratinocytes. Con- tory cytokines typical of SASP. sistent with its impact in fibroblasts, HPyV6 and 7 sTs also caused senescence in keratinocytes based on the pronounced change of cell The LSD motif is required for sT-induced transcriptional morphology and increased SA-b-gal positivity (Supplementary changes, including SASP Fig. S2D). The LSD motif of MCPyV sT stabilizes the large T antigen and is Because Rat2 fibroblasts show spontaneous loss or mutation of essential to the transforming properties of sT (6). The LSD motif p53 (30), we tested whether HPyV6 and 7 induced senescence in inhibits FBXW7, which is critical to the stabilization of cellular primary fibroblasts, but not Rat2 cells, due to differences in p53 status. oncogenes like c-Myc. However, the knockdown of FBXW7 alone Indeed, HPyV6 and 7 sTs failed to induce senescence phenotype in does not promote the transforming activity of the LSD mutant MCPyV Huh7 cells, which possess a p53 loss-of-function mutation (Supple- sT (6). Thus, its precise role in promoting fibroblast transformation mentary Fig. S2E), but did induce senescence in p53 WT A549 cells remains an area of active investigation (34–36). The LSD motif is not (Supplementary Fig. S2F; ref. 31, 32). To confirm directly that HPyV6 conserved in HPyV6/7 sT (Supplementary Fig. S1), suggesting that it and 7 sTs-induced senescence required p53, we used three shRNAs to might contribute to the phenotypic differences caused by HPyV6/7 knockdown p53 in BJ fibroblasts by at least 50% (Supplementary and MCPyV sT expression. To better define the role of the LSD motif, Fig. S2G). Knockdown of p53 rescued morphologic changes (Fig. 1L; we also performed RNA-seq on a MCPyV sT with a mutation of the Supplementary Fig. S2H) and significantly reduced the number of LSD domain (LSD; amino acids 91–95, LKDYM!AAAAA) cells SA-b-gal–positive cells (Fig. 1M and N, Supplementary Fig. S2I). (Supplementary Fig. S1, S7A). RNA-seq revealed that LSD- Thus, in contrast to the transforming properties of MCPyV sT, expressing fibroblasts were notably distinct from WT-infected cells HPyV6, and 7 sTs induce p53-dependent cellular senescence. and were more similar to vector control cells (Fig. 2A, I, and J). GSEA analyses revealed that Myc target activation by wild-type MCPyV sT HPyV6, HPyV7, and MCPyV sT all induce SASP gene expression was largely reversed by the LSD motif mutation (Supplementary To broaden our analysis of the differences between HPyV6, HPyV7, Fig. S8A). The effect of sT expression on c-Myc expression was and MCPyV sT, we performed RNA-seq on sT-expressing dermal examined. In contrast to MCPyV sT, HPyV6/7 sT, which lack the fibroblasts. Principal component analysis (PCA) revealed that MCPyV LSD motif, did not stabilize c-Myc protein levels when expressed in BJ sT possessed the most distinct transcriptomic profile compared with cells (Supplementary Fig. S8B). MCPyV sT has been reported to both EV control and HPyV6 and 7 sTs (Fig. 2A). MCPyV sT showed decrease c-Myc turnover. Using cycloheximide treatment, c-Myc significantly more differentially regulated genes compared with turnover was assessed in 293T cells cotransfected with c-Myc and a HPyV6 and 7 (Supplementary Fig. S4C and S4D). Some of the genes vector control, HPyV6 sT, HPyV7 sT, wild-type MCPyV sT, or LSD- most highly upregulated by MCPyV sT expression included IL6, IL1B, mutant MCPyV sT. Only wild-type MCPyV decreased the turnover of and IL8, cytokines which have previously been linked to the SASP, the c-Myc in an LSD motif–dependent manner (Supplementary Fig. S8C). secretory program adopted by senescent cells (Fig. 2B). To extend this We next assessed whether the LSD motif might also contribute to finding, we quantified the expression of six classical SASP genes SASP gene expression. Indeed, compared with WT sT, the LSD mutant including CCL26, CXCL10, CSF2 (GM-CSF), IL1B, IL6, and IL8. showed notable downregulation of the same SASP genes that were HPyV6 and 7 sT significantly upregulated each of these well- upregulated by WT (Fig. 2K; Supplementary Fig. S5A). GSEA analysis established SASP factors as might be expected on the basis of their also revealed that most of the previously upregulated signaling path- ability to induce senescence in fibroblasts. Despite the absence of ways were also rescued by the introduction of LSD motif, including features of senescence, MCPyV sT–expressing fibroblasts also signif- SASP and NFkB pathway (Fig. 2J; Supplementary Fig. S7B–S7E). icantly upregulated these genes, with some (i.e., CSF2, IL1B, IL6, and Using qRT-PCR for multiple SASP genes and ELISA for IL8, we IL8) being upregulated 10- to 100-fold higher than HPyV6 and 7 sT validated the rescue of SASP activation by the LSD mutant (Fig. 2K; (Fig. 2C). Consistent with their distinct transcriptomic profiles, Supplementary Fig. S7F). Specific matrix metalloproteinase genes (e.g., HPyV6, 7, and MCPyV sT did not show uniform upregulation of all MMP1, MMP3, and MMP10), previously reported to be part of the reported SASP factors (Supplementary Fig. S5A). Time course analyses SASP program, which were significantly upregulated by MCPyV sT revealed that expression of the SASP factors remained high up to were largely rescued by the LSD mutant (Supplementary Fig. S7G). 15 days after transduction (Supplementary Fig. S5B–S5D). ELISA of One notable exception to pathways rescued by the LSD mutation was conditioned media confirmed that IL8 secretion was significantly IFNg signaling. Specifically, wild-type sT, like HPyV6/7, inhibited upregulated by all sT expression, with MCPyV sT inducing IL8 cellular IFN signaling pathways (Supplementary Fig. S4E–I). The LSD secretion at levels 100-fold higher than either HPyV6 and 7 sT mutant induced IFNa, but not IFNg signaling, as assessed by (Fig. 2D). In addition to highlighting Myc as being highly upregulated GSEA analysis (Supplementary Fig. S7C). Notably, the LSD mutant by MCPyV sT, gene set enrichment analysis (GSEA) confirmed SASP did not induce cellular senescence as assessed by cell morphology to be one of the most enriched signatures after MCPyV sT expression (Supplementary Fig. S7H), cell proliferation as assessed by MTT (Fig. 2E, F, and H). One reported mechanism underlying the activa- (Supplementary Fig. S7I), or BrdU incorporation (Supplementary tion of SASP program is the activation of NFkB signaling (33). Indeed, Fig. S7J–S7K). In summary, the LSD motif of MCPyV sT drives many GSEA of the RNA-seq data also demonstrated that the NFkB pathway of the transcriptional changes induced by MCPyV sT, including both is highly enriched in MCPyV sT cells (Fig 2E, G, and H). To rule out NFkB pathway activation and the induction of SASP genes. any potential contributions by the lentiviral expression vectors, we knocked down MCPyV sT expression with a sT-specific shRNA, which HPyV6 and 7 and MCPyV sT activate distinct NFkB pathways showed approximately 90% knockdown efficiency (Supplementary The NFkB pathway can be activated in two ways, referred to as Fig. S6A-B). All SASP gene expression was significantly rescued canonical (classical) or noncanonical (alternative) NFkB signal- (Supplementary Fig. S6C), confirming that the observed SASP gene ing (37, 38). While both types of NFkB can induce inflammatory expression is specific to sT. Thus, HPyV6, HPyV7, and MCPyV sT– gene activation, canonical NFkB signaling promotes senescence while

