Nucleotide depletion reveals the impaired ribosome biogenesis checkpoint as a barrier against DNA damage
2020; Springer Nature; Volume: 39; Issue: 13 Linguagem: Inglês
10.15252/embj.2019103838
ISSN1460-2075
AutoresJoffrey Pelletier, Ferran Riaño‐Canalias, Eugènia Almacellas, Caroline Mauvezin, Sara Samino, Sònia Feu, Sandra Menoyo, Ana Domostegui, Marta Garcia‐Cajide, Ramón Salazar, Constanza Cortés, Ricard Marcos, Albert Tauler, Óscar Yanes, Neus Agell, Sara C. Kozma, Antonio Gentilella, George Thomas,
Tópico(s)RNA and protein synthesis mechanisms
ResumoArticle2 June 2020Open Access Transparent process Nucleotide depletion reveals the impaired ribosome biogenesis checkpoint as a barrier against DNA damage Joffrey Pelletier Corresponding Author [email protected] orcid.org/0000-0001-9174-044X Laboratory of Cancer Metabolism, ONCOBELL Program, Institut d'Investigació Biomèdica de Bellvitge—IDIBELL, L'Hospitalet de Llobregat, Spain Search for more papers by this author Ferran Riaño-Canalias Laboratory of Cancer Metabolism, ONCOBELL Program, Institut d'Investigació Biomèdica de Bellvitge—IDIBELL, L'Hospitalet de Llobregat, Spain Search for more papers by this author Eugènia Almacellas orcid.org/0000-0002-7726-8430 Laboratory of Cancer Metabolism, ONCOBELL Program, Institut d'Investigació Biomèdica de Bellvitge—IDIBELL, L'Hospitalet de Llobregat, Spain Search for more papers by this author Caroline Mauvezin orcid.org/0000-0003-4220-7272 Laboratory of Cancer Metabolism, ONCOBELL Program, Institut d'Investigació Biomèdica de Bellvitge—IDIBELL, L'Hospitalet de Llobregat, Spain Search for more papers by this author Sara Samino Metabolomics Platform, IISPV & University Rovira i Virgili, Tarragona, Spain Spanish Biomedical Research Center in Diabetes and Associated Metabolic Disorders (CIBERDEM), Madrid, Spain Search for more papers by this author Sonia Feu Department of Biomedicine, Faculty of Medicine, IDIBAPS Biomedical Research Institute, Hospital Clinic, University of Barcelona, Barcelona, Spain Search for more papers by this author Sandra Menoyo Laboratory of Cancer Metabolism, ONCOBELL Program, Institut d'Investigació Biomèdica de Bellvitge—IDIBELL, L'Hospitalet de Llobregat, Spain Search for more papers by this author Ana Domostegui Laboratory of Cancer Metabolism, ONCOBELL Program, Institut d'Investigació Biomèdica de Bellvitge—IDIBELL, L'Hospitalet de Llobregat, Spain Search for more papers by this author Marta Garcia-Cajide Laboratory of Cancer Metabolism, ONCOBELL Program, Institut d'Investigació Biomèdica de Bellvitge—IDIBELL, L'Hospitalet de Llobregat, Spain Search for more papers by this author Ramon Salazar Laboratory of Cancer Metabolism, ONCOBELL Program, Institut d'Investigació Biomèdica de Bellvitge—IDIBELL, L'Hospitalet de Llobregat, Spain Catalan Institute of Oncology (ICO), Barcelona, Spain Search for more papers by this author Constanza Cortés Department of Genetics and Microbiology, Faculty of Biosciences, Autonomous University of Barcelona, Barcelona, Spain Search for more papers by this author Ricard Marcos Department of Genetics and Microbiology, Faculty of Biosciences, Autonomous University of Barcelona, Barcelona, Spain Search for more papers by this author Albert Tauler Laboratory of Cancer Metabolism, ONCOBELL Program, Institut d'Investigació Biomèdica de Bellvitge—IDIBELL, L'Hospitalet de Llobregat, Spain Department of Biochemistry and Physiology, Faculty of Pharmacy, University of Barcelona, Barcelona, Spain Search for more papers by this author Oscar Yanes Metabolomics Platform, IISPV & University Rovira i Virgili, Tarragona, Spain Spanish Biomedical Research Center in Diabetes and Associated Metabolic Disorders (CIBERDEM), Madrid, Spain Search for more papers by this