Rapalog-Mediated Repression of Tribbles

Published OnlineFirst April 3, 2020; DOI: 10.1158/0008-5472.CAN-19-2366

CANCER RESEARCH | MOLECULAR CELL BIOLOGY

Rapalog-Mediated Repression of Tribbles Pseudokinase 3 Regulates Pre-mRNA Splicing Bojana Stefanovska1,2,3, Cecile Edith Vicier1,2,3, Thibault Dayris2,4, Vasily Ogryzko5,†, Veronique Scott1,2,3, Ibrahim Bouakka1,2,3, Suzette Delaloge6, Anna Rocca1,2,3, Olivia Le Saux7, Olivier Tredan 7, Thomas Bachelot7, Fabrice Andre1,2,6, and Olivia Fromigue1,2,3

ABSTRACT ◥ Rapalogs have become standard-of-care in patients with induced deregulation of RNA splicing. Conversely, overexpres- metastatic breast, kidney, and neuroendocrine cancers. Never- sion of TRIB3 in a panel of cancer cell lines abolished theless, tumor escape occurs after several months in most the cytotoxic effects of rapalogs. These findings identify TRIB3 patients, highlighting the need to understand mechanisms of as a key component of the spliceosome, whose repression resistance. Using a panel of cancer cell lines, we show that contributes significantly to the mechanism of resistance to rapalogs downregulate the putative protein kinase TRIB3 (trib- rapalog therapy. bles pseudokinase 3). Blood samples of a small cohort of patients with cancer treated with rapalogs confirmed down- Significance: Independent of mTOR signaling, rapalogs induce regulation of TRIB3. Downregulation of TRIB3 was mediated cytoxicity by dysregulating spliceosome function via repression of by LRRFIP1 independently of mTOR and disrupted its inter- TRIB3, the loss of which may, in the long term, contribute to action with the spliceosome, where it participated in rapalog- therapeutic resistance.

Introduction development of some derivatives overcame issues related to poor solubility and pharmacokinetics of the original rapamycin molecule. Tumor progression is a complex process resulting from the accu- In that way, temsirolimus and everolimus (also known as rapalogs) mulation of genetic alterations like chromosomal rearrangements, improved patient outcome in a broad range of cancers. As illustration, mutations, silencing of transcription through methylation, or defects the phase III trials BOLERO-2, RECORD-1, and RADIANT-3 dem- in posttranscriptional modifications such as mRNA splicing, mRNA onstrated that everolimus could improve progression-free survival of stabilization, and translational regulation. Thus, tumor cells synthesize patients with metastatic hormone receptor–positive breast cancer, and accumulate proteins altered in their structure or function, mod- metastatic renal cell carcinoma, or advanced pancreatic neuroendo- ifying some signaling pathways. One of the key proteins involved in crine tumors, respectively (6–8). Rapalogs are currently administered tumor progression is mTOR (mechanistic target of rapamycin), to patients presenting hormone receptor–positive breast cancers, because it integrates signals from nutrients, hormones, and growth metastatic kidney cancers, and neuroendocrine tumors (9). While the factors to regulate cell metabolism, growth, proliferation, and surviv- benefit for patients is matter of consensus, resistance to therapy occurs al (1). The mTOR signaling pathway is hyperactivated in up to 70% of usually after several months of treatment. Resistance can often occur human tumors (2). The macrolide rapamycin, originally identified for through the activation of mTOR-regulated feedback loops or by the its antifungal properties, forms a gain-of-function complex with the acquisition of new mutations (10, 11). Nevertheless, these mechanisms immunophilin FKBP12 (FK506-binding protein 1A, 12 kDa) that acts explain only a few percentage of resistance, and there is a need to as an allosteric inhibitor of mTOR (3). Rapamycin exhibits immuno- identify new mechanisms of treatment escape. suppressive properties, but is also used as anticancer therapy (4, 5). The In this study, we aim to identify new mechanisms of action for rapalogs and we have discovered that tribbles pseudokinase 3 (TRIB3) 1Inserm, UMR981, Villejuif, France. 2Gustave Roussy, Villejuif, France. 3Universite encoded by the tribbles pseudokinase-3 gene mediates effects of Paris Sud, Orsay, France. 4Inserm, US23, CNRS, UMS3655, Villejuif, France. rapalogs on splicing, in an mTOR-independent manner. 5CNRS, UMR8126, Villejuif, France. 6Department of Medical Oncology, Gustave Roussy, Villejuif, France. 7Department of Medical Oncology, Centre Leon Berard, Materials and Methods Lyon France. Chemicals and reagents Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Rapamycin was purchased from Sigma-Aldrich. Everolimus [40-O-(2-hydroxy)ethyl-rapamycin] was purchased from Novartis. B. Stefanovska and C.E. Vicier contributed equally to this article as co-first authors. Rabbit anti-TRIB3, anti-GFP, anti-CDC5L, anti-FKBP25, and mouse anti-LRRFIP1 antibodies were purchased from Abcam. F. Andre and O. Fromigue contributed equally to this article as co-last authors. Mouse anti-b-actin antibody was purchased from Sigma-Aldrich. † Deceased. Rabbit anti-phospho-Akt and anti-Akt antibodies were purchased Corresponding Author: Olivia Fromigue, Inserm UMR981, Gustave Roussy, Rue from Cell Signaling Technology. Camille Desmoulins, Villejuif 94805, France. Phone: 331-4211-4211; Fax: 331-4211- 6094; E-mail: [email protected] Plasmids Lentiviral vector for TRIB3 overexpression was generated by Gate- Cancer Res 2020;80:2190–203 way cloning from pDONR223-TRIB3 into pLX303, both purchased doi: 10.1158/0008-5472.CAN-19-2366 from Addgene. Prof. I.C. Eperon (University of Leicester, Leicester, 2020 American Association for Cancer Research. United Kingdom), and Dr. Y. Itoh (Nagoya City University, Nagoya,