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A C D IL8 ELISA J vector MCPyV-WT Vector WT LSD HPyV6-sT MCPyV-LSD CCL26 CXCL10 GM-CSF **** HPyV7-sT **** rep 123123 123 100 0 100 **** 4 100 2.5 *** ****

3 3 **** 10 A **** 2.0 **** **** 3 **** 2 **** 1.5 **** **** 2 **** 2 **** 1.0 0 1 1 **** Fold change 0.5 1 10 0 3CP 0 0.0 fold change 0 -0.5 PC2 -1 -1 Concentration log -100 V-sT -1 -sT Vector PC1 100 log mRNA Vector Vector 0 Vector HPyV6-sTHPyV7-sTMCPy HPyV6-sTHPyV7-sTMCPyV-sT HPyV6-sTHPyV7-sTMCPyV-sT 100 HPyV6-sTHPyV7-sTMCPyV

IL1B IL6 IL8 B I MCPyV-LSD vs. MCPyV-WT MCPyV-WT vs. vector **** **** ** 5 **** IL1B 200 **** 2.0 ** **** 300 IL8 IL1B 4 **** **** IL6 150 IL6 4 IL8 **** 1.5 **** 100 100 3 *** **** 25 50 **** 3 CXCL1 CXCL14 CSF3 1.0 ** CXCL14 2 2 adj P 20 CXCL1 Fold change 0.5 CSF3

1 10

10 1 adj MLL2 0.0 0 10 P CSF2 SETD1A/SETD1B 0 -Log 10 10 CSF2 -0.5 -1 MLL4 -sT -1 -sT -sT V Log NFKB2 V Vector RELB log mRNA Vector Vector HPyV6-sTHPyV7-sTMCPy 0 HPyV6-sTHPyV7-sTMCPyV HPyV6-sTHPyV7-sTMCPy 0 −8 −4 0 4 −6 −3 0 36 Log Fold change 2 Log2 fold change -2-1 0 1 2 EFHVector MCPyV-WT CXCL6 K rep IL7 Top enriched signatures 123123 CCL26 CXCL10 IL1B SASP CXCL10 Downregulated Upregulated MMP13 4 1.5 *** *** 3 **** **** 0.4 NES = 1.99 MMP1 3 10.0 P **** * NOM -val < 0.001 CXCL1 2 0.2 q 1.0 FDR -val < 0.001 CXCL3 2 G −M Checkpoint 0.0 2 CXCL2 Fold change 0.5 1 1 Myc -0.2 CXCL5 10 0.0 7.5 Targets IL1B 0 0 E2F Targets IL1A -0.5 -1 EV WT LSD Enrichment score EV WT LSD -1 EV WT LSD

Interferon alpha response log mRNA NF-κB Up in Down in MMP12 Signaling MCPyV vs. EV WT vs. EV MMP3 -logFDR 5.0 IL6 GM-CSF IL6 IL8 NFκB signaling IL8 2.0 **** **** G 2.0 4 SASP 0.4 IL11 ******** ******** 1.5 Unfolded protein CSF3 1.5 3 2.5 Interferon gamma response 0.2 CSF2 1.0 response NES = 2.26 1.0 2 NOM P-val < 0.001 EGF OXPHOS Fold change 0.5 q 10 FDR -val < 0.001 PLAUR 0.5 1 Reactive oxygen species 0.0

FDR = 0.25 CCL3 log 0.0 0.0 0 ICAM1

Enrichment score -0.5 EV WT LSD EV WT LSD mRNA EV WT LSD −2 024 CCL11 -0.5 -1 Up in Down in CCL26 Net enrichment score (NES) WT vs. EV WT vs. EV -2 -1 0 1 2

