MYC disrupts transcriptional and metabolic circadian oscillations in cancer and promotes enhanced biosynthesis

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Abstract

The molecular circadian clock, which controls rhythmic 24-hour oscillation of genes, proteins, and metabolites in healthy tissues, is disrupted across many human cancers. Deregulated expression of the MYC oncoprotein has been shown to alter expression of molecular clock genes, leading to a disruption of molecular clock oscillation across cancer types. It remains unclear what benefit cancer cells gain from suppressing clock oscillation, and how this loss of molecular clock oscillation impacts global gene expression and metabolism in cancer. We hypothesized that MYC or its paralog N-MYC (collectively termed MYC herein) suppress oscillation of gene expression and metabolism to upregulate pathways involved in biosynthesis in a static, non-oscillatory fashion. To test this, cells from distinct cancer types with inducible MYC were examined, using time-series RNA-sequencing and metabolomics, to determine the extent to which MYC activation disrupts global oscillation of genes, gene expression pathways, and metabolites. We focused our analyses on genes, pathways, and metabolites that changed in common across multiple cancer cell line models. We report here that MYC disrupted over 85% of oscillating genes, while instead promoting enhanced ribosomal and mitochondrial biogenesis and suppressed cell attachment pathways. Notably, when MYC is activated, biosynthetic programs that were formerly circadian flipped to being upregulated in an oscillation-free manner. Further, activation of MYC ablates the oscillation of nutrient transporter proteins while greatly upregulating transporter expression, cell surface localization, and intracellular amino acid pools. Finally, we report that MYC disrupts metabolite oscillations and the temporal segregation of amino acid metabolism from nucleotide metabolism. Our results demonstrate that MYC disruption of the molecular circadian clock releases metabolic and biosynthetic processes from circadian control, which may provide a distinct advantage to cancer cells.

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  1. Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

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    Reply to the reviewers

    We thank all Reviewers for their detailed and helpful comments and suggestions for this manuscript. The overall goal of this study is to interrogate which transcriptional and metabolic pathways lose oscillation when MYC is amplified or activated. We have now added additional replicates to our RNA-sequencing and nutrient transporter expression analyses, and have demonstrated that MYC disrupts oscillation of metabolic and biosynthetic gene expression, nutrient transporter oscillation, and metabolite pathways. On the suggestion of Reviewer #3, we have also strengthened this work by directly contrasting the transcriptional oscillations we observe in cancer …

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    Referee #3

    Evidence, reproducibility and clarity

    Review of "MYC Disrupts Transcriptional and Metabolic Circadian Oscillations in Cancer and Promotes Enhanced Biosynthesis"
    DeRollo et al. attempt to find commonalities in how MYC affects transcriptional and metabolic programming by examining MYC-switchable U2OS, SHEP, and SKNAS cell lines. They claim that oncogenic MYC both represses transcriptional oscillation of many genes and supports rhythmic expression of other genes. They use RNA-Seq and UPLC-MS/MS with the appropriate bioinformatics analyses. In some cases, they employ qPCR and immunoblotting. In the three different cell lines, they observed that MYC either statically …

  3. Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #2

    Evidence, reproducibility and clarity

    Major comments:

    1. The 3 cell lines used in this paper, what is the expression levels of MYC protein under -OFF and -ON conditions? It is important to demonstrate this information through the western blot data. Since the 4-hydroxy tamoxifen was used to activate MYC, what is the vehicle/control for MYC OFF cells? Otherwise, it will be difficult to assess with everything observed on this manuscript under MYC-ON could be due to 4-hydroxy tamoxifen treatment.
    2. In Fig. 1A, it is crucial to demonstrate that the circadian synchronization protocol is working by performing statistical analysis with at least 3 biological replicates. This should be …
  4. Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #1

    Evidence, reproducibility and clarity

    This is an interesting paper from the Altman, Weljie and Dang labs that furthers their previous publications looking at the effect of "oncogenic" Myc levels have on circadian gene expression. They provide compelling data that in neuroblastoma and osteosarcoma models of Myc amplification circadian gene expression and metabolite fluctuation are lost. The data is convincing and comprehensive and should be of broad general interest. There are a few major issues that need to be addressed.

    1. Is there a control (done here or in prior literature) showing that simply adding tam to cells doesn't change circadian gene expression?
    2. All of the …