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Japan) kindly provided the pTN23 (12, 13) and pTRB3-Luc1 reporter trypan blue solution. Experiments were conducted with 6 106 viable vector, respectively. The derived plasmid pTRB3-Luc1 D136-145 was cells per condition. generated by the QuikChange II XL Site-Directed Mutagenesis Kit Whole blood samples were derived from the ongoing clinical trial (Stratagene). The mutagenic oligonucleotides were: 50-TGGAGGCT- NCT02536625 (RAPANK; Supplementary Table S1). Blood samples CTGGCGCCCGGGCC-30 (forward) and 50-GGCCCGGGCGCCA- were collected before and one month after everolimus initiation. GAGCCTCCA-30 (reverse). The pEZX-PG02-TRIB3 promoter Written informed consent was collected from all patients, according reported plasmid (HPRM13929) was purchased from Tebu-Bio. The to the principles of the Declaration of Helsinki. pCH110 plasmid was purchased from Addgene. RNA extraction Minigene reporter assay Total RNAs were isolated using TRIzol reagent (Thermo Fisher Cells at 65%–70% confluence were cotransfected with pCH110 and Scientific) for cell lines or PBMC, and using TRIzol-LS reagent pTRB3-Luc1, pEZX-PG02-TRB3 or pTRB3-Luc1 D136-145 using (Thermo Fisher Scientific) for whole blood samples. The quality of Lipofectamine2000 reagent (Thermo Fisher Scientific). Transcription- RNA samples was checked using Bioanalyzer. Mean values of RNA al activity was evaluated using Luciferase Assay Kit (Promega) or integrity numbers were 9.1 0.3. Gaussia Luciferase Flash Assay Kit (Thermo Fisher Scientific) and transfection efficiency by b-gal gene Reporter Assay Kit (Roche). Comparative microarray analyses Transcriptional profiles were determined using Agilent SurePrint Cell culture G3 Human GE 8 60K V2 microarrays (AMADID 39494; Agilent All cell lines (except SUM52PE cells, kindly provided by Dr Turner Technologies), and analyzed with Agilent Feature Extraction software from The Institute of Cancer Research, London, UK) were purchased version 10.7.3.1 using the default settings. Raw data files were pro- from the ATCC (LGC Standards) between 2007 and 2011. Each ATCC cessed as follows: control probes were systematically removed and cell line was first amplified to generate a cell master bank. All flagged probes were set to NA. Arrays were normalized by quantile experiments were carried out from this master bank with cells kept normalization. Missing values were inferred using KNN algorithm in continuous culture for less than 6 months. No further authentication (package “impute” from R). was performed. HCC827, SKBR3, MKN45, and SUM52PE cells were cultured in GlutaMAX containing RPMI1640 medium supplemented RNA-seq analysis with 10% heat-inactivated FBS (Thermo Fisher Scientific), 1 mmol/L Libraries were made using the TruSeq Stranded mRNA Library sodium pyruvate, and 1% HEPES. A375 cells were cultured in Glu- Preparation Kit (Illumina). Equimolar pool of libraries were sequenced taMAX containing DMEM supplemented with 10% FBS, 1 mmol/L on a Illumina HiSeq2500 machine using paired-end reads (2 75 bp), sodium pyruvate, and 1% HEPES. MDA-MB-468 cells were cultured and high-output run mode allowing to get 40 millions of raw reads per in GlutaMAX containing DMEM/Nutrient Mix F-12 medium sup- sample. The quality of reads was assessed using FastQC. Quality plemented with 10% FBS, 1 mmol/L sodium pyruvate, and 1% HEPES. reports were gathered with MultiQC. Abundance estimation was MCF7 and T47D cells were cultured in DMEM/F12 medium supple- achieved with Salmon (15) with the arguments keepDuplicates mented with 10% FBS, 1 mmol/L sodium pyruvate, and 10 mg/mL --gencode --perfectHash in indexing step, and numBootstraps 100 insulin. The human embryonic kidney 293T (HEK293T) cells were --validateMappings --gcBias --seqBias in quantification step. Paired- cultured in GlutaMAX containing DMEM supplemented with 10% end clean reads were mapped to the GRCh38 human reference genome FBS. Cells were maintained under Mycoplasma free conditions, and assembly (version V27). Aggregation was achieved with an in-house regularly tested using the MycoAlert Mycoplasma Detection Kit script. The whole quantification process and pipeline can be found (Lonza). online on https://bitbucket.org/tdayris/rna-count-salmon/src/master, powered by both Snakemake and SnakemakeWrappers (16). Both Cell transduction differential transcript expression and differential gene expression were Lentiviral particles containing vector-encoding LRRFIP1, mTOR, assessed with Sleuth (17) following the SwimmingDownstream guide- or scrambled control short hairpin RNA (shRNA) were purchased lines (18). In-house Sleuth scripts are available on https://github.com/ from Santa Cruz Biotechnology. New stable cell lines were established tdayris/yawn/tree/master/Scripts/. as described previously (14). Among differentially expressed transcripts, we applied the following filters to keep only splicing variants. First, we identified the Cytotoxicity assay transcripts with differential expression status and/or b-value opposing WST-1 solution (Sigma-Aldrich) was added for the last 2 hours of to the ones of their respective gene. Same parameters and thresholds as cell culture. Formazan dye content was quantified by measuring its above were applied for Sleuth analysis to identify differentially absorbance (450 nm). expressed targets. Transcripts and genes were then divided into two classes: upregulated if q-value ≤ 0.001 and b-value ≥ 1; and down- Peripheral blood mononuclear cell and whole blood samples regulated if q-value ≤ 0.001 and b-value < 1. Not differentially Peripheral blood mononuclear cells (PBMC) were isolated from expressed genes (q-value > 0.001) were excluded from tables and blood samples of 9 patients with various cancer types (Supplementary graphs. Second, we identified transcripts sharing the same transcrip- Table S1). The protocol used in the study was approved by the tion start site (TSS). Transcripts with different TSSs were excluded Institutional Review Board of Gustave Roussy. Written informed from tables and graphs. consent was received from all patients, according to the principles of Specific junction differential coverage was performed on reads the Declaration of Helsinki. Blood samples (10 mL) were collected in mapped against Gencode GRCH38 version V27 with STAR version EDTA tubes, diluted in PBS, layered onto Ficoll in SepMate tubes 2.7.1, using LeafCutter version 0.2.7 (19). After conversion into Bed4 (Stemcell Technologies), and centrifuged at 1,200 g for 10 minutes at format with in-house scripts, comparison of sets of splicing regions room temperature. The viability of PBMC was evaluated with 1% was achieved using bedtools. Finally, LeafCutter computes percentage

