Abstract
The Reproducibility Project: Cancer Biology seeks to address growing concerns about reproducibility in scientific research by conducting replications of selected experiments from a number of high-profile papers in the field of cancer biology. The papers, which were published between 2010 and 2012, were selected on the basis of citations and Altmetric scores (Errington et al., 2014). This Registered Report describes the proposed replication plan of key experiments from "IDH mutation impairs histone demethylation and results in a block to cell differentiation" by Lu and colleagues, published in Nature in 2012 (Lu et al., 2012). The experiments that will be replicated are those reported in Figures 1B, 2A, 2B, 2D and 4D. Lu and colleagues demonstrated that expression of mutant forms of IDH1 or IDH2 caused global increases in histone methylation and increased levels of 2 hydroxyglutarate (Figure 1B). This was correlated with a block in differentiation (Figures 2A, B and D). This effect appeared to be mediated by the histone demethylase KDM4C (Figure 4D). The Reproducibility Project: Cancer Biology is a collaboration between the Center for Open Scienceand Science Exchange, and the results of the replications will be published by eLife.
Highlights
Mutations in the metabolic proteins IDH1 and IDH2 are associated with gliomas, acute myeloid leukemias, chondrosarcomas, intrahepatic cholangiocarcinomas, lymphomas, melanomas and colon, thyroid and prostate cancers
Transfection of 3T3-L1 cells with the mutant forms of IDH1 and IDH2 that produce 2HG lead to an increase in global methylation levels and prevented normal in vitro differentiation into adipocytes
The 2HG-sensitive histone demethylase KDM4C appeared to be required for this process, as knockdown of KDM4C recapitulated the phenotype of 2HG production
Summary
Mutations in the metabolic proteins IDH1 and IDH2 are associated with gliomas, acute myeloid leukemias, chondrosarcomas, intrahepatic cholangiocarcinomas, lymphomas, melanomas and colon, thyroid and prostate cancers (for review, see Krell et al, 2013). They confirmed that introduction of the mutated forms of IDH1 and IDH2 correlated with increased intracellular levels of the oncometabolite 2HG. Western blot analysis and Oil-Red-O staining confirmed that loss of KDM4C increased global methylation levels and inhibited differentiation This key finding will be replicated in Protocol 3. This effect may even hold true for human patients, as there is a marked increase in H3K9me levels associated with IDH mutations in oligodendromas and high grade astrocytomas (Venneti et al, 2013)
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