RAS and PP2A activities converge on epigenetic gene regulation

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Abstract

RAS-mediated human cell transformation requires inhibition of the tumor suppressor Protein Phosphatase 2A (PP2A). Both RAS and PP2A mediate their effects by phosphoregulation, but phosphoprotein targets and cellular processes in which RAS and PP2A activities converge in human cancers have not been systematically analyzed. Here, based on mass spectrometry phosphoproteome data we discover that phosphosites co-regulated by RAS and PP2A are enriched on proteins involved in epigenetic gene regulation. As examples, RAS and PP2A co-regulate the same phosphorylation sites on HDAC1/2, KDM1A, MTA1/2, RNF168 and TP53BP1. Mechanistically, we validate co-regulation of NuRD chromatin repressor complex by RAS and PP2A. Consistent with their known synergistic effects in cancer, RAS activation and PP2A inhibition resulted in epigenetic reporter de-repression and activation of oncogenic transcription. Notably, transcriptional de-repression by PP2A inhibition was associated with increased euchromatin and decrease in global DNA methylation. Further, targeting of RAS- and PP2A-regulated epigenetic proteins decreased viability of KRAS-mutant human lung cancer cells. Collectively the results indicate that epigenetic protein complexes involved in oncogenic gene expression constitute a significant point of convergence for RAS hyperactivity and PP2A inhibition in cancer. Further, the results provide a rich source for future understanding of phosphorylation as a previously unappreciated layer of regulation of epigenetic gene regulation in cancer, and in other RAS/PP2A-regulated cellular processes.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    Modulation of protein phosphorylation level is a critical mechanism in the regulation of different cellular processes, whose dysregulation is associated with disease, including cancers. Protein phosphatase (PP)2A is a central phosphatase involved in multiple cellular pathways, including cell cycle, metabolism, and regulation of gene expression. In addition, inactivation of PP2A is required for RAS-mediated human cell transformation, making reactivation of PP2A as a potential therapeutic approach. Aakula, Sharma et al investigated whether RAS and PP2A could co-regulate cellular processes involved in tumorigenesis via the modulation of protein phosphorylation. The authors re-analysed their previously published phosphoproteomics datasets performed after knockdown with siRNAs of RAS (H/K/N), PP2A-A, or PP2A inhibitory proteins (CIP2A, PME1, SET). The authors found a set of phosphosites commonly regulated by RAS and PP2A, which is enriched for proteins involved in the epigenetic regulation of gene expression, including DNA methylation, chromatin remodelling, and chromatin modifications. The authors then investigated how modulating RAS and PP2A activities (by siRNA or small molecule inhibitors) affect the chromatin recruitment of the HDAC1 and HDAC2 proteins, which are part of the NuRD chromatin-remodelling complex. Modulation of RAS and PP2A activities also affects transcription, both with a single GFP gene construct and by RNA-seq, with knockdown of RAS mostly decreasing gene expression while knockdown of PP2A generally associated with increased gene expression. The authors then investigated the genome-wide effects of knocking PP2A on DNA methylation and chromatin accessibility (ATAC-seq) and found a limited number of sites affected.

    Major comments:

    1. The investigation and characterization of the phosphosites that are common to both RAS and PP2A is an important question, as stated by the authors. However, the authors hardly investigated the potential roles of these common phosphosites (only CHD3 S713 has been partially investigated) but rather relied on knockdown by siRNAs of the factors, which limits the conclusions of the manuscript as it remains unknown whether these phosphosites have any effect on protein activity and/or interactions.
    2. The major technical limitation of the manuscript is the dependence on siRNAs to investigate RAS and PP2A. Knockdown by siRNAs takes a long time, which limits the conclusions that can be drawn as the results are going to be a mixture of direct (loss of RAS/PP2A) and indirect (cellular responses to the direct effects) effects. Typically, changes in gene expression, DNA methylation, and chromatin accessibility could be explained, at least in part, by indirect effects of the knockdown (changes in cell cycle, cellular responses to stress induced by the knockdown...). I think it will be important to confirm on some target genes that the main results of the manuscript are direct effects by using known small molecule inhibitors with short treatment time.
    3. The genome-wide data do not seem to have been submitted to the GEO (or I could not find the information), which also means that it is not clear how many biological replicates have been performed.
    4. Generally, the authors should put more information in the Legends/Methods as several key information are missing (see Minor Comments).
    5. The authors should integrate more their RNA-seq, RRBS, and ATC-seq data as these datasets have been generated in the same cell line (I suppose RRBS is also in HeLa, see Minor Comment 2). Do the authors see consistent changes on RRBS/ATAC-seq for the upregulated/downregulated genes?