author Neus Agell orcid.org/0000-0002-1205-6074 Department of Biomedicine, Faculty of Medicine, IDIBAPS Biomedical Research Institute, Hospital Clinic, University of Barcelona, Barcelona, Spain Search for more papers by this author Sara C Kozma Laboratory of Cancer Metabolism, ONCOBELL Program, Institut d'Investigació Biomèdica de Bellvitge—IDIBELL, L'Hospitalet de Llobregat, Spain Search for more papers by this author Antonio Gentilella Laboratory of Cancer Metabolism, ONCOBELL Program, Institut d'Investigació Biomèdica de Bellvitge—IDIBELL, L'Hospitalet de Llobregat, Spain Department of Biochemistry and Physiology, Faculty of Pharmacy, University of Barcelona, Barcelona, Spain Search for more papers by this author George Thomas Corresponding Author [email protected] orcid.org/0000-0002-1236-1714 Laboratory of Cancer Metabolism, ONCOBELL Program, Institut d'Investigació Biomèdica de Bellvitge—IDIBELL, L'Hospitalet de Llobregat, Spain Department of Physiological Sciences, Faculty of Medicine and Health Science, University of Barcelona, Barcelona, Spain Search for more papers by this author Joffrey Pelletier Corresponding Author [email protected] orcid.org/0000-0001-9174-044X Laboratory of Cancer Metabolism, ONCOBELL Program, Institut d'Investigació Biomèdica de Bellvitge—IDIBELL, L'Hospitalet de Llobregat, Spain Search for more papers by this author Ferran Riaño-Canalias Laboratory of Cancer Metabolism, ONCOBELL Program, Institut d'Investigació Biomèdica de Bellvitge—IDIBELL, L'Hospitalet de Llobregat, Spain Search for more papers by this author Eugènia Almacellas orcid.org/0000-0002-7726-8430 Laboratory of Cancer Metabolism, ONCOBELL Program, Institut d'Investigació Biomèdica de Bellvitge—IDIBELL, L'Hospitalet de Llobregat, Spain Search for more papers by this author Caroline Mauvezin orcid.org/0000-0003-4220-7272 Laboratory of Cancer Metabolism, ONCOBELL Program, Institut d'Investigació Biomèdica de Bellvitge—IDIBELL, L'Hospitalet de Llobregat, Spain Search for more papers by this author Sara Samino Metabolomics Platform, IISPV & University Rovira i Virgili, Tarragona, Spain Spanish Biomedical Research Center in Diabetes and Associated Metabolic Disorders (CIBERDEM), Madrid, Spain Search for more papers by this author Sonia Feu Department of Biomedicine, Faculty of Medicine, IDIBAPS Biomedical Research Institute, Hospital Clinic, University of Barcelona, Barcelona, Spain Search for more papers by this author Sandra Menoyo Laboratory of Cancer Metabolism, ONCOBELL Program, Institut d'Investigació Biomèdica de Bellvitge—IDIBELL, L'Hospitalet de Llobregat, Spain Search for more papers by this author Ana Domostegui Laboratory of Cancer Metabolism, ONCOBELL Program, Institut d'Investigació Biomèdica de Bellvitge—IDIBELL, L'Hospitalet de Llobregat, Spain Search for more papers by this author Marta Garcia-Cajide Laboratory of Cancer Metabolism, ONCOBELL Program, Institut d'Investigació Biomèdica de Bellvitge—IDIBELL, L'Hospitalet de Llobregat, Spain Search for more papers by this author Ramon Salazar Laboratory of Cancer Metabolism, ONCOBELL Program, Institut d'Investigació Biomèdica de Bellvitge—IDIBELL, L'Hospitalet de Llobregat, Spain Catalan Institute of Oncology (ICO), Barcelona, Spain Search for more papers by this author Constanza Cortés Department of Genetics and Microbiology, Faculty of Biosciences, Autonomous University of Barcelona, Barcelona, Spain Search for more papers by this author Ricard Marcos Department of Genetics and Microbiology, Faculty of Biosciences, Autonomous University of Barcelona, Barcelona, Spain Search for more papers by this author Albert Tauler Laboratory of Cancer Metabolism, ONCOBELL Program, Institut d'Investigació Biomèdica de Bellvitge—IDIBELL, L'Hospitalet de