## Replication Study: The Common Feature of Leukemia-associated IDH1 and IDH2 Mutations is a Neomorphic Enzyme Activity Converting Alpha-Ketoglutarate to 2-Hydroxyglutarate <br> **Abstract:** In 2016, as part of the [Reproducibility Project: Cancer Biology][1], we published a Registered Report ([Feign et al., 2016][2]), 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" ([Ward et al., 2010][3]). 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; [Ward et al., 2010][4]). 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; [Ward et al., 2010][5]). 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; [Ward et al., 2010][6]). Finally, we report meta-analyses for each result. ---------- ### Contents **Reports:** Read the [Replication Study][16], or view the [preprint versions][7]. To reproduce the Replication Study manuscript text run this in [R Studio][8] (note: downloads R markdown file directly from osf.io): library(httr) library(rmarkdown) GET("https://osf.io/ywphk/?action=download", write_disk("Replication_Study_16.Rmd", overwrite = T)) render("Replication_Study_16.Rmd", output_format = "word_document") <br> Also, explore the [Registered Report][9] and materials related to the Registered Report [here][10]. <br> **Data and Material Availability:** - 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, [http://www.metabolomicsworkbench.org][17]), where it has been assigned a Metabolomics Workbench Project ID: ST000548. The data is directly accessible at [http://www.metabolomicsworkbench.org/data/DRCCMetadata.php?Mode=Study&StudyID=ST000548][18]. [17]: http://www.metabolomicsworkbench.org [18]: http://www.metabolomicsworkbench.org/data/DRCCMetadata.php?Mode=Study&StudyID=ST000548 - Plasmids generated during this study are deposited and available at [Addgene][2]: pcDNA3.1-IDH2WT (plasmid# [87926][3]) and pcDNA3.1-IDH2R172K (plasmid# [87927][4]) - All other associated data, protocols, analysis scripts, and other digital materials are available within this OSF project. [2]: https://www.addgene.org [3]: https://www.addgene.org/87926/ [4]: https://www.addgene.org/87927/ <br> **Experiments replicated:** Reproduce and explore the figures, analyses, data, and methods generated in this replication attempt. - [Assessing the isocitrate-dependent NADPH production and alpha-ketoglutarate-dependent NADPH consumption of wild-type or R172K mutant IDH2][11] - Replication of Figures 2A-C in Ward et al., (2010). - [Production of 2-HG from IDH2 WT and mutant transfected cells][12] - Replication of Figures 3A-D in Ward et al., (2010). - [Assessing 2HG levels in AML patient samples][13] - Replication of Figure 5 in Ward et al., (2010). <br> **Meta-analyses:** As a measure of evaluating reproducibility a meta-analysis of each effect was performed. The forest plots, analyses, and data are available [here][14]. <br> Questions about the project can be directed to [contact+rpcb@cos.io][15]. [1]: https://osf.io/e81xl/wiki/home/ [2]: https://elifesciences.org/content/5/e12626 [3]: http://www.cell.com/cancer-cell/abstract/S1535-6108%2810%2900036-X [4]: http://www.cell.com/cancer-cell/abstract/S1535-6108%2810%2900036-X [5]: http://www.cell.com/cancer-cell/abstract/S1535-6108%2810%2900036-X [6]: http://www.cell.com/cancer-cell/abstract/S1535-6108%2810%2900036-X [7]: https://osf.io/ywb88/ [8]: https://www.rstudio.com [9]: https://elifesciences.org/content/5/e12626 [10]: https://osf.io/9jkpg/wiki/home/ [11]: https://osf.io/6ve4d/wiki/home/ [12]: https://osf.io/9ge2a/wiki/home/ [13]: https://osf.io/smdfr/wiki/home/ [14]: https://osf.io/4m3n8/wiki/home/ [15]: http://mailto:contact+rpcb@cos.io [16]: https://elifesciences.org/articles/26030