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<h2>Replication Study: The Common Feature of Leukemia-associated IDH1 and IDH2 Mutations is a Neomorphic Enzyme Activity Converting Alpha-Ketoglutarate to 2-Hydroxyglutarate</h2> <p><br> <strong>Abstract:</strong></p> <p>In 2016, as part of the <a href="https://osf.io/e81xl/wiki/home/" rel="nofollow">Reproducibility Project: Cancer Biology</a>, we published a Registered Report (<a href="https://elifesciences.org/content/5/e12626" rel="nofollow">Feign et al., 2016</a>), that described how we intended to replicate selected experiments from the paper "The common feature of leukemia-associated IDH1 and IDH2 mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutarate" (<a href="http://www.cell.com/cancer-cell/abstract/S1535-6108%2810%2900036-X" rel="nofollow">Ward et al., 2010</a>). Here we report the results of those experiments. We found that cells expressing R172K mutant IDH2 did not display isocitrate-dependent NADPH production above vector control levels, in contrast to the increased production observed with wild-type IDH2. Conversely, expression of R172K mutant IDH2 resulted in increased alpha-ketoglutarate-dependent consumption of NADPH compared to wild-type IDH2 or vector control. These results are similar to those reported in the original study (Figure 2; <a href="http://www.cell.com/cancer-cell/abstract/S1535-6108%2810%2900036-X" rel="nofollow">Ward et al., 2010</a>). Further, expression of R172K mutant IDH2 resulted in increased 2HG levels within cells compared to the background levels observed in wild-type IDH2 and vector control, similar to the original study (Figure 3D; <a href="http://www.cell.com/cancer-cell/abstract/S1535-6108%2810%2900036-X" rel="nofollow">Ward et al., 2010</a>). In primary human AML samples, 2HG levels were increased in samples with mutant IDH1 or IDH2 status above the levels observed in samples without an IDH mutation; analogous to the observations reported in the original study (Figure 5C; <a href="http://www.cell.com/cancer-cell/abstract/S1535-6108%2810%2900036-X" rel="nofollow">Ward et al., 2010</a>). Finally, we report meta-analyses for each result.</p> <hr> <h3>Contents</h3> <p><strong>Reports:</strong> Read the <a href="https://elifesciences.org/articles/26030" rel="nofollow">Replication Study</a>, or view the <a href="https://osf.io/ywb88/" rel="nofollow">preprint versions</a>. </p> <p>To reproduce the Replication Study manuscript text run this in <a href="https://www.rstudio.com" rel="nofollow">R Studio</a> (note: downloads R markdown file directly from <a href="http://osf.io" rel="nofollow">osf.io</a>):</p> <pre class="highlight"><code>library(httr) library(rmarkdown) GET(&quot;<a href="https://osf.io/ywphk/?action=download" rel="nofollow">https://osf.io/ywphk/?action=download</a>&quot;, write_disk(&quot;Replication_Study_16.Rmd&quot;, overwrite = T)) render(&quot;Replication_Study_16.Rmd&quot;, output_format = &quot;word_document&quot;)</code></pre> <p><br> Also, explore the <a href="https://elifesciences.org/content/5/e12626" rel="nofollow">Registered Report</a> and materials related to the Registered Report <a href="https://osf.io/9jkpg/wiki/home/" rel="nofollow">here</a>.</p> <p><br> <strong>Data and Material Availability:</strong></p> <ul> <li> <p>The metabolomics data generated during this study are available at the NIH Common Fund's Data Repository and Coordinating Center (supported by NIH grant, U01-DK097430) website, <a href="http://www.metabolomicsworkbench.org" rel="nofollow">http://www.metabolomicsworkbench.org</a>), where it has been assigned a Metabolomics Workbench Project ID: ST000548. The data is directly accessible at <a href="http://www.metabolomicsworkbench.org/data/DRCCMetadata.php?Mode=Study&StudyID=ST000548" rel="nofollow">http://www.metabolomicsworkbench.org/data/DRCCMetadata.php?Mode=Study&StudyID=ST000548</a>.</p> </li> <li> <p>Plasmids generated during this study are deposited and available at <a href="https://elifesciences.org/content/5/e12626" rel="nofollow">Addgene</a>: pcDNA3.1-IDH2WT (plasmid# <a href="http://www.cell.com/cancer-cell/abstract/S1535-6108%2810%2900036-X" rel="nofollow">87926</a>) and pcDNA3.1-IDH2R172K (plasmid# <a href="http://www.cell.com/cancer-cell/abstract/S1535-6108%2810%2900036-X" rel="nofollow">87927</a>)</p> </li> <li> <p>All other associated data, protocols, analysis scripts, and other digital materials are available within this OSF project.</p> </li> </ul> <p><br> <strong>Experiments replicated:</strong> Reproduce and explore the figures, analyses, data, and methods generated in this replication attempt.</p> <ul> <li><a href="https://osf.io/6ve4d/wiki/home/" rel="nofollow">Assessing the isocitrate-dependent NADPH production and alpha-ketoglutarate-dependent NADPH consumption of wild-type or R172K mutant IDH2</a><ul> <li>Replication of Figures 2A-C in Ward et al., (2010). </li> </ul> </li> <li><a href="https://osf.io/9ge2a/wiki/home/" rel="nofollow">Production of 2-HG from IDH2 WT and mutant transfected cells</a><ul> <li>Replication of Figures 3A-D in Ward et al., (2010).</li> </ul> </li> <li><a href="https://osf.io/smdfr/wiki/home/" rel="nofollow">Assessing 2HG levels in AML patient samples</a><ul> <li>Replication of Figure 5 in Ward et al., (2010).</li> </ul> </li> </ul> <p><br> <strong>Meta-analyses:</strong> As a measure of evaluating reproducibility a meta-analysis of each effect was performed. The forest plots, analyses, and data are available <a href="https://osf.io/4m3n8/wiki/home/" rel="nofollow">here</a>.</p> <p><br> Questions about the project can be directed to <a href="http://mailto:contact+rpcb@cos.io" rel="nofollow">contact+rpcb@cos.io</a>.</p>
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