Llobregat, Spain Department of Biochemistry and Physiology, Faculty of Pharmacy, University of Barcelona, Barcelona, Spain Search for more papers by this author Oscar Yanes Metabolomics Platform, IISPV & University Rovira i Virgili, Tarragona, Spain Spanish Biomedical Research Center in Diabetes and Associated Metabolic Disorders (CIBERDEM), Madrid, Spain Search for more papers by this author Neus Agell orcid.org/0000-0002-1205-6074 Department of Biomedicine, Faculty of Medicine, IDIBAPS Biomedical Research Institute, Hospital Clinic, University of Barcelona, Barcelona, Spain Search for more papers by this author Sara C Kozma Laboratory of Cancer Metabolism, ONCOBELL Program, Institut d'Investigació Biomèdica de Bellvitge—IDIBELL, L'Hospitalet de Llobregat, Spain Search for more papers by this author Antonio Gentilella Laboratory of Cancer Metabolism, ONCOBELL Program, Institut d'Investigació Biomèdica de Bellvitge—IDIBELL, L'Hospitalet de Llobregat, Spain Department of Biochemistry and Physiology, Faculty of Pharmacy, University of Barcelona, Barcelona, Spain Search for more papers by this author George Thomas Corresponding Author [email protected] orcid.org/0000-0002-1236-1714 Laboratory of Cancer Metabolism, ONCOBELL Program, Institut d'Investigació Biomèdica de Bellvitge—IDIBELL, L'Hospitalet de Llobregat, Spain Department of Physiological Sciences, Faculty of Medicine and Health Science, University of Barcelona, Barcelona, Spain Search for more papers by this author Author Information Joffrey Pelletier *,1,†,‡, Ferran Riaño-Canalias1,‡, Eugènia Almacellas1, Caroline Mauvezin1, Sara Samino2,3, Sonia Feu4, Sandra Menoyo1, Ana Domostegui1, Marta Garcia-Cajide1, Ramon Salazar1,5, Constanza Cortés6, Ricard Marcos6, Albert Tauler1,7, Oscar Yanes2,3, Neus Agell4, Sara C Kozma1, Antonio Gentilella1,7 and George Thomas *,1,8 1Laboratory of Cancer Metabolism, ONCOBELL Program, Institut d'Investigació Biomèdica de Bellvitge—IDIBELL, L'Hospitalet de Llobregat, Spain 2Metabolomics Platform, IISPV & University Rovira i Virgili, Tarragona, Spain 3Spanish Biomedical Research Center in Diabetes and Associated Metabolic Disorders (CIBERDEM), Madrid, Spain 4Department of Biomedicine, Faculty of Medicine, IDIBAPS Biomedical Research Institute, Hospital Clinic, University of Barcelona, Barcelona, Spain 5Catalan Institute of Oncology (ICO), Barcelona, Spain 6Department of Genetics and Microbiology, Faculty of Biosciences, Autonomous University of Barcelona, Barcelona, Spain 7Department of Biochemistry and Physiology, Faculty of Pharmacy, University of Barcelona, Barcelona, Spain 8Department of Physiological Sciences, Faculty of Medicine and Health Science, University of Barcelona, Barcelona, Spain †Present address: Colorectal Cancer Laboratory, Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain ‡These authors contributed equally to this work *Corresponding author. Tel: +34 260 7138; E-mail: [email protected] *Corresponding author. Tel: +34 260 7138; E-mail: [email protected] EMBO J (2020)39:e103838https://doi.org/10.15252/embj.2019103838 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Many oncogenes enhance nucleotide usage to increase ribosome content, DNA replication, and cell proliferation, but in parallel trigger p53 activation. Both the impaired ribosome biogenesis checkpoint (IRBC) and the DNA damage response (DDR) have been implicated in p53 activation following nucleotide depletion. However, it is difficult to reconcile the two checkpoints operating together, as the IRBC induces p21-mediated G1 arrest, whereas the DDR requires that cells enter S phase. Gradual inhibition of inosine monophosphate dehydrogenase (IMPDH), an enzyme required for de novo GMP synthesis, reveals a hierarchical organization