    Minor comments:

    1. Did the authors performed a total (with rRNA depletion) or a poly(A)+ RNA-seq?
    2. In the Methods section for the RRBS, it is written that the DNA was isolated from the same samples. Is it the same samples as the RNA-seq? More precision is required.
    3. It would also be useful to put in the legends the cell line used in each experiment.
    4. Figure 3, Figure 4, and Figure S5: I could not find any information on the treatment time and the concentrations of the small molecule inhibitors used. These information need to be added to the legends.
    5. Figure 3B: the authors need to performed qRT-PCR to show that the overexpression is similar between the different conditions. Right now, the differences could be explained by a difference in transcription between the different constructs.
    6. Also, do the mutations affect CHD3 chromatin association or interaction with other NuRD components? This kind of straightforward experiments would clearly improve the interest of the manuscript as it will provide information on the potential roles of phosphosites.
    7. Figure 3C, E, G, and I: A nuclear loading control is required for each experiment. Also, western blots on whole cell extracts are required to see if the changes in nuclear/chromatin level are not just explained by a change in the total expression of HDAC1 and HDAC2 following siRNA treatment.
    8. Lines 552-555: I am not convinced that the presence of DOT1L among the regulators associated with open promoter regions provides a direct link between the phosphoproteome and ATAC-seq data. DOT1L is a methyltransferase associated with transcription initiation and transcription elongation and therefore it is not surprising to find this protein in open promoter regions. In addition, to claim a direct link would require data showing that protein phosphorylation of DOT1L regulates its recruitment to promoter regions.
    9. Figure 7F/G: Are the overlaps significantly enriched?

    Referees cross-commenting

    If the manuscript is clearly presented as a ressource paper, I agree with reviewer 1. My major comments 1 and 2 (knockdown of total proteins rather than looking at phosphoresidues, RNAi) can be addressed in the discussion rather than experimentally.

    Significance

    The mechanistic roles of phosphosites remain generally an understudied area of research while kinases and phosphatases are known to be frequently dysregulated in disease. The generation of a list of phosphosites common to RAS proteins and PP2A is therefore of interest as this will provide targets for further investigation. The authors tested some of the targets by using a siRNA approach, which confirmed the involvement of PP2A in the regulation of gene expression, DNA methylation, and chromatin remodelling/accessibility and of RAS in the regulation of gene expression and chromatin accessibility. However, the authors focused on the proteins rather than the phosphosites, which limits the significance of the work as it remains unclear whether the effects the authors are observing are mediated by changes in phosphorylation level (in addition to the potential issues of indirect effects due to the siRNA approach).

    Context:

    Loss of PP2A phosphatase activity is required for human cell transformation while RAS is a known oncogene (Chen et al, 2004; Rangarajan et al, 2004). The manuscript investigated which proteins were commonly phospho-regulated by RAS and PP2A activities and found an overrepresentation of proteins involved in transcriptional regulation and epigenetics, which confirms and expands previous observations. PP2A, as part of the INTAC complex that is composed of Integrator and PP2A, has been found to regulate nascent transcription (Vervoort et al, 2021; Zheng et al, 2020). In addition, PP2A activity has also been linked to DNA methylation (Hausser et al, 2006; Kundu et al, 2020; Sunahori et al, 2013) and nuclear localization of several histone deacetylases (HDAC) (Tinsley & Allen-Petersen, 2022).

    Audience:

    The reported findings will be of interest to people working on the RAS/PP2A-associated cancers, and more generally in the fields of regulation of gene expression, chromatin remodelling, and epigenetics.

    Field of expertise:

    transcription, chromatin, RNA polymerase II, transcriptional kinases and phosphatases.