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of spliced in (PSI) values that reflect the proportion of usage of a given PAG magnetic beads for 90 minutes at þ4C, then washed twice with junction in relation to all other possible junctions. The difference in PSI each of the low salt, high salt, LiCl immune complex wash buffers, and (dPSI) is the difference in this usage proportion between two TE buffer. The protein-DNA complexes were reverse cross-linked conditions. according to the IPure Kit v2 protocol (Diagenode). The recovered Gene ontology analysis to identify biological processes likely mod- DNA was amplified by qRT-PCR using nonoverlapping primer ulated was achieved with Database for Annotation, Visualization, and pairs of about 70 to 150 bp apart for each region of interest: 50- Integrated Discovery (DAVID) tools. CACCCCGCCGAGTACCTC-30 and 50-CACCCCGCCGAGTAC- CTC-30 for TRIB3; 50-ACTCAGTCTGGGTGGAAGGT-30 and 50- RT-PCR CCCATTGCATTTGTTGGGGG-30 for MYC; 50-GACATAACCCC- Total RNA (1 mg) were reverse transcribed using Superscript-II CACCCCTTG-30 and 50-AAGGAAGCAGCCTCTCAACC-30 for Reverse Transcriptase (Thermo Fisher Scientific) and random DNMT3B set1; 50-TAAGGGAGGAGCAAGGGAGG-30 and 50-TG- hexamers. GGGCTGCAATAATGAGGG-30 for DNMT3B set2. Real-time quantitative PCR (qPCR) was performed on a ViiA7 apparatus with the SYBR-green master mix (Thermo Fisher Scientific). LC/MS-MS Gene-specific primers sequences are 50-GTCTTCGCTGACCGT- For trypsin digestion, the beads with immune complexes were GAGA-30 and 50-CAGTCAGCACGCAGGAGTC-30 for TRIB3; 50- preincubated with 7 mL of 15 ng/mL trypsin and ambic at RT for CGGCTACCACATCCAAGGAA-30 and 50-GCTGGAATTACCGC- 10 minutes. Afterward, 25 mL of 50 mmol/L ammonium bicarbonate GGCT-30 for 18S. The cycling protocol comprised 45 cycles of 95C for was added and the samples were incubated at 37C for 16 hours. The 15 seconds, 60C for 60 seconds, and 72C for 15 seconds. The relative supernatants were dried at 56C by SpeedVac for 30 minutes, then DDC amounts of RNA were calculated by the 2 t method. resuspended in 20 mL of solution containing 0.05% formic acid and 3% Semiquantitative PCR amplification was performed using GoTaq acetonitrile. G2 Hot Start Master mix (Promega), and specific primers designed on The resulting peptide mixtures were analyzed with a qExactive- the constitutive exons: 50-CATAGGATCCAGCTACCGGG-30 50- Orbitrap High Resolution and Accuracy Mass Spectrometer TGTCTTCTTGGAGCTGACGA-30 for SIN3B; 50-TGAGGACAT- (HRAM; Thermo Fisher Scientific) equipped with a nanoelectros- AGCTCGCCTTT-30 50-ACCATTCCTGATCCCTGTGG-30 for CC- pray source and coupled to a NanoLC Esay nLC 1000 (Thermo DC50; 50-CTGCCAGCATCGACCTTCT-30 50-GTCCTCCGGCCT- Fisher Scientific). The peptides were separated for 30 minutes on TCCACT-30 for DENND1A; 50-GGGGCAGATGGTGGATGAG-30 a gradient of 5% to 90% acetonitrile. The fragmentation voltage 50-AGGCAGTTCTCGTGGGATC-30 for PSD4; 50-TCTTTACGG- was 1.3 V. The mass spectrometer acquired successive sets of four CTCTGGGGATC-30 50-GCTTGTCACCTGTCTCCTCA-30 for SL- scan modes consisting of one full scan MS over the range of 200– C2A11; 50-GTGTTGCTGGCACCAATATCA-30 50-GAGGTCGCC- 2,200 m/z, followed by three data-dependent MS-MS scans on the CATTTTCACAG-30 for ADAM22. The cycling protocol comprised three most abundant ions in the full scan. Peak lists (mgf) were 35 cycles of 95C for 30 seconds, 55C for 30 seconds, and 72C for 30 generated from the raw data files using Proteome Discoverer seconds. The PCR products were separated onto 2% agarose gel. PSI application (Thermo Fisher Scientific) and proteins were identified was calculated as the ratio of the density of the short isoform signal with MASCOT search engine. Datasets were submitted to the versus that of the sum of the long and short isoform. The dPSI was Contaminant Repository for Affinity Purification (CRAPome; calculated between PSI in rapamycin-treated versus that of the vehicle. ref. 20). Putative candidates were discarded if (i) the anti-GFP–related score Western blotting assay was higher than 15% of any anti-TRIB3–related scores, (ii) they were Proteins (30 mg) from total cell lysates were resolved by SDS-PAGE not detected in all the four tested cell lines, and (iii) they were described as described previously (14). as background contaminants by the CRAPome interface. The protein subfamily functional classification was performed using the bioinfor- Immunoprecipitation matics tool DAVID. No cutoff was applied before submission of the list Cell lysates were prepared as for Western blotting analysis except to DAVID application. that Nonidet-P40 (Igepal) concentration was reduced to 0.1%. Lysates were precleared using recombinant fusion protein A/G (PAG) coupled Promoter analysis for transcription factor–binding sites to magnetic microbeads (Ademtech) for 1 hour. Aliquots of 2 mg of In silico analysis of the promoter region of the human TRIB3 gene protein were incubated ON at þ4C under weak agitation with 40 mL (Gene ID: 57761) was performed using Genomatix software. The of PAG magnetic beads and 4 mg of indicated antibody. The immu- region conserved in 8 of 12 orthologs loci and corresponding to noprecipitated complexes were washed thrice with lysis buffer, ali- 380117-381145 (GRCh38 Chromosome 20 contig NC_000020.11) quoted and stored at 80C. was selected for the identification of putative promoters, and TSSs. We focused on the region from 400 bp upstream to 200 bp Chromatin immunoprecipitation downstream of the main TSS to select common transcription Cells were incubated in 1% formaldehyde for 10 minutes at room factor–binding sites using the MatInspector tool of the Genomatix temperature. After three washes in PBS, cells (2107) were sonicated software. for 20 minutes (30% of maximum power) on ice, then centrifuged at 17,000 g for 2 minutes at þ4C. DNA shearing efficacy was validated Statistical analyses on a 2% agarose gel. Preclearing of chromatin immunoprecipitation All quantitative data are presented as mean SD unless otherwise (ChIP) lysates was carried out using PAG magnetic beads (Ademtech) specified. Two-sided Student t tests were used for comparisons of the for 1 hour at þ4C. Specific and nonspecific IPs were carried out means of data between two groups. For multiple independent groups, overnight using mouse anti-LRRFIP1 or IgG2a Isotype Control Anti- one-way or two-way ANOVA was used. A level of P < 0.05 was bodies (Abcam) at 10 mg/mL. Immune complexes were collected with considered significant.