Figure 2. MCPyV sT induces SASP and NFkB signaling in an LSD motif–dependent manner. A, Principal component analysis (PCA) of empty vector (EV), HPyV6 sT, HPyV7 sT, MCPyV wild-type sT, and MCPyV LSD–mutant sT transcriptomes (7 days) reveals distinct clustering of MCPyV sT compared with vector control and other PyV sTs. B, Volcano plot (single gene) of MCPyV sT compared with empty vector control highlights the upregulation of multiple SASP genes. C, qRT-PCR analysis of HPyV6, HPyV7, and MCPyV sT–transduced cells (7 days) confirms the significant upregulation of CCL26, CXCL10, GM-CSF, IL1B, IL6, and IL8 by PyV sT expression compared with vector control. For GM-CSF, IL1B, IL6, and IL8, expression induced by MCPyV sT is significantly higher than HPyV6/7. Data are normalized by empty vector control and scaled to log10 fold change. D, ELISA measurement of IL8 secreted by BJ fibroblasts after sT transduction (7 days). All PyVs induced IL8 secretion compared with the vector; MCPyV sT induced even greater IL8 secretion than HPyV6/7 sT. Data are normalized by empty vector and scaled to log10 fold change. E, Volcano plot of gene signatures induced by MCPyV sT compared with empty vector control. F, GSEA analysis of SASP signaling comparing BJ cells stably transduced with MCPyV sT to vector control. G, GSEA analysis of NFkB signaling comparing BJ cells stably transduced with MCPyV sT to vector control signaling by MCPyV sT compared with vector control. H, Heatmap of significant genes in SASP and NFkB signaling gene sets of MCPyV sT compared with empty vector control. Gene list (right) indicates a subset of transcripts induced by SASP induction. I, Volcano plot (single gene) of MCPyV sT LSD mutant compared with wild-type MCPyV sT reveals that many of the highly induced genes are reverted to levels comparable with the vector control (B). J, Heatmap comparing all significant genes in vector control, MCPyV sT LSD mutant, and wild-type MCPyV sT highlights the rescue of transcriptional changes by the LSD mutant. K, qRT-PCR analysis of SASP genes confirms that the MCPyV sT LSD mutant rescues changes in SASP cytokines induced by wild-type MCPyV sT (7 days). Data are normalized by vector control and scaled to log10 fold change. In C, D,andK, data are presented as mean SD. ¸ P < 0.05; , P < 0.01; , P < 0.001; , P < 0.0001. Significance assessed by one-way ANOVA followed by Tukey post hoc analysis.

noncanonical NFkB signaling is able to evade it (13–17). To test canonical NFkB pathway activation. We next tested whether whether HPyV6/7 and MCPyV sT might differ in their senescence MCPyV sT increased levels of noncanonical NFkBsignalingcom- response due to differential activation of NFkB pathways, we assessed ponents. Notably, in contrast to vector and HPyV6/7 sT–expressing NFkB signaling components by Western blot analysis. We first cells, MCPyV sT resulted in the marked accumulation of both assessed the activation of the canonical NFkBsignalingpathway. immature NFKB2 (p100) and its active p52 isoform and RELB HPyV6and7sT–expressing cells showed increased levels of (Fig. 3A). Because the LSD mutant reversed signatures of NFkB phosphor-p65 (RelA), which has been shown to be critical for the activation and SASP, we tested whether the LSD motif was neces- recruitment of CBP/p300 transcriptional coactivator complex and sary for ncNFkB pathway activation. Notably, the MCPyV sT-LSD canonical NFkB activation (39), consistent with activation of mutant reversed immature NFKB2 (p100) accumulation, p52 pro- canonical NFkB signaling. In contrast, we found that MCPyV sT cessing, and RELB levels (Fig. 3B). In addition, phosphorylated decreased levels of phospho-p65 (RelA; Fig. 3A). Thus, consistent IKKa, an additional marker suggestive of noncanonical NFkB with studies showing that MCPyV sT specifically targets the IKK pathway activation, was elevated by WT MCPyV sT, but not the kinase adaptor protein NEMO (11), we find that MCPyV inhibits LSD mutant; total IKKa and IKKb remained unchanged (Fig. 3B).

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Figure 3. MCPyV sT activates ncNFkB signaling through both transcriptional and posttranslational pathways. A, Western blot analyses of NFkB components in PyV sT–transduced BJ cells (7 days) reveals induction of NFkB by HPyV6/7 sT and induction of ncNFkB by MCPyV sT. Arrows indicate mature p52/NFKB2 (top) and p50/NFKB1 (bottom) proteins. HSP90, loading control. B, Western blot analyses of NFkB components in wild-type and LSD mutant MCPyV sT-transduced BJ fibroblasts (7 days) reveals induction of ncNFkB by MCPyV sT requires the LSD domain. Arrow indicates mature p52 protein processed from immature p100 (NFKB2). HSP90, loading control. C, qRT-PCR analysis reveals significant upregulation of central ncNFkB pathway genes, NFKB2 and RELB, in wild-type, but not LSD mutant, MCPyV sT–expressing BJ cells (5 days). Data are normalized by empty vector control and represent three biological replicates. D, qRT-PCR analysis reveals significant upregulation of H3K4me3 histone methyltransferases—MLL2, MLL4, SETD1A, and SETD1B—in wild-type, but not LSD mutant, MCPyV sT–expressing BJ cells (5 days). Data are normalized by empty vector control and represent three biological replicates. E, Western blot analyses reveals levels of of H3K4me3 are elevated in wild- type, but not LSD mutant, MCPyV sT–expressing BJ fibroblasts (5 days). HSP90, loading control. F, Subcellular fractionation of transduced BJ cells (5 days) reveals that wild-type, but not LSD mutant, MCPyV sT is present in the chromatin fraction. HSP90, cytoplasmic fraction control; HDAC2, nucleoplasmic control; H3, chromatin fraction control. G, Schematic indicating the location of putative MCPyV sT binding sequences in the NFKB2 and RELB promoter regions. H, FLAG or H3K4me3 ChIP- qPCR analyses reveal the enrichment of NFKB2 and RELB promoter regions in wild-type, but not LSD mutant, MCPyV sT–expressing BJ cells (5 days). Data are presented as percentage input and represent three biological replicates. I, Cells were first transduced with vector control or FBXW7 (5 days) and then vector or MCPyV sT (7 days) followed by Western blot analyses for indicated proteins. FBXW7 partially rescues increases in NFKB2 and RELB induced by sT. In D, E,andH, data are presented as mean SD. , P < 0.05; , P < 0.01; , P < 0.001; , P < 0.0001. Significance assessed by one-way ANOVA followed by Tukey post hoc analysis.

