Replication Study: The common feature of leukemia-associated IDH1 and IDH2 mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutarate

In 2016, as part of the Reproducibility Project: Cancer Biology, we published a Registered Report (<xref ref-type="bibr" rid="bib14">Fiehn et al., 2016</xref>), 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). 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). 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). In primary human AML samples, the 2HG levels observed in samples with mutant IDH1 or IDH2 status were higher than those observed in samples without an IDH mutation, similar to what was observed in the original study (Figure 5C; Ward et al., 2010). Finally, we report meta-analyses for each result.

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Registered report: The common feature of leukemia-associated IDH1 and IDH2 mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutarate

eLife ◽

10.7554/elife.12626 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 5

Author(s):

Oliver Fiehn ◽

Megan Reed Showalter ◽

Christine E Schaner-Tooley ◽

Keyword(s):

Enzyme Activity ◽

Myeloid Leukemia ◽

Cancer Biology ◽

Open Science ◽

Wild Type ◽

High Profile ◽

Isocitrate Dehydrogenase 2 ◽

The Common ◽

Idh1 And Idh2 Mutations ◽

Acute Myeloid

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 (<xref ref-type="bibr" rid="bib3">Errington et al., 2014</xref>). This Registered Report describes the proposed replication plan of key experiments from “The common feature of leukemia-associated IDH1 and IDH2 mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutarate” by Ward and colleagues, published in Cancer Cell in 2010 (<xref ref-type="bibr" rid="bib16">Ward et al., 2010</xref>). The experiments that will be replicated are those reported in Figures 2, 3 and 5. Ward and colleagues demonstrate the mutations in isocitrate dehydrogenase 2 (IDH2), commonly found in acute myeloid leukemia (AML), abrogate the enzyme’s wild-type activity and confer to the mutant neomorphic activity that produces the oncometabolite 2-hydroxyglutarate (2-HG) (Figures 2 and 3). They then show that elevated levels of 2-HG are correlated with mutations in IDH1 and IDH2 in AML patient samples (Figure 5). The Reproducibility Project: Cancer Biology is a collaboration between the Center for Open Science and Science Exchange and the results of the replications will be published by eLife.

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Replication Study: Coding-independent regulation of the tumor suppressor PTEN by competing endogenous mRNAs

eLife ◽

10.7554/elife.56651 ◽

2020 ◽

Vol 9 ◽

Author(s):

Hongyan Wang ◽

Hanna S Radomska ◽

Mitch A Phelps ◽

Elizabeth Iorns ◽

Rachel Tsui ◽

...

Keyword(s):

Cell Proliferation ◽

Tumor Suppressor ◽

Cancer Biology ◽

Hct116 Cell ◽

Original Study ◽

Wild Type ◽

Pten Protein ◽

Protein Levels ◽

Meta Analyses ◽

Hct116 Cells

As part of the Reproducibility Project: Cancer Biology, we published a Registered Report (Phelps et al., 2016) that described how we intended to replicate selected experiments from the paper ‘Coding-independent regulation of the tumor suppressor PTEN by competing endogenous mRNAs’ (Tay et al., 2011). Here, we report the results. We found depletion of putative PTEN competing endogenous mRNAs (ceRNAs) in DU145 cells did not impact PTEN 3’UTR regulation using a reporter, while the original study reported decreased activity when SERINC1, VAPA, and CNOT6L were depleted (Figure 3C; Tay et al., 2011). Using the same reporter, we found decreased activity when ceRNA 3’UTRs were overexpressed, while the original study reported increased activity (Figure 3D; Tay et al., 2011). In HCT116 cells, ceRNA depletion resulted in decreased PTEN protein levels, a result similar to the findings reported in the original study (Figure 3G,H; Tay et al., 2011); however, while the original study reported an attenuated ceRNA effect in microRNA deficient (DicerEx5) HCT116 cells, we observed increased PTEN protein levels. Further, we found depletion of the ceRNAs VAPA or CNOT6L did not statistically impact DU145, wild-type HCT116, or DicerEx5 HCT116 cell proliferation. The original study reported increased DU145 and wild-type HCT116 cell proliferation when these ceRNAs were depleted, which was attenuated in the DicerEx5 HCT116 cells (Figure 5B; Tay et al., 2011). Differences between the original study and this replication attempt, such as variance between biological repeats, are factors that might have influenced the results. Finally, we report meta-analyses for each result.