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Results cell lines. The overexpression of TRIB3 did not significantly interfere Rapamycin reduced TRIB3 gene expression in cancer cell lines with the stimulatory effect of rapamycin. and patient samples We therefore assessed other potential mechanisms of action of TRIB3. To characterize the TRIB3 protein interaction network, we We first determined the cell response in a panel of eight cancer cell isolated protein complexes by IP with antibodies raised against N-term lines to increasing doses of rapamycin (0.01–1,000 nmol/L) using the and C-term domains of TRIB3. We identified partner candidates by WST1 colorimetric assay. Rapamycin induced cell cytotoxicity in all LC/MS-MS–based targeted proteomics. First screening on whole tested cell lines in a dose-dependent manner (13% to 31% in cell lysates resulted with the detection of 350 putative partners of cell viability, P < 0.05 vs. vehicle; Supplementary Fig. S1A). All the TRIB3 in common between the four tested cell lines (HCC827, MCF7, following experiments were performed with 10 nmol/L rapamycin. MDA-MB-468, and SUM52PE). We identified 22 functional annota- Using gene expression arrays, we identified genes whose expres- tion clusters based on Gene Ontology (GO) categories and Kyoto sion was modulated by rapamycin. The subsequent analyses using Encyclopedia of Genes and Genomes (KEGG) pathway enrichment |log2(FC)|>1andP < 0.05 cutoff revealed that TRIB3 is the only analyses. The cluster with the highest enrichment score (ES) suggested gene downregulated in common in all tested cell lines treated that the majority of the identified partners are related to the pre-mRNA with rapamycin (Fig. 1A and B; Supplementary Table S2). The splicing process (Supplementary Fig. S3A). We performed a second full list of detected genes is available in the ArrayExpress database at round of IP on whole cell lysates pretreated with RNase-A to avoid EMBL-EBI (www.ebi.ac.uk/arrayexpress) under accession number precipitation of proteins present onto RNA molecules but not related E-MTAB-8727. Quantitative real-time PCR and Western blotting to TRIB3-containing complexes. This second screening shortened the analysis confirmed that rapamycin decreased TRIB3 mRNA (36% list to 209 putative partners for TRIB3 (Supplementary Table S3). Four to 62%, P < 0.05 vs. vehicle; Fig. 1C)andprotein(37% to out of the top five clusters with the highest ES still designated the 71%; Fig. 1E) expression level in all tested cell lines. Moreover, mRNA splicing process (Fig. 3A), and 41 of the 209 putative partners everolimus also decreased TRIB3 mRNA expression level in all mapped with the spliceosome complexes (Supplementary Table S3). tested cell lines (30% to 65%, P < 0.05 vs. vehicle; Fig. 1D)with To better identify the spliceosomal components that interact with the same magnitude as rapamycin. TRIB3, we performed enrichment analysis using a published gene To confirm the effect of rapamycin or everolimus on TRIB3 set (25) compiling 404 spliceosomal proteins identified in previous expression ex vivo, PBMC collected from patients with various malig- studies (26, 27) and/or reported in the spliceosome database (http:// nancies (Supplementary Table S1) were incubated for 24 hours with or spliceosomedb.ucsc.edu). We refined this gene set for components that without rapamycin or everolimus. Quantitative real-time PCR con- are shared by the three sources (total 149 hits). Among the 16 identified firmed that rapamycin decreased (average 36%, P < 0.0002 vs. spliceosomal core proteins, 63% (10/16) belong to the B or Bact vehicle; Supplementary Fig. S1B) TRIB3 expression level. Accordingly, complexes. Ranking TRIB3 putative interactors according to the everolimus also decreased (average 31%, P < 0.001 vs. vehicle; relative score revealed enrichment in proteins present in the PRP19 Supplementary Fig. S1C) TRIB3 expression level. complex, and the U2 small nuclear ribonucleoproteins complex (U2 To estimate the effect of everolimus on TRIB3 expression in vivo, snRNP) at the top of the list (Supplementary Table S3). Hence we chose whole blood cells were collected from patients with breast cancer CDC5L (cell division dycle 5 like) and SF3B1 (splicing factor 3B before and one month after everolimus treatment initiation. (Supple- subunit 1A), key components of the splicing machinery (28), to mentary Table S1). Quantitative real-time PCR showed that the confirm that TRIB3 interacts with the PRP19 and U2 complexes. treatment decreased TRIB3 expression in 10 of 16 (63%) samples Reciprocal coimmunoprecipitations (co-IP) and Western blotting (Fig. 1F), leading to an average of 33% vs. beginning of treatment analyses confirmed that TRIB3 interacts with CDC5L and SF3B1 (Fig. 1G). (Fig. 3B and C, respectively), reinforcing the hypothesis that TRIB3 These data suggest that TRIB3 expression is downregulated by could be involved in pre-mRNA splicing modulation. rapalogs, leading to the hypothesis that it could mediate at least part of These findings led to the hypothesis that rapalogs could modulate the activity of this drug family. splicing via TRIB3 downregulation.

TRIB3 overexpression prevents the cytotoxic effects of Rapamycin inhibits pre-mRNA splicing process through rapamycin in vitro downregulation of TRIB3 expression fl To evaluate whether TRIB3 expression levels in uence the response To evaluate splicing efficiency in the presence of rapamycin and the to rapamycin, we established new cell lines by lentiviral transduction. possible contribution of TRIB3 in the pre-mRNA splicing process, we As expected, the stable integration of the TRIB3 coding sequence into used a double-reporter vector previously reported and validated to the cancer cell genome led to a stably increased TRIB3 protein level assess splicing (13, 29). This vector encodes b-galactosidase and compared with cells transduced with empty vector (Supplementary luciferase in the same reading frame when splicing occurs. When Fig. S2). TRIB3 overexpression prevented or at least markedly reduced there is no splicing, they are separated by an intronic region containing the cytotoxic effect of rapamycin on all tested cancer cell lines by 64% a stop codon (Fig. 3D). Rapamycin reduced the splicing efficiency up in average (range: 39% to 86%; Fig. 2). to 55% (P < 0.05 vs. vehicle; Fig. 3E). For A375, MCF7, SKBR3, and SUM52PE cell lines, where the splicing is impaired the most, we TRIB3 interacts with pre-mRNA splicing machinery observed that the overexpression of TRIB3 abolishes or markedly Inhibiting mTOR with rapamycin abolishes negative feedback-loop reduces the inhibitory effect of rapamycin on splicing activity (Fig. 3F). signals and leads to the reactivation of PI3K/Akt pathway (21). Several These data suggest that rapamycin treatment affected the splicing publications suggest that TRIB3 directly interacts with Akt and inhibits process, and that TRIB3 expression level could modulate this effect. its phosphorylation (22–24). Western blotting analyses confirmed that Because this double-reporter is an artificial system that evaluates rapamycin increased (1.79-fold vs. vehicle; Supplementary Fig. S2) Akt particular splicing context, we performed RNA-seq analyses to deter- phosphorylation level on Ser473 and/or Thr308 residues in all tested mine whether rapamycin affects splicing genome-wide. The complete

