Registered report: Widespread potential for growth factor-driven resistance to anticancer kinase inhibitors

Thank you for sending your work entitled “Registered report: Widespread potential for growth-factor-driven resistance to anticancer kinase inhibitors” for consideration at eLife. Your article has been favorably evaluated by Tony Hunter (Senior editor) and 3 reviewers, one of whom is a member of our Board of Reviewing Editors.

The Reviewing editor and the other reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.

1) The experimental design to test the reproducibility of Wilson et al. (2012) is thorough and well-articulated, with some exceptions. First, it will be important to perform positive controls to assess the performance of the growth factors or the kinase inhibitors that will be used.

2) Second, given that the western blots in the original manuscript are not quantified and that quantification is derived from the published work, the authors should describe how they are going to determine whether the data are “reproducible” or not.

3) Third, it is not immediately clear whether the distribution of the data (IC50 for Protocol 1 and band intensity for Protocol 2) will exhibit any skew. Therefore, at the time of analysis, it may be useful to plot histograms of the data to examine their distributions, and, if necessary, consider suitable transformations (for example, the Box–Cox family of transformations) of the data to obtain (approximately) symmetric distributions so that the testing procedures are valid.

4) Lastly, the authors should either include or explain the reason for excluding in the replication study the role of HGF-MET signaling in resistance to BRAF inhibition that was observed in some melanomas in the original study and other reports.

1) The experimental design to test the reproducibility of Wilson et al (2012) is thorough and well-articulated, with some exceptions. First, it will be important to perform positive controls to assess the performance of the growth factors or the kinase inhibitors that will be used.

We agree that verifying the activity of the reagents prior to their use in our experiments is an important step. We have three classes of reagent: primary RTK inhibitors, growth factor ligands, and secondary RTK inhibitors. Each cohort includes a positive control where the cell line of interest is treated solely with its cognate primary RTK inhibitor. This should demonstrate that the drug is active as anticipated, and the quality control data (for both primary and secondary RTK inhibitors) provided by the manufacturers will be included in the materials publicly available through the Open Science Framework. However, as indicated by the reviewers, there is known lot-to-lot variation in growth factors, so we have added steps to test the growth factors we are using for activity. In Protocol 2, we have added in additional cell lines that have a known response to treatment with the ligand alone, as evidenced by phosphorylation of downstream targets. We will treat these positive control cell lines with the growth factors and assess phosphorylation of their cognate target by Western blot. The manuscript has been updated to reflect this additional work.

2) Second, given that the western blots in the original manuscript are not quantified and that quantification is derived from the published work, the authors should describe how they are going to determine whether the data are “reproducible” or not.

We will present both the original data and replication data for side-by-side comparison. We will plot the mean value of our replication data along with the 95% confidence interval. We will then include the original data point (IC₅₀ or quantified Western blot band intensity) on the same plot to demonstrate if the original data falls within the 95% confidence interval of the replication data. We have also updated the language of the manuscript to reflect this change.

3) Third, it is not immediately clear whether the distribution of the data (IC50 for Protocol 1 and band intensity for Protocol 2) will exhibit any skew. Therefore, at the time of analysis, it may be useful to plot histograms of the data to examine their distributions, and, if necessary, consider suitable transformations (for example, the Box–Cox family of transformations) of the data to obtain (approximately) symmetric distributions so that the testing procedures are valid.

Thank you for this suggestion. At the time of analysis, we will generate a histogram of all the data to determine if it follows a Gaussian distribution or not. If it is skewed, we will perform the appropriate transformation in order to proceed with the proposed statistical analysis. We will note any changes or transformations made. We have also updated the manuscript to address this point.

4) Lastly, the authors should either include or explain the reason for excluding in the replication study the role of HGF-MET signaling in resistance to BRAF inhibition that was observed in some melanomas in the original study and other reports.

We agree that all of the experiments included in the original study are important, and choosing which experiments to replicate has been one of the great challenges of this project. In this case, the RP:CB core team felt that the most impactful information in Wilson et al., 2012 was that bypassing RTK inhibition by ligand-mediated activation of parallel signaling pathways was a mechanism applicable to many different types of cancer, each with its own constellation of addictive mutations and cognate inhibitors. The experiments addressing the role of HGF in activating MET signaling to bypass EGFR inhibition provide a more detailed exploration of this mechanism in one specific cancer type scenario, and support the larger conclusion drawn from the experiments we chose for replication. As such, we will restrict our analysis to the experiments being replicated and will not include discussion of experiments not being replicated in this study.