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Replication Study: RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth

10.1101/2021.11.30.470372 ◽

2021 ◽

Author(s):

Steven Pelech ◽

Curtis Gallagher ◽

Catherine Sutter ◽

Lambert Yue ◽

John Kerwin ◽

...

Keyword(s):

Cell Proliferation ◽

Cancer Biology ◽

Mapk Pathway ◽

Mek Inhibitor ◽

Original Study ◽

Braf V600e ◽

Cancer Cell Proliferation ◽

Wild Type ◽

Differential Binding ◽

Meta Analyses

As part of the Reproducibility Project: Cancer Biology, we published a Registered Report (Bhargava et al., 2016) that described how we intended to replicate selected experiments from the paper "RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth" (Hatzivassiliou et al., 2010). Here we report the results. We found two unrelated RAF inhibitors, PLX4720 or GDC-0879, selectively inhibited BRAF(V600E) cell proliferation, while the MEK inhibitor, PD0325901, inhibited BRAF(V600E), wild-type RAF/RAS, and mutant RAS cancer cell proliferation, similar to the original study (Figure 1A; Hatzivassiliou et al., 2010). We found knockdown of CRAF, but not BRAF, in mutant RAS cells attenuated the phospho-MEK induction observed after PLX4720 treatment, similar to the original study (Figure 2B; Hatzivassiliou et al., 2010). The original study reported analogous results with GDC-0879, which was not observed in this replication, although unexpected control results confound the interpretation. We also attempted a replication of an assay with recombinant proteins to test the differential effect of RAF inhibitors on BRAF-CRAF heterodimerization (Figure 4A; Hatzivassiliou et al., 2010). Although we were unable to conduct the experiment as planned, we observed differential binding of BRAF by RAF inhibitors; however, it was between BRAF and beads, independent of CRAF. While these data were unable to address whether, under the conditions of the original study, the same observations could be observed, we discuss key differences between the original study and this replication that are important to consider for further experiments. Finally, where possible, we report meta-analyses for each result.

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Replication Study: Coadministration of a tumor-penetrating peptide enhances the efficacy of cancer drugs

eLife ◽

10.7554/elife.17584 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 34

Author(s):

Christine Mantis ◽

Irawati Kandela ◽

Fraser Aird ◽

Keyword(s):

Cancer Biology ◽

Xenograft Model ◽

Original Study ◽

Cancer Drug ◽

Tunel Staining ◽

Positive Staining ◽

Cancer Drugs ◽

Tumor Weight ◽

Significant Difference ◽

Meta Analyses

In 2015, as part of the Reproducibility Project: Cancer Biology, we published a Registered Report (Kandela et al., 2015) that described how we intended to replicate selected experiments from the paper “Coadministration of a tumor-penetrating peptide enhances the efficacy of cancer drugs“ (Sugahara et al., 2010). Here we report the results of those experiments. We found that coadministration with iRGD peptide did not have an impact on permeability of the chemotherapeutic agent doxorubicin (DOX) in a xenograft model of prostate cancer, whereas the original study reported that it increased the penetrance of this cancer drug (Figure 2B; Sugahara et al., 2010). Further, in mice bearing orthotopic 22Rv1 human prostate tumors, we did not find a statistically significant difference in tumor weight for mice treated with DOX and iRGD compared to DOX alone, whereas the original study reported a decrease in tumor weight when DOX was coadministered with iRGD (Figure 2C; Sugahara et al., 2010). In addition, we did not find a statistically significant difference in TUNEL staining in tumor tissue between mice treated with DOX and iRGD compared to DOX alone, while the original study reported an increase in TUNEL positive staining with iRGD coadministration (Figure 2D; Sugahara et al., 2010). Similar to the original study (Supplemental Figure 9A; Sugahara et al., 2010), we did not observe an impact on mouse body weight with DOX and iRGD treatment. Finally, we report meta-analyses for each result.

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Replication Study: Inhibition of BET recruitment to chromatin as an effective treatment for MLL-fusion leukaemia

eLife ◽

10.7554/elife.25306 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 8

Author(s):

Xiaochuan Shan ◽

Juan Jose Fung ◽

Alan Kosaka ◽

Gwenn Danet-Desnoyers ◽

Keyword(s):