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Figure 1. Rapalogs reduce TRIB3 expression level in cancer cells and patient samples. A, Volcano plot of differentially transcribed genes identiﬁed by microarray analysis. The

average difference in mRNA expression level (log2-fold change) for all genes are plotted against the log10 of the adjusted P value. Thresholds are shown as dashed lines. B, Box-and-whisker plot showing the minimum, ﬁrst quartile, median, third quartile, and maximum fold change in TRIB3 mRNA expression level. C and D, Expression pattern of TRIB3 mRNA after 24-hour incubation with 10 nmol/L rapamycin (C) or everolimus (D), assessed by qRT-PCR. The relative mRNA levels are

expressed as log2 (fold change) versus vehicle. E, Expression pattern of TRIB3 protein, assessed by Western blotting analysis. Actin was used as loading control. F, Expression pattern of TRIB3 mRNA in whole blood samples (see characteristics of patients in Supplementary Table S1), assessed by qRT-PCR. The relative expression

levels are expressed as log2 (fold change). G, Box-and-whisker plot showing the minimum, ﬁrst quartile, median, third quartile, and maximum average log2 (fold change) in TRIB3 expression level in whole blood cells. FC, fold change.

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Figure 2. TRIB3 overexpression prevents the rapamycin inhibitory effect on cancer cells viability. Cell viability was determined after 24-hour incubation in the presence of rapamycin (1–100 nmol/L) or vehicle, assessed by the WST-1 assay. Results are expressed as mean of percentage of inhibition compared with vehicle SEM (three independent experiments; n ¼ 8–10 replicates per experiment). a, P < 0.05 versus vehicle; b, P < 0.05 versus pLX-empty vector.

list of the detected transcripts is available in the ArrayExpress database Fig. S3E). These results are in agreement with the distribution of at EMBL-EBI under accession number E-MTAB-8726. alternative splicing events landscape described in reference annota- To avoid a bias due to transcriptional regulation, we discarded tions of the human genome (RefSeq, Gencode, and EnsEMBL; ref. 30), alternative transcripts that do not share the same TSS. We then focused and indicate that the overexpression of TRIB3 did not induce a on transcripts with differential or opposing expression level compared significant shift in alternative splicing patterns. To gain insights into with their respective genes (q-value ≤ 0.001). This multilevel analysis the most relevant GO-terms and bio-pathways that underwent alter- allowed us to identify 3,161 transcripts with modulated expression native splicing under rapamycin treatment, we performed a DAVID level between rapamycin- and vehicle-treated cells (Fig. 4A). Among functional annotation clustering analysis of the 1,847 impacted genes. those transcripts, 1,365 (43%) were modulated in both pLX-empty and The top four most enriched clusters refer to (i) cell division, (ii) pLX-TRIB3 cells, whereas 844 transcripts (27%) were specifically kinetochore, (iii) sterol biosynthesis and metabolism, and (iv) trans- modulated in pLX-empty cells, and 952 (30%) in pLX-TRIB3 cells. lation and ribosome (Fig. 4D). The corresponding genes are listed in Supplementary Table S2 and To identify the regions affected by splicing modulations, we per- their intersections are presented in Supplementary Fig. S3B. The formed a differential splice junction coverage (Supplementary modulations were consistent between the pLX-empty and pLX-TRIB3 Table S2). We identified 13,417 genes that contain at least one cells: 704 (52%) upregulated transcripts and 660 (48%) downregulated differential spliced region. Among them, 4,182 are in common transcripts, and only one transcript was modulated in an opposing between the pLX-empty and pLX-TRIB3 cells whereas 8,923 are sense (Fig. 4C). Interestingly, we observed that the overexpression of present only and pLX-TRIB3 cells and 312 only in pLX-empty cells TRIB3 led to the modulation of expression of just 16 transcripts in the (Supplementary Fig. S3F). The intersections of the splicing modula- absence of rapamycin (Fig. 4B), encoding for 13 genes (Supplementary tions between the modulation (Up or Down) and the stable cell line Fig. S3C), whereas the overexpression of TRIB3 combined with the modification (pLX-empty or pLX-TRIB3) are presented in Supple- rapamycin treatment modulated only 65 transcripts (56 genes). mentary Fig. S3G. Among the 232 differential spliced regions exhibit- In summary, these data suggest that the overexpression of TRIB3 ing a dPSI ≥ 20% (Supplementary Table S2), we investigated high alone modulated a very limited number of transcripts. The vast confident examples by semiquantitative RT-PCR. Differential splic- majority of the splicing modulations were dependent on the presence ing was confirmed for all tested examples (Fig. 5A and B). The of rapamycin and the TRIB3 overexpression prevents some of these prevention mediated by the overexpression of TRIB3 was also splicing modulations. confirmed. For instance, we observed similar splicing modulations According to the alternative splicing patterns (Supplementary byrapamycininbothpLX-emptyandpLX-TRIB3cellsfor Fig. S3D), we identified skipped exon as the major pattern followed CCDC50 (coiled-coil domain containing 50), PSD4 (Pleckstrin and by alternative first exon (AF), and alternative 30 splice site (A3), Sec7 domain containing 4), and SLC2A11 (solute carrier family 2 whereas retained intron (RI), mutually exclusive exons (MX), and member 11). Furthermore, we confirmed a prevention of the alternative last exon (AL) occurred with a lower frequency. No splicing modulations by rapamycin in pLX-TRIB3 cells for SIN3B difference was detectable in the pattern distribution between the (SIN3 transcription regulator family member B), DENND1A pLX-empty cells and the TRIB3 overexpressing cells (Supplementary (DENN domain containing 1A), and ADAM22 (ADAM

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Figure 3. TRIB3 overexpression counterbalances the rapamycin inhibitory effects on pre-mRNA splicing efficiency. A, Classification of identified proteins with DAVID. The top five functional association clusters ranked by the ES are shown. B and C, Whole SUM-52PE cell lysate was subjected to CDC5L, SF3B1, or TRIB3 IP. Anti-GFP antibody served as negative IP control. D, Schematic representation of the principle of the assay using the pTN23 minigene reporter construct. A segment of the b-galactosidase gene and an intervening intron are cloned in-frame with the luciferase coding sequence. If correct splicing occurs, both b-galactosidase and luciferase activities are detectable. In case of altered splicing, the stop codon (dotted line) present in the retained intron prevents the expression of luciferase. E and F, Relative luciferase activity determined after 24-hour incubation. Results are expressed as fold change SD (three independent experiments; n ¼ 4–5 replicates per experiment). , P < 0.05 versus vehicle. ns, nonspecific.