    References

    Chen W, Possemato R, Campbell KT, Plattner CA, Pallas DC, Hahn WC (2004) Identification of specific PP2A complexes involved in human cell transformation. Cancer Cell 5: 127-136

    Hausser A, Link G, Hoene M, Russo C, Selchow O, Pfizenmaier K (2006) Phospho-specific binding of 14-3-3 proteins to phosphatidylinositol 4-kinase III beta protects from dephosphorylation and stabilizes lipid kinase activity. J Cell Sci 119: 3613-3621

    Kundu A, Shelar S, Ghosh AP, Ballestas M, Kirkman R, Nam H, Brinkley GJ, Karki S, Mobley JA, Bae S et al (2020) 14-3-3 proteins protect AMPK-phosphorylated ten-eleven translocation-2 (TET2) from PP2A-mediated dephosphorylation. J Biol Chem 295: 1754-1766

    Rangarajan A, Hong SJ, Gifford A, Weinberg RA (2004) Species- and cell type-specific requirements for cellular transformation. Cancer Cell 6: 171-183

    Sunahori K, Nagpal K, Hedrich CM, Mizui M, Fitzgerald LM, Tsokos GC (2013) The catalytic subunit of protein phosphatase 2A (PP2Ac) promotes DNA hypomethylation by suppressing the phosphorylated mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)/phosphorylated ERK/DNMT1 protein pathway in T-cells from controls and systemic lupus erythematosus patients. J Biol Chem 288: 21936-21944

    Tinsley SL, Allen-Petersen BL (2022) PP2A and cancer epigenetics: a therapeutic opportunity waiting to happen. NAR Cancer 4: zcac002

    Vervoort SJ, Welsh SA, Devlin JR, Barbieri E, Knight DA, Offley S, Bjelosevic S, Costacurta M, Todorovski I, Kearney CJ et al (2021) The PP2A-Integrator-CDK9 axis fine-tunes transcription and can be targeted therapeutically in cancer. Cell 184: 3143-3162 e3132

    Zheng H, Qi Y, Hu S, Cao X, Xu C, Yin Z, Chen X, Li Y, Liu W, Li J et al (2020) Identification of Integrator-PP2A complex (INTAC), an RNA polymerase II phosphatase. Science 370

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    Referee #1

    Evidence, reproducibility and clarity

    In this resource manuscript by Aakula et al., the authors reanalyze existing phosphoproteomic datasets to find areas of convergence of proteins and sites regulated by RAS activation on the one hand, and PP2A inhibition on the other. They identify a number of such sites. The validation is relatively modest, showing effects on HDAC1/2 localization, the silencing of an artificial promoter, and then focus on epigenetic regulation in more general experiments. They provide plentiful data and correlations that will be useful for others interested in mechanism of regulation by protein phosphorylation. The major limitation, acknowledged by the authors, is that this is a resource rather than a deep validation of the overlaps.

    I have only a few minor specific comments.

    The overlap of PP2A and RAS regulated phosphoproteins in the gene ontology networks is made up of small numbers - 3/6 in term 0070087. When only 3 genes are in a category, given the reliability of GO terms, it doesn't generate much excitement.

    Likewise the effect of knockdowns of putative targets in NSCLC cells was modest, with 10- 20% decrease in cell viability. I suspect many gene knockdowns might give a similar effect.

    Line 299 starts a >1.5 pages long paragraph about CHD3 and HDAC1/2; it would be easier to read if this were two or three shorter paragraphs.

    The pulldown data (S5A) is done with over-expressed proteins and shows a weak interaction. Without evidence for endogenous protein interaction, the conclusion that there is a substantial in vivo physiologic interaction between B56α and HDAC1 must be qualified.

    Referees cross-commenting

    I agree with reviewer 2 that there are shortcomings. If this is viewed as a resource, and not a strong conclusion paper, my feeling is that additional confirmation experiments would not add much. I agree they should be careful to discuss the limitations of the RNAi approach.

    Significance

    The data suggest that two major cancer mutations converge to influence epigenetic regulators. The data is correlative and will assist future mechanistic studies.