Growth Inhibition ◽

Effective Treatment ◽

Cell Line ◽

Cancer Biology ◽

Vehicle Control ◽

Original Study ◽

Selective Growth ◽

Significant Difference ◽

K 562 Cells ◽

Meta Analyses

In 2015, as part of the Reproducibility Project: Cancer Biology, we published a Registered Report (Fung et al., 2015), that described how we intended to replicate selected experiments from the paper "Inhibition of BET recruitment to chromatin as an effective treatment for MLL-fusion leukaemia" (Dawson et al., 2011). Here, we report the results of those experiments. We found treatment of MLL-fusion leukaemia cells (MV4;11 cell line) with the BET bromodomain inhibitor I-BET151 resulted in selective growth inhibition, whereas treatment of leukaemia cells harboring a different oncogenic driver (K-562 cell line) did not result in selective growth inhibition; this is similar to the findings reported in the original study (Figure 2A and Supplementary Figure 11A,B; Dawson et al., 2011). Further, I-BET151 resulted in a statistically significant decrease in BCL2 expression in MV4;11 cells, but not in K-562 cells; again this is similar to the findings reported in the original study (Figure 3D; Dawson et al., 2011). We did not find a statistically significant difference in survival when testing I-BET151 efficacy in a disseminated xenograft MLL mouse model, whereas the original study reported increased survival in I-BET151 treated mice compared to vehicle control (Figure 4B,D; Dawson et al., 2011). Differences between the original study and this replication attempt, such as different conditioning regimens and I-BET151 doses, are factors that might have influenced the outcome. We also found I-BET151 treatment resulted in a lower median disease burden compared to vehicle control in all tissues analyzed, similar to the example reported in the original study (Supplementary Figure 16A; Dawson et al., 2011). Finally, we report meta-analyses for each result.

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Decision letter: Registered report: The common feature of leukemia-associated IDH1 and IDH2 mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutarate

10.7554/elife.12626.002 ◽

2016 ◽

Keyword(s):

Enzyme Activity ◽

The Common ◽

Idh1 And Idh2 Mutations

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Decision letter: Replication Study: The common feature of leukemia-associated IDH1 and IDH2 mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutarate

10.7554/elife.26030.009 ◽

2017 ◽

Keyword(s):

Enzyme Activity ◽

Replication Study ◽

The Common ◽

Idh1 And Idh2 Mutations

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Replication Study: Melanoma genome sequencing reveals frequent PREX2 mutations

eLife ◽

10.7554/elife.21634 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 19

Author(s):

Stephen K Horrigan ◽

Pascal Courville ◽

Darryl Sampey ◽

Faren Zhou ◽

Steve Cai ◽

...

Keyword(s):

Genome Sequencing ◽

Cancer Biology ◽

Tumor Formation ◽

Original Study ◽

Free Survival ◽

Tumor Onset ◽

Study Evaluation ◽

Significant Difference ◽

Meta Analyses

In 2015, as part of the Reproducibility Project: Cancer Biology, we published a Registered Report (Chroscinski et al., 2014) that described how we intended to replicate selected experiments from the paper "Melanoma genome sequencing reveals frequent PREX2 mutations" (Berger et al., 2012). Here we report the results of those experiments. We regenerated cells stably expressing ectopic wild-type and mutant phosphatidylinositol-3,4,5-trisphosphate-dependent Rac exchange factor 2 (PREX2) using the same immortalized human NRASG12D melanocytes as the original study. Evaluation of PREX2 expression in these newly generated stable cells revealed varying levels of expression among the PREX2 isoforms, which was also observed in the stable cells made in the original study (Figure S6A; Berger et al., 2012). Additionally, ectopically expressed PREX2 was found to be at least 5 times above endogenous PREX2 expression. The monitoring of tumor formation of these stable cells in vivo resulted in no statistically significant difference in tumor-free survival driven by PREX2 variants, whereas the original study reported that these PREX2 mutations increased the rate of tumor incidence compared to controls (Figure 3B and S6B; Berger et al., 2012). Surprisingly, the median tumor-free survival was 1 week in this replication attempt, while 70% of the control mice were reported to be tumor-free after 9 weeks in the original study. The rapid tumor onset observed in this replication attempt, compared to the original study, makes the detection of accelerated tumor growth in PREX2 expressing NRASG12D melanocytes extremely difficult. Finally, we report meta-analyses for each result.

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

eLife ◽

10.7554/elife.30274 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 16

Author(s):

L Michelle Lewis ◽

Meredith C Edwards ◽

Zachary R Meyers ◽

C Conover Talbot ◽

Haiping Hao ◽

...

Keyword(s):

Gene Expression ◽

Tumor Cells ◽

Cancer Biology ◽

Statistical Significance ◽

Digital Gene Expression ◽

Original Study ◽

Silent Genes ◽

Before And After ◽

Meta Analyses ◽

Transcriptional Amplification

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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