Article Nucleotide depletion reveals the impaired ribosome biogenesis checkpoint as a barrier against DNA damage Joffrey Pelletier1,*,†,‡ , 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 & George Thomas1,8,** Abstract Introduction Many oncogenes enhance nucleotide usage to increase ribosome The reprogramming of metabolic networks is a hallmark of cancer, content, DNA replication, and cell proliferation, but in parallel trig- which is initiated by the activation of oncogenes or the loss of tumor ger p53 activation. Both the impaired ribosome biogenesis check- suppressors, acting to drive and sustain tumor cell proliferation point (IRBC) and the DNA damage response (DDR) have been (Hanahan & Weinberg, 2011). One of the most studied oncogenes is implicated in p53 activation following nucleotide depletion. c-Myc, whose dysregulation or amplification has been implicated in However, it is difficult to reconcile the two checkpoints operating > 70% of all cancers (Murphy et al, 2008). c-Myc is a master regula- together, as the IRBC induces p21-mediated G1 arrest, whereas the tor of both cancer initiation and progression (Stine et al, 2015), DDR requires that cells enter S phase. Gradual inhibition of inosine through its ability to control the transcription of a wide range of monophosphate dehydrogenase (IMPDH), an enzyme required for genes (Kress et al, 2015). A critical set of these genes promote de novo GMP synthesis, reveals a hierarchical organization of these anabolic metabolism, including the synthesis of lipids, amino acids, two checkpoints. We find that the IRBC is the primary nucleotide and nucleotides, required for tumor cell proliferation (Kress et al, sensor, but increased IMPDH inhibition leads to p21 degradation, 2015; Stine et al, 2015). However, c-Myc appears to be distinct from compromising IRBC-mediated G1 arrest and allowing S phase entry other oncogenes, many of which fail to upregulate anabolic genes and DDR activation. Disruption of the IRBC alone is sufficient to involved in nucleotide metabolism (Bester et al, 2011; Aird et al, elicit the DDR, which is strongly enhanced by IMPDH inhibition, 2013), including Rb-E2F (Bester et al, 2011) and RAS-RAF (Aird suggesting that the IRBC acts as a barrier against genomic et al, 2013). In those cases, oncogene activation can lead to a instability. decrease in nucleotide pools, replicative stress, and genomic insta- bility, argued to be an initial step in tumorigenesis (Bester et al, Keywords IMPDH; IRBC; nucleotides; p21; p53 2011; Aird et al, 2013). Indeed, ectopic expression of c-Myc in Rb- Subject Categories Cell Cycle; DNA Replication, Recombination & Repair; E2F tumorigenic model re-establishes the nucleotide pools (Bester Signal Transduction et al, 2011), an effect partially recapitulated by the ectopic expres- DOI 10.15252/embj.2019103838 | Received 26 October 2019 | Revised 7 March sion of its target gene IMPDH2 (Mannava et al, 2008). Both IMPDH1 2020 | Accepted 14 April 2020 | Published online 2 June 2020 and 2 catalyze the rate limiting step in GMP synthesis, the oxidation The EMBO Journal (2020) 39: e103838 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 1 Laboratory of Cancer Metabolism, ONCOBELL Program, Institut d’Investigació Biomèdica de Bellvitge—IDIBELL, L’Hospitalet de Llobregat, Spain 2 Metabolomics Platform, IISPV & University Rovira i Virgili, Tarragona, Spain 3 Spanish Biomedical Research Center in Diabetes and Associated Metabolic Disorders (CIBERDEM), Madrid, Spain 4 Department of Biomedicine, Faculty of Medicine, IDIBAPS Biomedical Research Institute, Hospital Clinic, University of Barcelona, Barcelona, Spain 5 Catalan Institute of Oncology (ICO), Barcelona, Spain 6 Department of Genetics and Microbiology, Faculty of Biosciences, Autonomous University of Barcelona, Barcelona, Spain 7 Department of Biochemistry and Physiology, Faculty of Pharmacy, University of Barcelona, Barcelona, Spain 8 Department of Physiological Sciences, Faculty of Medicine and Health Science, University of Barcelona, Barcelona, Spain *Corresponding author. Tel: +34 260 7138; E-mail: jpelletier@idibell.cat **Corresponding author. Tel: +34 260 7138; E-mail: gthomas@idibell.cat † These authors contributed equally to this work ‡ Present address: Colorectal Cancer Laboratory, Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain ª 2020 The Authors. Published under the terms of the CC BY 4.0 license The EMBO Journal 39: e103838 | 2020 1 of 19 The EMBO Journal Joffrey Pelletier et al synthetic lethality (Stine et al, 2015), a concept supported by recent IRBC, leading to G1 arrest in a p53 wild-type setting (Sun et al, studies showing that a subset of small-cell lung carcinomas (SCLCs), 2008), and replicative stress. characterized by c-Myc and IMPDH upregulation, are highly vulner- Given the wide use of nucleotide synthesis inhibitors in cancer able to IMPDH inhibitors (Huang et al, 2018). therapy, it is important to understand the cellular checkpoints Although nucleotides are essential for DNA replication and repair induced by these anticancer agents. Here, we set out to address the (Tong et al, 2009; Aird et al, 2013), they are largely consumed in contribution of the IRBC and replicative stress in driving p53 stabi- the production of ribosomes, with ribosomal RNA (rRNA) represent- lization upon guanine nucleotide imbalance in sporadic colorectal ing ~80% of