Thus, in contrast to HPyV6/7, MCPyV activates noncanonical, and tion of MLL2, MLL4, SETD1A, and SETD1B were confirmed by inhibits canonical, NFkB signaling in an LSD-dependent manner. qRT-PCR (Fig. 3D). In addition, global levels of H3K4me3 were markedly upregulated by wild-type MCPyV sT, but not LSD Activation of ncNFkB signaling through H3K4me3 and FBXW7 mutant, expressing cells (Fig. 3E). inhibition Because previous reports have demonstrated a role for sT in The RNA-seq data suggested that noncanonical NFkBpathway transcriptional regulation through chromatin remodeling (42), we members, NFKB2 (p100) and RELB, might be upregulated at the used subcellular fractionation assays to verify that WT sT was found in transcriptional level (Fig. 2B). qRT-PCR confirmed that that cytoplasmic, nucleoplasmic, and chromatin-bound fractions (Fig. 3F). NFKB2 and RELB transcripts were upregulated by WT sT cells, Notably, despite higher levels of expression in this system, we found but not the LSD mutant (Fig. 3C). Alterations in histone modifica- that the LSD mutant abrogated the ability of sT to associate with tions have been implicated in the pathogenesis of MCC (40–42). chromatin (Fig. 3F). We identified multiple potential sT-binding Our GSEA confirmed chromatin and histone modification path- motifs in the NFKB2 and RELB promoter regions (Fig. 3G). To ways to be upregulated compared with vector, HPyV6/7 sT, and determine whether sT might bind the NFKB2 and RELB promoters LSD mutant MCPyV sT controls (Supplementary Fig. S9). Specif- and promote H3K4me3 modification through direct binding, we ically, histone methyltransferases involved in H3K4 trimethylation performed ChIP-qPCR against both H3K4me3 and FLAG tagged sT (H3K4me3) were highly upregulated by GSEA (Fig. 2B). Upregula- antigen. In sT-expressing cells, both WT ST and the H3K4me3 mark