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Figure 4. Rapamycin modulates splicing genome- wide. A–C, UpSet plot of intersections between sets of shared splicing events (transcripts) in pLX-empty and pLX- TRIB3 cells incubated for 24 hours in the presence of 10 nmol/L rapamycin or vehicle. The bar chart on the left indicates the total number of events. The top bar chart indicates the intersection size between sets of events shared with one or more condition(s). Dark connected dots indicate which conditions are considered for each intersection. D, Func- tional annotation of transcripts with modulated splicing after 24-hour rapamycin treatment was performed with DAVID. The top four functional association clusters ranked by the ES are shown.

metallopeptidase domain 22). The variation in dPSI values deter- Rapamycin downregulates TRIB3 expression through an mined from signal on agarose gels (Fig. 5B) were similar to those mTOR-independent mechanism via LRRFIP1-dependent determined from RNA-seq (Fig. 5C), conﬁrming that treatment of inhibition of promoter activity pLX-empty cells with rapamycin impacts splicing process, and that We then assessed the molecular mechanisms involved in the TRIB3 overexpression could prevent some events. regulation of TRIB3 expression by rapalogs. Because rapamycin is an Altogether, these data suggest that rapamycin modulates the pre- allosteric mTOR inhibitor, we ﬁrst investigated the role of this mRNA splicing process, and that part of this effect is dependent on the signaling pathway in the regulation of TRIB3 expression. Targeting downregulation of TRIB3. mTOR with the ATP-competitive inhibitor Torin-1 dose dependently

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Figure 5. RT-PCR validation of high conﬁdence alternative splicing events identiﬁed by RNA-seq. A, Left, diagram of alternative splicing pattern. Arrows, primers; boxes, exons; lines, introns. Right, agarose gel electrophoresis of RT-PCR products from pLX-empty and pLX-TRIB3 cells incubated for 24 hours with 10 nmol/L rapamycin or vehicle. B, Graphical comparison in the dPSI between pLX-empty and pLX-TRIB3 cells incubated for 24 hours with rapamycin or vehicle, assessed by RT-PCR. C, Graphical comparison in dPSI between pLX-empty and pLX-TRIB3 cells incubated for 24 hours with rapamycin or vehicle, assessed by RNA-seq.

reduced phosphorylation level of key effectors of mTOR signaling (Leucine rich repeat in flightless-1 interacting protein 1), also known pathway such as Akt or S6RP but did not affect TRIB3 protein level as GCF2 (GC-binding factor 2) was the only DNA-template negative (Fig. 6A). regulator of transcription. ChIP experiments performed on SUM52PE In silico analyses of TRIB3 promoter region (chromosome location cells, followed by RT-qPCR detected specific LRRFIP1 binding on the 20p13-p12.2; contig NC_000020.11; GRCh38 reference primary TRIB3 promoter region upon rapamycin treatment (Fig. 6B). No assembly) identified six unique putative promoters, but three of LRRFIP1 binding was detectable on nonspecific target sequences, them without assigned transcript. Among the three remaining namely the promoters of MYC or DNMT3B (DNA methyltransferase putative promoters (Supplementary Fig. S4A), promoter #1 allowed 3 beta). generating a transcript encoding a truncated protein (60 aa). In The inhibitory effect of rapamycin on the TRIB3 promoter activity contrast, promoter #2 and #3 allowed generating 10 putative was evaluated using a gene reporter assay. Three vectors were used encoding transcripts ultimately corresponding to the two major (Supplementary Fig. S4B): pTRB3-Luc1, which contains the LRRFIP1 isoforms of TRIB3 protein (358 aa and 385 aa). A minor truncated binding site located upstream of TSS, pTRB3-Luc1 D136-145, where cds (130 aa) was also proposed. the LFFRIP1 binding site was deleted by site-directed mutagenesis A broad spectrum screening for putative transcription factor– (Supplementary Fig. S4C), and pEZX-PG02 TRB3, which contains a binding sites in the region covering the promoters #2 and #3 detected region upstream to the LRRFIP1 binding site. Rapamycin reduced 267 matches for sequences corresponding to 109 putative different (20% to 32%, P < 0.05 vs. vehicle; Fig. 6C) luciferase activity in cells transcription factors (multiple match score). Applying a threshold transfected with the pTRB3-Luc1 vector but did not affect luciferase adjusted for P value < 0.05 restricted the list to 12 candidates activity in cells transfected with the pTRB3-Luc1 D136-145 vector or (Supplementary Table S4). Among those 12 candidates, LRRFIP1 with the pEZX-PG02 TRB3 vector.

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Figure 6. Rapamycin reduces TRIB3 expression through a LRRFIP1-dependent repression of promoter activity, independently of mTOR. A, Expression pattern of indicated proteins after 24-hour incubation with Torin-1 (2–250 nmol/L), assessed by Western blotting analysis. Actin was used as loading control. B, ChIP-qPCR analysis using LRRFIP1 or IgG control antibodies, and primers against TRIB3 promoter region, or MYC and DNMT3B promoter regions as negative controls. Results are expressed as fold enrichment SD (n ¼ 3 independent ChIP). C, Relative luciferase activity determined after 24-hour incubation. Results are expressed as percentage of control after normalization for b-galactosidase expression SD (three independent experiments; n ¼ 4–5 replicates per experiment). a, P < 0.05 versus vehicle. D, Expression pattern of LRRFIP1 protein in cells transduced with shRNA-control or shRNA-LRRFIP1 vector, assessed by Western blotting analysis. Actin was used as loading control. E, Relative luciferase activity determined after 24-hour incubation. Results are expressed as percentage of control after normalization for b-galactosidase expression SD (two independent experiments; n ¼ 4–5 replicates per experiment). a, P < 0.05 versus vehicle; b, P < 0.05 versus sh-Control cells. F, Expression pattern of LRRFIP1 and TRIB3 proteins in sh-Control or sh-LRRFIP1 cells after 24-hour rapamycin treatment (0.1–100 nmol/L), assessed by Western blotting analysis. Actin was used as loading control.