of these two checkpoints. We find that the IRBC is the primary nucleotide sensor, but increased IMPDH inhibition leads to p21 degradation, compromising IRBC-mediated G1 arrest and allowing S phase entry and DDR activation. Disruption of the IRBC alone is sufficient to elicit the DDR, which is strongly enhanced by IMPDH inhibition, suggesting that the IRBC acts as a barrier against genomic instability. Synopsis How S-phase DNA damage responses and G1 arrest via the impaired ribosome synthesis checkpoint (IRBC) cooperate in p53 activation upon nucleotide depletion has remained unclear. Different levels of inosine monophosphate dehydrogenase (IMPDH) inhibition now reveal a hierarchical organization of these two genomic instability barriers. Gradual inhibition of GMP synthesis by IMPDH inhibitors elicits primarily the IRBC, and secondarily ATR/Chk1 activation. Enhanced proteasomal p21 degradation upon severe nucleotide depletion overcomes IRBC-dependent G1 arrest, leading to replicative stress. Cells exposed to prolonged IMPDH inhibition and replicative stress experience DNA damage. IRBC disruption alone can elicit DNA damage, which is strongly enhanced by IMPDH inhibition. Introduction The reprogramming of metabolic networks is a hallmark of cancer, which is initiated by the activation of oncogenes or the loss of tumor suppressors, acting to drive and sustain tumor cell proliferation (Hanahan & Weinberg, 2011). One of the most studied oncogenes is c-Myc, whose dysregulation or amplification has been implicated in > 70% of all cancers (Murphy et al, 2008). c-Myc is a master regulator of both cancer initiation and progression (Stine et al, 2015), through its ability to control the transcription of a wide range of genes (Kress et al, 2015). A critical set of these genes promote anabolic metabolism, including the synthesis of lipids, amino acids, and nucleotides, required for tumor cell proliferation (Kress et al, 2015; Stine et al, 2015). However, c-Myc appears to be distinct from other oncogenes, many of which fail to upregulate anabolic genes involved in nucleotide metabolism (Bester et al, 2011; Aird et al, 2013), including Rb-E2F (Bester et al, 2011) and RAS-RAF (Aird et al, 2013). In those cases, oncogene activation can lead to a decrease in nucleotide pools, replicative stress, and genomic instability, argued to be an initial step in tumorigenesis (Bester et al, 2011; Aird et al, 2013). Indeed, ectopic expression of c-Myc in Rb-E2F tumorigenic model re-establishes the nucleotide pools (Bester et al, 2011), an effect partially recapitulated by the ectopic expression of its target gene IMPDH2 (Mannava et al, 2008). Both IMPDH1 and 2 catalyze the rate limiting step in GMP synthesis, the oxidation of inosine monophosphate (IMP) to xanthosine monophosphate (XMP) (Huang et al, 2018). The identification of the IMPDHs as key targets of c-Myc has opened a potential therapeutic window for synthetic lethality (Stine et al, 2015), a concept supported by recent studies showing that a subset of small-cell lung carcinomas (SCLCs), characterized by c-Myc and IMPDH upregulation, are highly vulnerable to IMPDH inhibitors (Huang et al, 2018). Although nucleotides are essential for DNA replication and repair (Tong et al, 2009; Aird et al, 2013), they are largely consumed in the production of ribosomes, with ribosomal RNA (rRNA) representing ~80% of the nucleic acids of the cell and ~ 15% of its biomass (Pelletier et al, 2018). The demand for nucleotides is further exacerbated in tumors driven by c-Myc, which coordinates the enhanced transcription of genes required for the hyperactivation of ribosome biogenesis, needed to increase protein synthetic capacity, cell proliferation, and ultimately malignancy (Santagata