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are significantly upregulated at NFKB2 and RELB promoters. The LSD proliferation in breast and prostate cancer in a paracrine manner mutant, which does not activate noncanonical NFkB, showed signif- through the secretion of SASP-associated cytokines (15, 48–50). icantly less accumulation at these regions (Fig. 3H; Supplementary Because many SASP cytokines have well-established, growth- Fig. S10A). promoting properties, we hypothesized that ncNFkB might indirectly Transcriptional upregulation of NFKB2 and RELB alone is insuf- stimulate MCC proliferation in an autocrine fashion. Cells expressing ficient to activate to NFKB2 ncNFkB signaling, as the NFKB2 p100 MCPyV sT have the ability to grow in the absence of serum, a hallmark must also be processed into its active p52 form (43). NFKB2 has been of cancer cells (24, 51). We confirmed that MCPyV sT fibroblasts, but reported to be a target of FBXW7-mediated degradation and the loss of not those transduced with the vector alone, could proliferate in low FBXW7 alone can result in the accumulation of the NFKB2 p100 serum (2% FBS) conditions (Fig. 5A). The ability to proliferate in low precursor protein and ectopic activation of ncNFkB pathway (44, 45). serum was dependent on ncNFkB activation, as shRNAs targeting To determine whether MCPyV sTs previously reported ability to RELB and NFKB2 markedly impaired proliferation in low serum, inhibit FBXW7 might contribute to ncNFkB activation, we over- more notably than it had been impaired in normal serum (Fig. 5B; expressed FBXW7 in sT-expressing cells. The overexpression of Supplementary Fig. S11C). The inhibition of ncNFkB signaling using FBXW7 decreased p100/NFKB2 accumulation and p52 processing cell-permeable inhibitor significantly slowed the growth of MCPyV (Fig. 3I). In addition, FBXW7 overexpression partially reversed sT–expressing BJ fibroblasts in low serum (Supplementary Fig. S12A). MCPyV sT SASP gene expression (Supplementary Fig. S10B). The In contrast, the chemical inhibition of canonical NFkB had no effect further expression of the LSD mutant completely blocked SASP (Supplementary Fig. S12B). To test whether secreted cytokines were cytokine expression, suggesting that the functions of the LSD motif important for cell proliferation, we blocked SASP cytokine processing were not entirely epistatic with FBXW7. In summary, we conclude that and secretion using a commercial Protein Tranport Inhibitor Cocktail sT activates ncNFkB signaling through promoting H3K4me3 meth- [PTI] (Thermo Fisher Scientific), containing a combination of Bre- ylation and transcriptional upregulation of NFKB2 and RELB and feldin A and Monensin. Cells treated with PTI are unable to secrete through inhibiting FBXW7-mediated degradation of NFKB2. cytokines and should, therefore, be unable to promote cell proliferation through autocrine signaling. Indeed, blocking protein secretion sig- Chemical and genetic inhibition of ncNFkB activation abrogates nificantly inhibited the proliferation of MCPyV sT–expressing cells in SASP activation low (2%) serum, but had no effect in full (10%) serum, presumably due To confirm that ncNFkB activation was required for SASP activa- to the presence of excess growth factors in the serum (Fig. 5C). To tion, we inhibited ncNFkB activation with both chemical and genetic directly test whether secreted factors present in the media were able to approaches. First, we employed calcitriol (1,25-dihydroxyvitamin D3), promote the proliferation of MCPyV-sT–expressing cells in low which inhibits ncNFkB activation by inhibiting RELB expres- serum, we next tested whether conditioned media could rescue the sion (46, 47). The treatment of sT-infected cells with calcitriol for growth of MCPyV sT–expressing cells in which protein secretion 7 days (Fig. 4A) reversed the induction of the SASP gene transcription had been blocked. Notably, conditioned media generated by (CCL26, CXCL10, GM-CSF, IL1B, IL6, and IL8) and secretion MCPyV sT–expressing BJ cells, but not vector control cells, (IL8; Fig. 4B and C). We also confirmed the suppression of RELB significantly rescued the growth of MCPyV sT fibroblasts, even in protein by Western blot analysis (Fig. 4D). Unexpectedly, calcitriol the presence of PTI (Fig. 5D). Because HPyV6 and 7 sT-expressing treatment also increased phospho-p65 (RELA) levels, suggesting that cells also induce a SASP response, we next tested whether the canonical NFkB signaling was activated by calcitriol (Fig. 4D). conditioned media of these cells might be substituted for the Because calcitriol functions as a vitamin D agonist, it has many off- conditioned media of MCPyV sT–expressing cells. Remarkably, target effects through its induction of vitamin D responsive (VDR) while BJ cells expressing the LSD-mutant MCPyV sT were unable genes. Therefore, we used a complementary genetic approach and used to proliferate efficiently in low serum, the addition of media har- multiple shRNAs to knockdown the ncNFkB signaling components vested from wild-type MCPyV sT or HPyV6/7 sT–expressing including IKKa, NIK, NFKB2, and RELB. Knockdowns were efficient fibroblasts all had the ability to significantly rescue the growth of and confirmed both qRT-PCR and Western blot analysis (Fig. 4E the LSD MCPyV sT–expressing cells (Fig. 5E), albeit with different and F). The knockdown of ncNFkB signaling impacted several features levels of efficacy. However, MCPyV sT fibroblasts still grew signif- of MCPyV sT expression. Knockdown of NIK, NFKB2, and RELB all icantly better than vector control fibroblasts treated with PyV resulted in a significant reduction of SASP gene expression, providing sT–conditioned media, indicating that the supplementation of additional evidence that the ncNFkB pathway was critical for the SASP cytokines alone was not sufficient to promote fibroblast activation of SASP by sT (Fig. 4G; Supplementary Fig. S11A). proliferation, consistent with the multiple transforming properties Decreased expression of EZH2 after knockdown of ncNFkB signaling of MCPyV sT (Supplementary Fig. S12C). Thus, the induction of components was also noted, consistent with their reported role in ncNFkB signaling by MCPyV sT promotes the secretion of SASP- EZH2 expression (refs. 16, 17; Supplementary Fig. S11B). Given a associated cytokines that promote autocrine signaling and cell reported role for EZH2 in impacting cell proliferation, we also assessed proliferation. whether the knockdown of ncNFkB could impact the proliferation of MCPyV sT-expressing BJ cells. The knockdown of NKFB2 or RELB in Noncanonical NFkB and SASP activation in MCC cell lines and these cells resulted in a significantly slower growth compared with an tumors shGFP control, but only at late time points (>5 days) in vitro (Sup- We next determined whether ncNFkB signaling occurred in plementary Fig. S11C). MCC cell lines. First, we assessed the activation of ncNFkBin VP-MCC (MKL-1, MKL-2, MS-1, and WaGa) and VN-MCC Activation of ncNFkB promotes the growth of sT-expressing (UISO, MCC13, MCC26) cell lines. VP-MCC, but not VN-MCC, cells lines showed increased levels of NFKB2, both activated p52 and its We sought to characterize the effects of ncNFkB on cell prolifer- precursor p100, and RELB (Fig. 6A). In addition, VP-MCC lines ation in greater detail. Senescent fibroblasts can promote cell show increased levels of global H3K4me3, consistent with its role in

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Figure 4. Chemical and genetic rescue of MCPyV sT induced ncNFkB activation. A, Schematic of calcitriol (RELB inhibitor) rescue experiment. BJ ﬁbroblasts were plated at approximately 50% conﬂuence 1 day before infection. Cells were then spin- infected and puromycin selection was started 24 hours post spin infection along with 50 nmol/L calcitriol or vehicle control. Cells and supernatant were harvested 7 days postinfection for qRT-PCR, West- ern blot, and ELISA. B, Chemical inhibition of ncNFkB with calcitriol reverses SASP gene induction by MCPyV sT as assessed by qRT-PCR. Data are normalized by vec-

tor þ vehicle control and scaled to log10 fold change. C, ELISA of IL8 secretion by BJ cells reveals the inhibition of sT-mediated IL8 secretion by calcitriol. Data are normalized by vector þ vehicle

control and scaled to log10 fold change. D, Western blot analysis of NFkB pathway components in calcitriol (50 nmol/L calcitriol) treated cells. Calcitriol induces canonical, but inhibits, noncanonical NFkB signaling. E, BJ cells were transduced with the indicated shRNA construct (7 days) and knockdown of ncNFkBpath- way genes—IKKA, NFKB2, MAP3K14,and RELB—were assessed by qRT-PCR. Data are normalized by shGFP control and presented as three biological replicates. F, Western blot validates shRNA knockdown (7 days) of ncNFkB pathway genes IKKA, NFKB2, MAP3K14,andRELB. G, BJ cells were ﬁrst transduced with MCPyV sT (5 days), then the indicated shRNA (7 days). Induction of SASP genes by sT is partially rescued by shRNA knockdown of ncNFkB pathway genes. Replicate shRNAs targeting the same gene were excluded for clarity and are detailed in Supplementary Fig. S9. Data are normal-

ized by shGFP control and scaled to log10 fold change. Data are presented as mean SD for B–D and G. , P < 0.05; , P < 0.01; , P < 0.001; , P < 0.0001. Signiﬁ- cance assessed by one-way ANOVA followed by Tukey post hoc analysis.