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To confirm that LRRFIP1 is a key factor for the inhibitory effect of tified among several response elements a putative binding site for rapamycin on TRIB3 promoter activity, we established new cell lines by LRRFIP1. LRRFIP1 is described as a transcriptional repressor over- lentiviral transduction of shRNA sequences targeting LRRFIP1. As expressed in cancer cell lines that decreases activity of EGFR (36, 37) expected, the stable integration of shRNA-LRRFIP1 sequences into the and other genes (38). Through ChIP, we confirmed the interaction of genome of the cell, markedly reduced LRRFIP1 protein level compared LRRFIP1 with TRIB3 proximal promoter region in our cancer cell with control cells transduced with nonrelevant shRNA sequences model. Site-directed mutagenesis and genetic modification of our cell (43% to 70% vs. sh-Ctrl; Fig. 6D). Rapamycin reduced luciferase lines fully confirmed that LRRFIP1 is a key factor in TRIB3 promoter activity in sh-Ctrl cells transfected with the pTRB3-Luc1 vector (20% activity repression by rapamycin. Further in silico analyses of protein– to 30%, P < 0.05 vs. vehicle; Fig. 6E), but did not affect luciferase protein association networks (interactome) and corresponding co-IPs activity in LRRFIP1-silenced cells. Furthermore, in LRRFIP1 silenced identified FKBP3 (FK506-binding protein) as a LRRFIP1 putative cells rapamycin did not reduce the level of endogenous TRIB3 protein partner. FKBP3 belongs to the same family of immunophilin as (Fig. 6F). FKBP12 and has been reported to bind also rapamycin (32). However, In silico analyses of LRRFIP1 putative partners using the general further investigations ought to be carried out to determine whether repository for interaction datasets BioGRID identified FKBP3 (FK506- both FKBP3 and LRRFIP1 are required for this complex to repress binding protein 3; alias FKBP25), a cis-trans prolylisomerase reported TRIB3 promoter activity, or on the contrary, if the dissociation is to bind with high affinity FK506 and rapamycin (31, 32; Supplemen- required to release LRRFIP1 for binding onto TRIB3 promoter tary Table S4). Reciprocal co-IP experiments confirmed that FKBP3 (Fig. 7A and B, respectively). In both models, the binding of LRRFIP1 robustly binds to LRRFIP1 in the presence of rapamycin (Supple- on the promoter of TRIB3, in proximity of the TSS is crucial to prevent mentary Fig. S4D–S4E). TRIB3 gene transcription. Altogether, these data suggest that rapamycin reduced TRIB3 IP of TRIB3-enriched complexes combined to mass spectrometry expression through LRRFIP1-dependent repression of promoter activ- and additional co-IP suggested the interaction between TRIB3 and ity in an mTOR-independent manner. some components of the spliceosome, a highly dynamic, multimega- dalton ribonucleoprotein complex that catalyzes the process of pre- mRNA splicing (28). The spliceosome is emerging as a potential Discussion hallmark of cancer progression (39). Our previous work highlighted In this study, we identified TRIB3 as a target gene of rapamycin in that the expression level of some spliceosome components differs cancer cells. We demonstrated that the rapamycin-driven regulation of between normal and cancer cells (40). Moreover, splicing factors TRIB3 expression relies rather on its transcriptional regulation via the expression level can also be deregulated in cancer cells and, in some repressor LRRFIP1 than the inhibition of the mTOR signaling path- cases, could even act as oncogenes, such as, for instance, SRSF1 (41). In way. Moreover, we showed that the overexpression of TRIB3 impairs addition, mutations in spliceosome coding genes like SF3B1 could be the cytotoxic effect of rapamycin in vitro. In addition, we identified a recurrent driver of cancer progression (42). Finally, all the modifica- novel role of TRIB3 as component of the spliceosome complex. We tions that lead to the disruption of the splicing process can result in the demonstrated that rapamycin modulates the rate of pre-mRNA production of aberrant end-products from cancer-critical genes splicing in cancer cells and that this effect is prevented if TRIB3 is (reviewed in ref. 43). The majority of the core spliceosomal factors overexpressed. Overall, these data suggest that rapalogs could have an that interact with TRIB3 are present in the U2, U5, PRP19, PRP19- mTOR-independent inhibitory effect on splicing machinery, and that related, and Bact complexes, thus suggesting that TRIB3 may operate LRRFIP1/TRIB3 mediate a large part of this effect. just prior to the catalytic activation of the spliceosome (Fig. 3C). In Targeting mTOR with rapamycin triggers feedback-loop signals addition, the RNA-seq-derived analysis indicated no difference and reactivation of PI3K/Akt pathway (21). Du and colleagues dem- between the splicing patterns in control cells and cells overexpressing onstrated that TRIB3 could bind to Akt and interfere with its phos- TRIB3, supporting the hypothesis that TRIB3 is required for the phorylation on Ser473 and Thr308, both required for Akt activa- catalytic activation of the spliceosome after the choice of the splice tion (22). The literature suggests that in fact TRIB3 inhibits cancer sites. The overexpression of TRIB3 did not markedly influence the initiation by limiting Akt-driven tumorigenesis (33). In addition, there splicing efficacy, suggesting that endogenous level of TRIB3 protein is are evidences that TRIB3 can interact with the rapamycin-insensitive sufficient for optimal function of the spliceosome. In contrast, down- mTORC2 complex, necessary for the complete activation of Akt (34). regulation of its endogenous level leads to impairment of the spliceo- As expected, rapamycin increased pAkt relative level, suggesting an some function. upstream reactivation of Akt signaling. However, TRIB3 overexpres- This study shows, for the first time to our knowledge, that part of sion did not significantly interfere with the rapamycin-induced Akt the antitumor activity of rapalogs is related to the deregulation of activation, while it abolished or limited the rapamycin cytotoxic spliceosome activity, independently from mTOR signaling. One effects. Thus, TRIB3 influences rapalogs action in a mechanism publication demonstrated that constitutive mTORC1 activation independent of the suppression of the negative-feedback loops and promotes de novo lipids biogenesis through SRPK2 nuclear trans- the reactivation of prosurvival Akt. location that phosphorylates SR proteins and favors the splicing of Schwarzer and colleagues have demonstrated that in vitro, in the lipogenic pre-mRNAs. In contrast, mTORC1 inhibition leads to absence of nutrients, TRIB3 expression is dependent on the PI3K intron retention and subsequent degradation of the lipogenic pre- activity in [PTEN / ] prostate carcinoma PC3 cells, and that the use of mRNAs (44). In accordance with these results, we detected that one the PI3K inhibitor LY294002 or rapamycin leads to TRIB3 down- of the major enriched functional annotations of the transcripts regulation (35). In our in vitro cancer cell models, the blockade of impacted by splicing modulations was the metabolism of lipids. mTOR signaling with the ATP-competitive inhibitor Torin-1 did not Moreover, we observed rapamycin-induced splicing modulations in modify the expression level of TRIB3, suggesting that the rapalogs- the same lipogenic genes. However, in addition to lipid metabolism, driven downregulation of TRIB3 is independent from the mTOR our results suggest that rapamycin can induce splicing modulations pathway. In silico exploration of the TRIB3 promoter sequence iden- in genes related to a variety of cellular processes. Thus, we can