et al, 2013). Given the dependency of such tumors on ribosome biogenesis (Barna et al, 2008; van Riggelen et al, 2010), a search for novel agents that specifically target this process has been initiated (Bywater et al, 2012; Pelletier et al, 2018). Importantly, insults to ribosome biogenesis, including c-Myc-induced oncogenic stress (Macias et al, 2010; Morcelle et al, 2019), also trigger a p53-mediated cell-cycle checkpoint, recently termed the impaired ribosome biogenesis checkpoint (IRBC) (Gentilella et al, 2017; Pelletier et al, 2018). The IRBC is mediated by a nascent pre-ribosomal complex containing RPL5 (or uL18), RPL11 (or uL5), and 5S rRNA, which upon insults to ribosome biogenesis, is redirected from its assembly into 60S ribosomes to the binding and inhibition of the p53-E3-ubiquitin ligase HDM2 (or MDM2) (Donati et al, 2013), leading to p53 stabilization (Kubbutat et al, 1997; Pelletier et al, 2018). In most cases, p53 stabilization by the IRBC leads to G1 cell-cycle arrest, largely mediated by the cyclin-dependent kinase inhibitor 1 (CDKN1A) or p21. This mechanism is distinct from that involving replicative stress and the DNA Damage Response (DDR), where phosphorylation of either HDM2 or p53 prevents their interaction, stabilizing p53 (Bode & Dong, 2004), independent of the IRBC (Macias et al, 2010). Because of the critical role of IMPDHs in proliferating tumor cells (Liu et al, 2008; Mannava et al, 2008), a number of studies have focused on the development of antimetabolites, which inhibit their function (Stine et al, 2015), including mycophenolic acid (MPA), mizoribine, and AVN-944. These agents are catalytic non-competitive inhibitors of IMPDH, which unlike nucleoside analogs that produce chromosome breaks or inhibit DNA repair enzymes, do not incorporate into the DNA (Allison & Eugui, 2000). However, the underlying mechanisms by which they lead to p53 stabilization are not clearly understood. Initial studies suggested that IMPDH inhibitors activate a reversible p53-dependent cell-cycle checkpoint (Linke et al, 1996), which was later attributed to the IRBC (Sun et al, 2008). However, others have reported that IMPDH inhibition leads to replicative stress and the DDR (Liu et al, 2008). This seeming contradiction was recently highlighted in studies of two human tumor types, in SCLCs characterized by the low expression of the Achaete-scute homolog-1 (ASCL1) and the upregulation of c-Myc (Huang et al, 2018) and in tuberous sclerosis complex 2 (TSC2)-deficient kidney tumors, where increased rRNA synthesis is driven by constitutive mTOR signaling (Valvezan et al, 2017). Both are sensitive to IMPDH inhibitors, but apparently through different mechanisms: the first through the inhibition of ribosome biogenesis (Huang et al, 2018) and the second through DDR (Valvezan et al, 2017). The differences in these findings are not clearly understood, as is the apparent incompatibility between the induction of the IRBC, leading to G1 arrest in a p53 wild-type setting (Sun et al, 2008), and replicative stress. Given the wide use of nucleotide synthesis inhibitors in cancer therapy, it is important to understand the cellular checkpoints induced by these anticancer agents. Here, we set out to address the contribution of the IRBC and replicative stress in driving p53 stabilization upon guanine nucleotide imbalance in sporadic colorectal cancer (sCRC) cell lines, as almost all CRCs are initiated by c-Myc dysregulation (TCGA, 2012) and are addicted to hyperactive ribosome biogenesis (Pelletier et al, 2018). We find that a reduction of guanine nucleotide pools first impairs ribosome biogenesis, leading to the induction of the IRBC, p53 stabilization, and G1 arrest. However, if nucleotide depletion becomes