ncNFkBactivation(Fig. 6B). We noted the expression of sT to be NFKB2 precursor (p100) and RELB expression, with four samples lower in this isolate of MS-1, compared with other VP-MCC lines showing strikingly higher expression of RELB. At least 6 of 7 (Supplementary Fig. S13A), perhaps explaining the lower levels of samples showed detectable processing of NFKB2/p100 precursor H3K4me3 and ncNFkB activation in this cell line. We next assessed protein (Fig. 6C). The variation in NFKB2/p52 and RELB levels whether ncNFkB activation might be similarly upregulated in suggests that in vivo factors, in addition to sT expression, may patient MCC tumors. We identified VP-MCC patient tumors and impact ncNFkB activation. The expression of 6 SASP genes (CCL26, confirmed expression of MCPyV sT in the tumors, but not normal CXCL10, GM-CSF, IL1B, IL6,andIL8)weresignificantly elevated in skin controls, with endpoint and qRT-PCR (Supplementary VP-MCC, compared with VN-MCC cell lines (Fig. 6D). All SASP Fig. S13A–S13C). Compared with the normal skin controls, all genes, except GM-CSF,werealsosignificantly elevated in patient seven patient samples were found to have increased levels of the tumors compared with normal skin (Fig. 6E).

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MCPyV sT Activates NonCanonical NFkB

A 2%FBS B 2%FBS C MCPyV sT 20 40 20 **** DMSO + 10%FBS **** MCPyV sT **** DMSO + 2%FBS 15 vector **** 30 shGFP 15 PTI + 10%FBS **** **** PTI + 2%FBS **** 10 20 RELB sh2 10 **** **** * RELB sh3 NFKB2 sh1 ** 5 10 **** 5 OD Fold change NFKB2 sh2 OD Fold change OD Fold change 0 0 0 12345 1234567 1234567 Time (days) Time (days) Time (days) 2%FBS + PTI 2%FBS DE30 25 + conditioned media ± conditioned media MCPyV sT **** 20 **** +WT sT media 20 MCPyV sT **** + sT media 15 +6sT media ** **** MCPyV sT **** MCPyV sT 10 +7sT media LSD mutant 10 + vector media LSD alone 5 OD Fold change OD Fold change 0 0 123456 123456 Time (days) Time (days)

Figure 5. MCPyV sT induced ncNFkB and SASP promotes proliferation through autocrine signaling. A, BJ fibroblasts were transduced with MCPyV sT or vector control (5 days). After selection, cells were plated in low serum (2% FBS) media and proliferation was assessed by MTT. B, BJ fibroblasts were first transduced with MCPyV sT (5 days) and then the indicated ncNFkB signaling component shRNA (7 days). After selection, cells were plated in low serum (2% FBS) media and proliferation was assessed by MTT. Knockdown of NFKB2 or RELB significantly inhibited the proliferation of MCPyV sT-expressing fibroblasts compared with a GFP shRNA control. C, BJ cells transduced with MCPyV sT (5 days). After selection, cells were cultured in low (2% FBS) or normal (10% FBS) serum in the presence of a protein transport inhibitor (PTI) cocktail (Thermo Fisher Scientific) or a vehicle (DMSO) control and proliferation was assessed by MTT. Proliferation of MCPyV sT cells was significantly slower in the low (2%) serum media in the presence of the PTI cocktail. D, BJ cells were transduced with vector control or MCPyV sT (5 days). These cells were plated in low (2% FBS) serum media and this conditioned media was collected (2 days). BJ cells were separately transduced with MCPyV sT (5 days). After selection, cells were cultured in low (2% FBS) serum, a PTI cocktail, and the indicated conditioned media. Media harvested from MCPyV sT expressing, but not vector control, BJ cells rescues the proliferation of MCPyV sT-expressing BJ cells in low (2%) serum þ PTI. E, BJ cells were transduced with HPyV6/7 sT or MCPyV sT (5 days). These cells were plated in low (2% FBS) serum media and this conditioned media was collected (2 days). BJ cells were separately transduced with wild-type or LSD mutant MCPyV sT (5 days). After selection, cells were cultured in low (2% FBS) serum, a PTI cocktail, and the indicated conditioned media. Media harvested from HPyV6 sT, HPyV7 sT, or MCPyV sT each significantly increases the proliferation of LSD mutant MCPyV sT-expressing BJ cells in low (2%) serum. For all growth curves, each timepoint was run as biological triplicates. , P < 0.05; , P < 0.01; , P < 0.001; , P < 0.0001. Significance assessed by one-way ANOVA followed by Tukey post hoc analysis.

