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      Replication Study: Transcriptional amplification in tumor cells with elevated c-Myc

      research-article
      1 , 1 , 1 , 2 , 2 , 1 , Reproducibility Project: Cancer Biology
      eLife
      eLife Sciences Publications, Ltd
      Reproducibility Project: Cancer Biology, replication, metascience, reproducibility, c-Myc, gene expression, Human

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          Abstract

          As part of the Reproducibility Project: Cancer Biology, we published a Registered Report (Blum et al., 2015), that described how we intended to replicate selected experiments from the paper ‘Transcriptional amplification in tumor cells with elevated c-Myc’ (Lin et al., 2012). Here we report the results. We found overexpression of c-Myc increased total levels of RNA in P493-6 Burkitt’s lymphoma cells; however, while the effect was in the same direction as the original study (Figure 3E; Lin et al., 2012), statistical significance and the size of the effect varied between the original study and the two different lots of serum tested in this replication. Digital gene expression analysis for a set of genes was also performed on P493-6 cells before and after c-Myc overexpression. Transcripts from genes that were active before c-Myc induction increased in expression following c-Myc overexpression, similar to the original study (Figure 3F; Lin et al., 2012). Transcripts from genes that were silent before c-Myc induction also increased in expression following c-Myc overexpression, while the original study concluded elevated c-Myc had no effect on silent genes (Figure 3F; Lin et al., 2012). Treating the data as paired, we found a statistically significant increase in gene expression for both active and silent genes upon c-Myc induction, with the change in gene expression greater for active genes compared to silent genes. Finally, we report meta-analyses for each result.

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          Most cited references24

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          NIH Image to ImageJ: 25 years of image analysis.

          For the past 25 years NIH Image and ImageJ software have been pioneers as open tools for the analysis of scientific images. We discuss the origins, challenges and solutions of these two programs, and how their history can serve to advise and inform other software projects.
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            The spliceosome is a therapeutic vulnerability in MYC-driven cancer

            c-MYC (MYC) overexpression or hyperactivation is one of the most common drivers of human cancer. Despite intensive study, the MYC oncogene remains recalcitrant to therapeutic inhibition. MYC is a transcription factor, and many of its pro-tumorigenic functions have been attributed to its ability to regulate gene expression programs 1–3 . Notably, oncogenic MYC activation has also been shown to increase total RNA and protein production in many tissue and disease contexts 4–7 . While such increases in RNA and protein production may endow cancer cells with pro-tumor hallmarks, this elevation in synthesis may also generate new or heightened burden on MYC-driven cancer cells to properly process these macromolecules 8 . Herein, we discover the spliceosome as a new target of oncogenic stress in MYC-driven cancers. We identify BUD31 as a MYC-synthetic lethal gene, and demonstrate that BUD31 is a component of the core spliceosome required for its assembly and catalytic activity. Core spliceosomal factors (SF3B1, U2AF1, and others) associated with BUD31 are also required to tolerate oncogenic MYC. Notably, MYC hyperactivation induces an increase in total pre-mRNA synthesis, suggesting an increased burden on the core spliceosome to process pre-mRNA. In contrast to normal cells, partial inhibition of the spliceosome in MYC-hyperactivated cells leads to global intron retention, widespread defects in pre-mRNA maturation, and deregulation of many essential cell processes. Importantly, genetic or pharmacologic inhibition of the spliceosome in vivo impairs survival, tumorigenicity, and metastatic proclivity of MYC-dependent breast cancers. Collectively, these data suggest that oncogenic MYC confers a collateral stress on splicing and that components of the spliceosome may be therapeutic entry points for aggressive MYC-driven cancers.
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              Selective transcriptional regulation by Myc in cellular growth control and lymphomagenesis.

              The c-myc proto-oncogene product, Myc, is a transcription factor that binds thousands of genomic loci. Recent work suggested that rather than up- and downregulating selected groups of genes, Myc targets all active promoters and enhancers in the genome (a phenomenon termed 'invasion') and acts as a general amplifier of transcription. However, the available data did not readily discriminate between direct and indirect effects of Myc on RNA biogenesis. We addressed this issue with genome-wide chromatin immunoprecipitation and RNA expression profiles during B-cell lymphomagenesis in mice, in cultured B cells and fibroblasts. Consistent with long-standing observations, we detected general increases in total RNA or messenger RNA copies per cell (hereby termed 'amplification') when comparing actively proliferating cells with control quiescent cells: this was true whether cells were stimulated by mitogens (requiring endogenous Myc for a proliferative response) or by deregulated, oncogenic Myc activity. RNA amplification and promoter/enhancer invasion by Myc were separable phenomena that could occur without one another. Moreover, whether or not associated with RNA amplification, Myc drove the differential expression of distinct subsets of target genes. Hence, although having the potential to interact with all active or poised regulatory elements in the genome, Myc does not directly act as a global transcriptional amplifier. Instead, our results indicate that Myc activates and represses transcription of discrete gene sets, leading to changes in cellular state that can in turn feed back on global RNA production and turnover.
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                Author and article information

                Contributors
                Role: Reviewing Editor
                Journal
                eLife
                Elife
                eLife
                eLife
                eLife Sciences Publications, Ltd
                2050-084X
                09 January 2018
                2018
                : 7
                : e30274
                Affiliations
                Science Exchange Palo AltoUnited States
                Science Exchange Palo AltoUnited States
                Center for Open Science CharlottesvilleUnited States
                Science Exchange Palo AltoUnited States
                Center for Open Science CharlottesvilleUnited States
                [1 ]University of Georgia, Bioexpression and Fermentation Facility GeorgiaUnited States
                [2 ]Johns Hopkins University, Deep Sequencing and Microarray Core Facility MarylandUnited States
                Howard Hughes Medical Institute, University of Massachusetts Medical School United States
                Howard Hughes Medical Institute, University of Massachusetts Medical School United States
                Author information
                https://orcid.org/0000-0002-3758-2425
                Article
                30274
                10.7554/eLife.30274
                5760205
                29313490
                7c9718ff-4771-412d-aa1b-e99f87dbcfbc
                © 2018, Lewis et al

                This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

                History
                : 10 July 2017
                : 16 November 2017
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/100009827, Laura and John Arnold Foundation;
                Award Recipient : Reproducibility Project: Cancer Biology
                The funder had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
                Categories
                Replication Study
                Biochemistry
                Cancer Biology
                Custom metadata
                This Replication Study has reproduced important parts of the original paper, but it also contains results that are not consistent with some parts of the original paper.

                Life sciences
                reproducibility project: cancer biology,replication,metascience,reproducibility,c-myc,gene expression,human

                Comments

                Readers may be interested in a related piece "Integrating Binder and Stencila" by Daniel Nust: https://stenci.la/blog/2018-11-20-stencila-binder/

                2019-02-20 22:46 UTC
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                2019-02-20 22:46 UTC
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