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Figure 7. Proposed models suggesting that rapalogs deregulate splicing of cancer cells by downregulating TRIB3. A, We propose a ﬁrst model, suggesting that in the presence of rapalogs (R), FKBP3 forms a complex with the transcriptional repressor LRRFIP1. The binding site for LRRFIP1 is proximal to the TSS, restricting the access of the RNA polymerase II to the promoter and thus leading to reduced transcription of TRIB3. In an alternative model, we propose that in the presence of rapalogs, FKBP3 releases the transcriptional repressor LRRFIP1, which in turn inhibits TRIB3 transcription. B, We propose that in the absence of rapalogs, the RNA polymerase II binds to the TRIB3 promoter and allows the transcription of TRIB3 gene. In this scenario, the basal level of TRIB3 is guaranteed and the spliceosome machinery works properly. C, When rapalogs are present, LRRFIP1 reduces the transcription of TRIB3 and the function of the spliceosome machinery is consequently impaired.

speculate that both antitumor activity of rapalogs and the mechan- machinery and overcome the resistance or insensitivity upon rapa- isms of resistance are highly complex. mycin treatment. Among the tested cancer cell lines, we detected two different In conclusion, the purpose of this study was to explore novel behaviors in terms of splicing alteration under rapamycin treatment: mechanism of action of rapalogs. We demonstrated that the one cluster of cell lines that is sensitive to rapamycin deregulation of rapamycin-driven downregulation of TRIB3 expression is inde- pre-mRNA splicing (A375, MCF7, SKBR3, and SUM52PE) and a pendent from the mTOR signaling, but relies on its transcriptional second one that is not sensitive (HCC827, MDA-MB-468, MKN45, regulation via the transcriptional repressor LRRFIP1 (proposed and T47D). We hypothesized that this phenomenon could be depen- models in Fig. 7A–C). We showed that rapalogs modulate pre- dent on the different genetic background of the cancer cell lines. mRNA splicing genome-wide. Finally, we demonstrated that the Among the known alterations, a common one concerns EGFR sig- overexpression of TRIB3 prevents the cytotoxic and splicing effects naling with either gene amplification or activating mutations for the of rapalogs. nonsensitive cells HCC827, MDA-MB-468, and MKN45 (45–47). We believe our findings have important implications in overcoming Zhou and colleagues have reported that a massive reprogramming of the resistance to rapalogs in clinic. We highlighted TRIB3 as a suitable alternative splicing occurs upon EGF treatment in HEK293T cells (48). biomarker to assess the efficacy of the rapalogs treatment by moni- Chettouh and colleagues demonstrated that EGFR stimulates splicing toring its expression. Moreover, TRIB3 could be suitable as a thera- factor expression, thus increases mRNA splicing of the insulin recep- peutic target to synergize with rapalogs. In addition, our research hints tors A and B in hepatocellular carcinoma cells (49). Further investiga- at the possibility of combining rapalogs and splicing modulators, like tions are required to evaluate the possible relationship between the for instance H3B-8800 (50), to improve the outcome of the rapalogs alterations in EGFR signaling that could overactivate the splicing treatment.

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Disclosure of Potential Conflicts of Interest Acknowledgments C.E. Vicier reports receiving a commercial research grant from BMS. O. Tredan The authors thank Dr. Y. Itoh (Nagoya City University, Nagoya, Japan) and has received speakers' bureau honoraria from Novartis, Roche, Pfizer, Lilly, Astra- Prof. I.C. Eperon (University of Leicester, Leicester, UK) for providing the pTRB3- Zeneca, MSD, and Sandoz. T. Bachelot is a board member for Novartis. F. Andreisan Luc1 and pTN23 reporter vectors, respectively. The authors thank Dr. N. Turner unpaid consultant/advisory board member for Astra Zeneca, Novartis, Pfizer, Lilly, (The Institute of Cancer Research, London, UK) for providing the SUM52PE cell and Roche. No potential conflicts of interest were disclosed by other authors. line.WethankDr.G.MeuriceandMrs.N.Pata-Merci(GustaveRoussy Functional Genomics Unit and Bioinformatic Core Facility, UMS AMMICA, Authors’ Contributions Villejuif, France) for microarray analyses. The authors thank Mrs. A. Vouillon (Gustave Roussy, Proteomic Platform, Villejuif, France) and Mrs. C. Thirant Conception and design: B. Stefanovska, C.E. Vicier, S. Delaloge, F. Andre, (INSERM U1170, Villejuif, France) for their technical help with proteomics and O. Fromigue ChIP experiments, respectively. Development of methodology: B. Stefanovska, C.E. Vicier, V. Ogryzko, V. Scott, This work was supported by INSERM, Gustave Roussy (taxe d'apprentissage), I. Bouakka, S. Delaloge, O. Fromigue Operation Parrains Chercheurs, Odyssea, Dassault Foundation, Breast Cancer Acquisition of data (provided animals, acquired and managed patients, provided Research Foundation, and La Ligue contre le Cancer (Rhone-69, and Allier-03). facilities, etc.): B. Stefanovska, C.E. Vicier, V. Ogryzko, V. Scott, I. Bouakka, C.E. Vicier was a recipient of a PhD fellowship from the French National Cancer S. Delaloge, O. Le Saux, O. Tredan, T. Bachelot, O. Fromigue Institute (INCa-INSERM Plan Cancer). Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): B. Stefanovska, C.E. Vicier, T. Dayris, V. Ogryzko, V. Scott, I. Bouakka, S. Delaloge, A. Rocca, F. Andre, O. Fromigue The costs of publication of this article were defrayed in part by the payment of advertisement Writing, review, and/or revision of the manuscript: B. Stefanovska, C.E. Vicier, page charges. This article must therefore be hereby marked in S. Delaloge, A. Rocca, O. Le Saux, O. Tredan, T. Bachelot, F. Andre, O. Fromigue accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): B. Stefanovska, C.E. Vicier, V. Scott, O. Fromigue Received August 1, 2019; revised January 27, 2020; accepted March 24, 2020; Study supervision: B. Stefanovska, V. Ogryzko, F. Andre, O. Fromigue published first April 3, 2020.

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Rapalog-Mediated Repression of Tribbles Pseudokinase 3 Regulates Pre-mRNA Splicing

Bojana Stefanovska, Cecile Edith Vicier, Thibault Dayris, et al.

Cancer Res 2020;80:2190-2203. Published OnlineFirst April 3, 2020.

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