Noncanonical NFkB signaling is required in MCC xenograft MCC. In contrast, ncNFkB inhibited MKL-1 and WaGa tumors were growth smaller, possessed increased amounts of eosinophilic cytoplasm, and To test the role of ncNFkB might in MCC in vivo, we used a specific, showed large areas with necrosis and loose stromal tissue (Fig. 7G; peptide inhibitor of ncNFkB signaling, SN52 (52). The SN52 peptide Supplementary Fig. S14D and S14E). penetrates cells and competitively inhibits NFKB2/p52 nuclear trans- location and ncNFkB activation through its consensus NFKB2/p52 NLS sequence (Supplementary Fig. S14A). It specifically inhibits Discussion ncNFkB signaling in both cancer cells and immune cells (52–54). In addition to promoting inflammation and regulating immune Consistent with the activation of ncNFkB by MCPyV sT, the SN52 cell development, noncanonical NFkB signaling can promote cell peptide slowed cell proliferation only in VP-MCC (MKL-1, MKL-2, proliferation and survival (55). Thus, many viruses have deviously MS-1, and WaGa), but not VN-MCC (MCC13, MCC26, and UISO) coopted the pathway to promote their own replication and dissem- cell lines (Fig. 7A; Supplementary Fig. S14B). Next, VP-MCC lines ination. Notably, the oncoproteins of several DNA tumors viruses (MKL-1 and WaGa) were xenografted onto NSG immunodeficient target ncNFkB, including the Tax protein of human T-cell leukemia mice and the graft sites injected biweekly with the ncNFkB inhibitor or virus, the LMP1 protein of the Epstein–Barr virus, and the vFLIP a control peptide for 30 days (Fig. 7B). The inhibition of ncNFkB protein of the Kaposi sarcoma virus (37, 43). Here, we describe the significantly impaired both MKL-1 and WaGa xenograft growth, as first example of a polyomavirus protein activating ncNFkB assessed by tumor size and weight (Fig. 7C–E). Consistent with the (Supplementary Fig. S15). Clearly, not all polyomaviruses share in vitro findings, the peptide inhibitor of ncNFkB also significantly the ability to activate ncNFkB signaling. Specifically, we find that inhibited SASP gene (CCL26, CXCL10, GM-CSF, IL1B, IL6, and IL8) HPyV6/7 sT activates canonical NFkB signaling, while MCPyV sT expression in the xenografts compared with the control peptide appears unique in its ability to inactivate canonical and activate (Fig. 7F; Supplementary Fig. S14C). Histologic analyses revealed that ncNFkB signaling. These differences are critical to the transforming control peptide–treated MKL-1 and WaGa xenografts showed sheets properties of MCPyV sT and suggest that the activation of ncNFkB of tightly packed, monotonous tumor cells, with large nuclei and scant may be a feature that is conserved among DNA viruses with cytoplasm, consistent with the “small blue cell” histology typical of transforming properties. In addition to the striking differences in

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Figure 6. ncNFkB signaling is activated in VP-MCC, but not VN-MCC, cells and tumors. A, Western blot analyses of NFKB2 and RELB in VN-MCC and VP-MCC cell lines reveals activation of ncNFkB in VP-MCC. Arrow indicates mature p52/NFKB2 processed from immature p100 precursor. HSP90, loading control. B, Western blot analyses of H3K4me3 levels of VN-MCC and VP-MCC lines. Cells were prepared as whole-cell lysates. H3, loading control. C, Western blot analyses of NFKB2 and RELB from patient VP-MCC tumor samples and normal skin controls. Arrow indicates mature p52 protein. HSP90, loading control. D, qRT-PCR analysis of relative expression of SASP genes (CCL26, CXCL10, GM-CSF, IL1B, IL6,andIL8) in VN-MCC and VP-MCC cancer cells. SASP genes are signiﬁcantly upregulated in VP-MCC. Data are normalized to b-actin expression and each group run as triplicates. E, qRT-PCR analysis of SASP genes (CCL26, CXCL10, GM-CSF, IL1B, IL6, and IL8) from patient VP-MCC tumor samples and unaffected control skin. Data are normalized by b-actin expression. Each point represents one individual tumor. Data are presented as mean SD for (D and E). , P < 0.05; , P < 0.01; , P < 0.001; , P < 0.0001. Signiﬁcance assessed by t test comparing virus-positive to virus-negative samples.

NFkB pathway choice, we also ﬁnd that HPyV6/7 sT, unlike This study provides a framework for understanding previously MCPyV sT, do not stabilize or activate Myc signaling. The regu- disparate observations about the biology of MCC tumors. Specif- lation of both ncNFkB signaling and c-Myc converge on the LSD ically, the speciﬁcity of MCPyV sT for only one arm of the NFkB motif present in MCPyV sT and reinforce the importance of this signaling pathway explains why ncNFkB pathway components (i.e., motif in promoting transformation. IKKa and MAP3K14) are among the most highly upregulated

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Figure 7. ncNFkB signaling is required for the growth of VP-MCC in vitro and in vivo. A, Proliferation of VP-MCC (MKL-1, MKL-2, MS-1, and WaGa) and VN-MCC (MCC13, MCC26) cell lines treated with a peptide inhibitor of ncNFkB inhibitor (SN52) or a control scrambled peptide (SN52mut). One-thousand cells plated per well; peptide (35 mg/mL) added on day 0. Proliferation assessed at the indicated time point by XTT assay. Each time point was run as triplicates. VP-MCC, but not VN-MCC, is sensitive to the inhibition of ncNFkB. B, Schematic of VP-MCC (MKL-1, WaGa) cancer cell xenograft and ncNFkB inhibitor (SN52) treatment in NSG immunodeficient mice. Cells (1 107 cells) were xenografted onto the flanks of NSG mice (n ¼ 8 per condition). C, Representative images of tumor-bearing mice and dissected MKL-1 and WaGa tumors (30 days) after xenograft and biweekly treatment with bearing mice images of MKL-1 and WaGa cell lines grown for 1 month receiving ncNFkB inhibitor or control peptide treatment subcutaneously. Inhibition of ncNFkB (SN52) significantly inhibits the growth of MKL-1 and WaGa xenografts. D, Weight of MKL-1 xenografted tumors receiving an ncNFkB inhibitor or control peptide. Each point represents an individual tumor. E, Weight of WaGa xenografted tumors receiving an ncNFkB inhibitor or control peptide. Each point represents an individual tumor. F, qRT-PCR analysis of a subset of SASP genes—IL1B, IL6, IL8—in MKL-1 and WaGa cells xenografts treated with an ncNFkB inhibitor or control peptide. SASP gene expression is significantly inhibited by the peptide ncNFkB inhibitor. Each point represents an individual tumor. Additional SASP genes in Supplementary Fig. S12. G, Representative images of H&E-stained tumors formed by MKL-1 and WaGa xenografts treated with an ncNFkB inhibitor (SN52) or control peptide (SN52mut). Scale bar, 200 mm. Data are presented as mean SD. , P < 0.05; , P < 0.01; , P < 0.001; , P < 0.0001. Significance assessed by one-way ANOVA followed by Tukey post hoc analysis for A and t test (D–F).