more severe, cells enter S phase and encounter replicative stress, which is paralleled by the decreased expression of p21. Although the levels of nascent 5S rRNA, an essential component of the IRBC complex decrease under these conditions, we find that this is not sufficient to impinge on complex formation nor the transcriptional induction of p21. Instead reduced p21 protein levels appear to be attributed to enhanced proteasome degradation. Moreover, S phase entry is further enhanced in MPA-treated p21−/− cells, with ectopic expression of p21 reversing this response. Consistent with this observation, the downregulation of the IRBC complex, reducing p21 transcription, facilitates MPA-induced S phase entry despite low guanine nucleotides levels, leading to further DNA damage. Unexpectedly, loss of the IRBC alone triggered DNA damage. These findings demonstrate that guanine nucleotide levels differentially control two distinct p53-checkpoints in a hierarchical manner and that the IRBC acts as a barrier against DNA damage. Results Distinct mechanisms mediate p53 activation in response to decreasing guanine nucleotides To identify the molecular mechanisms by which guanine nucleotide depletion regulates p53, we carried out a dose-response analysis with the IMPDH non-competitive catalytic inhibitor, MPA. As reported earlier in U2OS osteosarcoma cells (Sun et al, 2008), 24 h treatment of HCT116 cells with MPA leads to a dose-dependent increase in p53 protein levels (Fig 1A). Similar maximal levels of p53 were observed following treatment with 5 nM Actinomycin D (ActD) (Fig 1A), which selectively inhibits Pol I-dependent rRNA transcription (Perry, 1963), leading to activation of the IRBC (Donati et al, 2013). However, as the concentration of MPA increased, the amount of p21 protein was reduced (Fig 1A and Appendix Fig S1A). Moreover, concomitant with the decrease in p21 protein levels, the phosphorylation of p53 S15, Chk1 S345, and BRCA1 S1524 was increased, markers of replicative stress and ATR activation, absent in cells treated with 1 μM MPA or ActD (Fig 1A). However, at these time points, MPA does not lead to the phosphorylation of the ATM targets Chk2 T68 or H2AX S139 (γ-H2AX), nor increases the number of γ-H2AX or 53BP1 foci, indicating that DNA double-strand breaks (DSBs) are not a primary consequence of GMP depletion (Fig 1A and Appendix Fig S1B–D, respectively). Similar findings were obtained with the non-competitive inhibitor of IMPDH, AVN944 (Appendix Fig S1E). In contrast, inhibition of topoisomerase II by etoposide, which generates DNA-double strand breaks, led to an increase in Chk2 T68 phosphorylation and γ-H2AX (Appendix Fig S1F). These results suggest that at lower concentrations of IMPDH inhibitors the IRBC is activated and p53 stabilized, whereas at higher concentrations ATR and p53 phosphorylation are induced. Figure 1. Inhibition of guanosine nucleotide synthesis elicits distinct p53 responses A. Western blots in HCT116 cells treated for 24 h with increasing concentrations of MPA or 5 nM Actinomycin D (ActD). GAPDH served as a loading control. B. HCT116 cells were treated for 24 h with vehicle alone (−), 1 μM MPA, 10 μM MPA, or the combination of 10 μM MPA with 400 μM guanosine. Guanylate nucleotide levels were measured by LC-MS and normalized to protein content. Mean ± SEM is representative of 3 independent experiments carried out in triplicate. C, D. Parental HCT116 cells expressing a stable tetracycline-inducible shRNA against IMPDH2 (IM2iKD) and either a stable non-targeting (NT) shRNA (IM2iKD-shNT) or an shRNA targeting IMPDH1 (IM2iKD—shIM1) were grown for 7 days in the absence or presence of doxycycline (2 μg/ml) (− dox or + dox, respectively). mRNAs of IMPDH1 and IMPDH2 were