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  1. Evaluation Summary:

    The potential mechanism of METTL3 N6-methyltransferase in the chemotherapeutic response is poorly defined. Herein, Li and colleagues describe a pathway where METTL3 promoted EGF expression through m6A modification, which further upregulated RAD51 expression, resulting in enhanced HR activity. METTL3 knockdown results in DNA damage accumulation, which renders breast cancer cells sensitive to adriamycin treatment.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

  2. Reviewer #1 (Public Review):

    In the manuscript by Li Enjie. et al., the authors determine that METTL3 is important for HR and deletion sensitizes MCF10A cells to Doxorubicin. Importantly, the authors show that METTL3 functions by regulating RAD51 via m6A modifications of RNA which changes how EGF expression factors regulating cellular invasion. This is important for cancer biology as m6A RNA methylation levels, METTL3 and RAD51 proteins are elevated in certain cancers and are targets for therapeutic intervention. The authors show that METTL3 knockdown in MCF-7 cells sensitize cells to the chemotherapeutic and topoisomerase II inhibitor Adriamycin (Doxorubicin). MCF-7 and MB-231 cells treated with Doxorubicin have elevated pro-apoptotic factors. The authors then go on to show that MCF-7 and MB-231 cells have increased gamma-H2AX protein levels and foci when METTL3 is knocked down, treated with Doxorubicin or Etoposide. Using a dr-GFP reporter assays the authors show that METTL3 knockdown decreases repair via HR. Using RNA-seq coupled with meRIP-qPCR the authors identified targets of METT3-mediated m6A modification to identify EGF. The authors then go on to show that EGF is a novel regulator of RAD51 expression. This is a significant finding as RAD51 is the key protein involved in HR that catalyzes homology search and strand exchange. The authors then went on to use EGF inhibitors to show in cells treated with ADR, METTL3 modulates HR via the EGF-RAD51 axis. They further define important m6A readers to include (YTHDC1) which regulates EGF mRNA stability and promotes EGF synthesis. The results of the study are potentially important for those interested in signaling and cancer invasion, as well as the DNA repair community, as the work characterizes transcript regulation by m6A binding and links it to HR and EGF signaling.

    • The authors identified a novel protein involved in HR whose depletion sensitizes cells to TOPII inhibitors.
    • The authors identified a new mechanism of regulation of key DNA repair proteins, mainly RAD51.
    • They also find that this protein is regulated by EGF a known protein involved in cancer progression as well as DNA Repair.

    • While doxorubicin was a standard therapy in breast cancer treatment, Olaparib is now commonly used in the clinic. It is unknown how this cancer treatment therapeutic would have on the mechanism proposed by the authors.
    • Gamma H2AX phosphorylation is a global marker of DSBs and stalled forks. The authors failed to note that H2AX phorylation is present and a marker of stalled replications forks.
    o PMID: 11673449, PMID: 20053681, doi:10.1101/gad.2053211, etc.

  3. Reviewer #2 (Public Review):

    In this study, the authors sought to define the influence of the METTL3 m6A methyltransferase on cellular resistance to Adriamycin (topo2 poison) and homologous recombination, along with studies aimed at linking such effects to METTL3 being important for expression of EGF, which in turn promotes expression of the RAD51 recombinase. A final set of experiments also examine the link of the m6A reader TYHDC1 to EGF expression.

    The authors use a series of DNA damage response assays, and find some interesting results with METTL3 overexpression and depletion, and EGF expression. Since homologous recombination is a major end point of the study, which is most active in S/G2, then cell cycle analysis needs to be employed to determine if the effects are due to cell proliferation changes. This is particularly relevant for experiments with growth factor manipulations, such as EGF expression. Also, while interesting results are shown with METTL3 overexpression and depletion, not many of the experiments test both manners of manipulating METTL3 levels, which is important for interpreting the results (namely are effects only observed with overexpression for some and only depletion for others?). Finally, a major mechanistic idea in the manuscript is centered on increased levels of the RAD51 recombinase, but then testing whether the phenotypes can be mimicked by overexpression of RAD51 should be tested, particularly since the literature is not consistent about whether elevated RAD51 indeed causes more homologous recombination.

  4. Reviewer #3 (Public Review):

    METTL3 is part of a N6-methyltransferase complex that methylates adenosine residues at the N6 position of some RNAs and regulates various vital processes including DNA damage response. To date, the potential mechanism of METTL3 in the chemotherapeutic response is poorly defined. A previous study reported that METTL3 participates in the regulation of homologous recombination (HR) based DNA damage repair. Interestingly, in this paper, Li and colleagues added a piece into a puzzle by describing a parallel pathway where METTL3 promoted EGF expression through m6A modification, which further upregulated RAD51 expression, resulting in enhanced HR activity. Additionally, the m6A reader YTHDC1, bound to the m6A modified EGF transcript promoted EGF synthesis, which enhanced HR and cell survival during Adriamycin treatment. Hence, METTL3 knockdown results in DNA damage accumulation, which renders breast cancer cells sensitive to ADR treatment. This study establish the rationale for the development of METTL3 inhibitors for cancer treatment. The paper is well-written and the authors' claims and conclusions are justified by their data. Some figures will benefit from editing and proper quantification of the data.