genes, while canonical NFkB(i.e.,IKKb and NEMO) are among the sis(41,58).Wefurtherpredictthat EZH2-overexpressing MCCs most significantly downregulated genes, in VP-MCC compared will overwhelmingly be virus-positive. Finally, telomerase activation with VN-MCC (56). It also helps to explain why NFkB signaling has also been identified as a downstream target of ncNFkBsignal- has been reported to be both inhibited and activated in different ing. Specifically, in cooperation with ETS1/2, NFKB2/p52 has studies (11, 12). Our findings also provide mechanistic insight to been shown to bind and promote the overexpression of the catalytic previous observations that MCPyV sT could increase inflammatory subunit of telomerase (TERT) in cells that contain the C250T cytokine expression when expressed in conjunction with MCC (-146C>T), but not the C228T(-124C>T), TERT promoter muta- tumor–derived LT (57). In concert with observations that the tion (59). MCCs possess a higher rate of C250T promoter mutations transcriptional upregulation of EZH2 is a key mechanism through than most other cancers (60–63). Thus, we suggest that the which NFKB2 is able to bypass senescence (16, 17), this study activation of ncNFkB signaling early in MCC tumorigenesis selects provides an explanation for EZH2 overexpression in many MCCs for the C250T promoter mutation at later stages of tumor and suggests why it may be associated with a worse progno- development.

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Given its oncogenic potential, it is not surprising that the signaling pathway in VP-MCC presents novel opportunities for activation of ncNFkB is tightly regulated. MCPyV sT induces global understanding and treating this deadly cancer. increases in H3K4me3 levels resulting in an increase in NFKB2 and RELB transcription. Although this transcriptional upregulation Disclosure of Potential Conflicts of Interest increases the pool of available NFKB2/p100 precursor and its No potential conflicts of interest were disclosed. binding partner RELB, processing of NFKB2 to its active p52 form is still necessary to activate ncNFkBsignaling.Wefind that MCPyV Authors’ Contributions sT’s ability to inhibit FBXW7 through its LSD domain and induce J. Zhao: Conceptualization, formal analysis, investigation, visualization, NFKB2 stabilization and cleavage is critical for activation of methodology, writing-original draft. Y. Jia: Resources. S. Shen: Formal analysis. J. Kim: Data curation, software, formal analysis. X. Wang: Resources. E. Lee: ncNFkB signaling in fibroblasts (Fig. 3I). However, MCC tumors Resources, investigation. I. Brownell: Resources. J.H. Cho-Vega: Resources. from patients showed marked variations in both RELB levels and C. Lewis: Resources. J. Homsi: Resources, writing-review and editing. NFKB2/p52 activation, suggesting that additional in vivo signals R.R. Sharma: Resources. R.C. Wang: Conceptualization, resources, data curation, contribute to the tuning of ncNFkBactivation. formal analysis, supervision, funding acquisition, investigation, visualization, While checkpoint inhibitors are effective in many cases of methodology, writing-original draft, project administration, writing-review and advanced MCC, new therapies are still needed for progressive or editing. recurrent disease. Inhibiting ncNFkB signaling could be a novel Acknowledgments therapeutic approach for VP-MCC tumors. While peptide inhibi- We thank Dr. Jerry Shay for BJ fibroblasts; Dr. Chris Buck for 1t1 antibody; Dr. tors of NFKB2 have been used in mouse models, small-molecule Shuyuan Zhang for Tet-on doxycycline-inducible vector; Drs. Masa Shuda, Paul inhibitors of ncNFkB, similar to those now available for canonical Nghiem, and Juergen Becker for the MCC cell lines. We thank Dr. Chris Buck for NFkB, would be more feasible in the clinical setting. These studies critically reviewing the manuscript. This work was supported by grants from the UT also revealed that the activation of vitamin D receptor (VDR) Southwestern Cary Council, ACS (RSG-18-058-01), and NIAMS (R01AR072655; to signaling with calcitriol could inhibit RELB and reduce the expres- R.C. Wang). sion of ncNFkB target genes. As low calcemic analogues of calcitriol have shown some benefits to patients with a wide variety of cancers The costs of publication of this article were defrayed in part by the payment of page in early-stage clinical trials (64, 65), it may be worth exploring charges. This article must therefore be hereby marked advertisement in accordance whether this pathway could be exploited in VP-MCC. Notably, with 18 U.S.C. Section 1734 solely to indicate this fact. MCCs express the VDR, and patients with vitamin D deficiency show significantly larger tumor size and worse prognosis (66). In Received July 2, 2020; revised July 16, 2020; accepted July 16, 2020; published first summary, the identification of ncNFkB activation as a novel August 4, 2020.

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Merkel Cell Polyomavirus Small T Antigen Activates Noncanonical NF κB Signaling to Promote Tumorigenesis

Jiawei Zhao, Yuemeng Jia, Shunli Shen, et al.

Mol Cancer Res Published OnlineFirst August 4, 2020.

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