quantified by real-time qPCR in 2 independent experiments carried out in triplicate and normalized to β-actin mRNA (C). Whole cell extracts were analyzed on Western blots with the indicated antibodies. GAPDH was used as a loading control. Quantification of band intensity of p53 and p21 is shown. Mean ± SD is representative of four independent experiments. (right panel) (D). E. Western blots in HCT116 cells treated for 24 h with the indicated concentration of MPA, in the presence of dimethyl sulfoxide (DMSO) or 400 μM guanosine. GAPDH was used as a loading control. Data information: In panels (B–D), data are presented as relative to control. *P < 0.05, **P < 0.01, ***P < 0.001 by two-tailed Student's t-test. Download figure Download PowerPoint Given the differential effects of IMPDH inhibitors on p53 stabilization and phosphorylation, we asked whether these differences were associated with changes in guanine nucleotides pools, as measured by liquid chromatography-mass spectrometry (LC-MS). Following 24 h treatment with 1 or 10 μM MPA, we observed a dose-dependent drop in all guanine ribonucleotides, whereas dGTP levels only decreased significantly in 10 μM MPA-treated cells (Fig 1B). Moreover, IMPDH inhibition leads to the accumulation of the substrate IMP (Appendix Fig S1G) but has no effect on adenine nucleotide levels (Appendix Fig S1H), pyrimidine nucleotide UTP levels (Appendix Fig S1I), or on the NAD+/NADH redox state, despite NAD+ being an IMPDH coenzyme (Appendix Fig S1J). The differential effects of increasing concentrations of MPA on guanine nucleotide levels appear to be compatible with IMPDH inhibition eliciting distinct effects on ribosome biogenesis and replicative stress. The induction of p53 by MPA is selectively mediated by IMPDH inhibition To ensure that the effects of MPA on p53 were mediated by IMPDH inhibition, we depleted cells of IMPDH1 and/or IMPDH2, the dominant isoform in most mammalian tissues (Carr et al, 1993; Senda & Natsumeda, 1994), and measured the responses above. We first generated a stable HCT116 cell line expressing a tetracycline (Tet)-inducible miR30 based shRNA (Zuber et al, 2011) against IMPDH2 (IM2iKD). The IM2iKD parental cell line was then transfected with either a non-targeting (NT) or IMPDH1 shRNA, to generate the IM2iKD-shNT and IM2iKD–shIM1 stable cell lines. In IM2iKD-shNT cells, tetracycline treatment did not affect IMPDH1 mRNA levels (Fig 1C, left panel), whereas IMPDH2 mRNA levels were reduced (Fig 1C, right panel). In IM2iKD–shIM1 cells, IMPDH1 mRNA levels were decreased (Fig 1C, left panel), with tetracycline treatment inducing a reduction in IMPDH2 mRNA levels (Fig 1C, right panel). The shRNA targeting of IMPDH1 had no effect on total IMPDH (see Materials and Methods), p53 or p21 protein levels, nor on Chk1 S345 phosphorylation (Fig 1D). Despite IMPDH2 depletion decreasing the levels of IMPDH2 and total IMPDH protein, while inducing p53 and p21 protein, it had no effect on Chk1 S345 phosphorylation (Fig 1D). Similar results were obtained with an independent shRNA sequence against IMPDH2 (Appendix Fig S1K and L). These findings argue that IMPDH2 is the predominant isoenzyme in HCT116 cells and that its depletion induces p53, but apparently independent of replicative stress. The failure of IMPDH depletion to induce Chk1 phosphorylation (Fig 1D) suggested that the decrease in IMPDH levels was not severe enough to lower nucleotide levels to the level achieved with MPA (Fig 1B). In agreement with this observation, addition of 10 μM MPA in cells depleted of IMPDH2 led to an increase in Chk1 S345 phosphorylation (Appendix Fig S1M) and a further decrease in guanine nucleotide levels (Appendix Fig S1N). We also assessed potential off-target effects of MPA by supplementing cells with exogenous guanosine, through which the purine salvage pathway can regenerate nucleotides from degradative intermediates. The addition of exogenous guanosine bypassed the effects of MPA on guanine nucleotide levels (Fig 1B), completely preventing the induction of p53, p21, and Chk1 S345 phosphorylation by 10 μM MPA (Fig 1E), with none of the other three nucleosides showing a similar effect on p53 (Appendix Fig S1O). These results support the notion that as guanine nucleotide levels decrease distinct responses are activated, which converge on p53. The IRBC and ATR contribute distinctly to the regulation of p53 The high demand of ribonucleotides for ribosome biogenesis (Pelletier et al, 2018) supports the concept that the p53 stabilization, by low concentrations of IMPDH inhibitors, is mediated by the IRBC. To test this hypothesis, we pretreated cells with a siRNA NT or a siRNA against RPL11 (siRPL11), an essential component of the IRBC (Pelletier et al, 2018). The results show that the upregulation of p53 by 1 or 10 μM MPA is reduced by depletion of RPL11; however, the levels of p53 are significantly higher in cells treated with 10 μM MPA (Fig 2A), despite an equivalent decrease in RPL11 mRNA levels (Appendix Fig S2A). To ensure that the second input to p53 is independent of the IRBC, we depleted cells of RPL7a (or eL8), an essential RP of the 60S ribosomal subunit whose depletion leads to the activation of the IRBC (Fumagalli et al, 2012), or co-depleted RPL7a and RPL11 (Fig 2B and Appendix Fig S2B), followed by treatment with 10 μM MPA for 6 h. The results show that depletion of RPL11 alone has no effect on p53 or p21 levels, whereas the strong induction of both responses following depletion of RPL7a is completely reversed by co-depletion of RPL11 (Fig 2B). However, p53 upregulation by MPA in control or RPL7a-depleted cells is only partially reversed by RPL11 co-depletion (Fig 2B). These findings support the role of the IRBC in the primary response to nucleotide depletion, and that there is a distinct secondary input to p53 when the inhibition of GMP synthesis is more severe. Figure 2. The IRBC and ATR distinctly regulate p53 stabilization upon nucleotide depletion HCT116 cells were transfected with either a NT or a siRNA against RPL11 for 24 h and treated with the vehicle alone (−) or the indicated concentration of MPA for 24 h. The levels of p53 and p21 were analyzed on Western blots. GAPDH was used as a loading control. Quantification of band intensity of p53 and p21 of four independent experiments is shown (right panel). HCT116 cells were transfected with the indicated siRNA and 24 h later treated with the vehicle alone or 10 μM MPA for 6 h, and the levels of p53 and p21 were analyzed on Western blots. GAPDH was used as a loading control. Quantification of band intensity of p53 of at least four independent experiments is shown (right panel). HCT116 cells were transfected with either a NT siRNA or a siRNA against RPL11 for 24 h and were preincubated for 30 min in the absence (−) or the presence (+) of the combination of 10 μM of the ATR inhibitor VE-821 (ATRi) and 10 μM the ATM inhibitor KU-55933 (ATMi), followed by addition of the vehicle alone (−) or MPA 10 μM for additional 24 h. The levels of p53 were analyzed on Western blots. GAPDH was used as a loading control. Quantification of band intensity of p53 of at least two independent experiments is shown (right panel). Data information: All data are presented as Mean ± SD, relative to control. *P < 0.05, **P < 0.01, ***P < 0.001, by two-tailed Student's t-test. Download figure Download PowerPoint As ATR-mediated p53 S15 phosphorylation (Fig 1A) impairs i
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