the nucleic acids of the cell and ~ 15% of its biomass cancer (sCRC) cell lines, as almost all CRCs are initiated by c-Myc (Pelletier et al, 2018). The demand for nucleotides is further exacer- dysregulation (TCGA, 2012) and are addicted to hyperactive ribo- bated in tumors driven by c-Myc, which coordinates the enhanced some biogenesis (Pelletier et al, 2018). We find that a reduction of transcription of genes required for the hyperactivation of ribosome guanine nucleotide pools first impairs ribosome biogenesis, leading biogenesis, needed to increase protein synthetic capacity, cell prolif- to the induction of the IRBC, p53 stabilization, and G1 arrest. eration, and ultimately malignancy (Santagata et al, 2013). Given However, if nucleotide depletion becomes more severe, cells enter S the dependency of such tumors on ribosome biogenesis (Barna et al, phase and encounter replicative stress, which is paralleled by the 2008; van Riggelen et al, 2010), a search for novel agents that decreased expression of p21. Although the levels of nascent 5S specifically target this process has been initiated (Bywater et al, rRNA, an essential component of the IRBC complex decrease under 2012; Pelletier et al, 2018). Importantly, insults to ribosome biogen- these conditions, we find that this is not sufficient to impinge on esis, including c-Myc-induced oncogenic stress (Macias et al, 2010; complex formation nor the transcriptional induction of p21. Instead Morcelle et al, 2019), also trigger a p53-mediated cell-cycle check- reduced p21 protein levels appear to be attributed to enhanced point, recently termed the impaired ribosome biogenesis checkpoint proteasome degradation. Moreover, S phase entry is further (IRBC) (Gentilella et al, 2017; Pelletier et al, 2018). The IRBC is enhanced in MPA-treated p21/ cells, with ectopic expression of mediated by a nascent pre-ribosomal complex containing RPL5 (or p21 reversing this response. Consistent with this observation, the uL18), RPL11 (or uL5), and 5S rRNA, which upon insults to ribo- downregulation of the IRBC complex, reducing p21 transcription, some biogenesis, is redirected from its assembly into 60S ribosomes facilitates MPA-induced S phase entry despite low guanine nucleo- to the binding and inhibition of the p53-E3-ubiquitin ligase HDM2 tides levels, leading to further DNA damage. Unexpectedly, loss of (or MDM2) (Donati et al, 2013), leading to p53 stabilization the IRBC alone triggered DNA damage. These findings demonstrate (Kubbutat et al, 1997; Pelletier et al, 2018). In most cases, p53 stabi- that guanine nucleotide levels differentially control two distinct p53- lization by the IRBC leads to G1 cell-cycle arrest, largely mediated checkpoints in a hierarchical manner and that the IRBC acts as a by the cyclin-dependent kinase inhibitor 1 (CDKN1A) or p21. This barrier against DNA damage. 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 & Results Dong, 2004), independent of the IRBC (Macias et al, 2010). Because of the critical role of IMPDHs in proliferating tumor cells Distinct mechanisms mediate p53 activation in response to (Liu et al, 2008; Mannava et al, 2008), a number of studies have decreasing guanine nucleotides focused on the development of antimetabolites, which inhibit their function (Stine et al, 2015), including mycophenolic acid (MPA), To identify the molecular mechanisms by which guanine nucleotide mizoribine, and AVN-944. These agents are catalytic non-competi- depletion regulates p53, we carried out a dose-response analysis tive inhibitors of IMPDH, which unlike nucleoside analogs that with the IMPDH non-competitive catalytic inhibitor, MPA. As produce chromosome breaks or inhibit DNA repair enzymes, do not reported earlier in U2OS osteosarcoma cells (Sun et al, 2008), 24 h incorporate into the DNA (Allison & Eugui, 2000). However, the treatment of HCT116 cells with MPA leads to a dose-dependent underlying mechanisms by which they lead to p53 stabilization are increase in p53 protein levels (Fig 1A). Similar maximal levels of not clearly understood. Initial studies suggested that IMPDH inhibi- p53 were observed following treatment with 5 nM Actinomycin D tors activate a reversible p53-dependent cell-cycle checkpoint (ActD) (Fig 1A), which selectively inhibits Pol I-dependent rRNA (Linke et al, 1996), which was later attributed to the IRBC (Sun transcription (Perry, 1963), leading to activation of the IRBC (Donati et al, 2008). However, others have reported that IMPDH inhibition et al, 2013). However, as the concentration of MPA increased, the leads to replicative stress and the DDR (Liu et al, 2008). This seem- amount of p21 protein was reduced (Fig 1A and Appendix Fig S1A). ing contradiction was recently highlighted in studies of two human Moreover, concomitant with the decrease in p21 protein levels, the tumor types, in SCLCs characterized by the low expression of the phosphorylation of p53 S15, Chk1 S345, and BRCA1 S1524 was Achaete-scute homolog-1 (ASCL1) and the upregulation of c-Myc increased, markers of replicative stress and ATR activation, absent (Huang et al, 2018) and in tuberous sclerosis complex 2 (TSC2)- in cells treated with 1 lM MPA or ActD (Fig 1A). However, at these deficient kidney tumors, where increased rRNA synthesis is driven time points, MPA does not lead to the phosphorylation of the ATM by constitutive mTOR signaling (Valvezan et al, 2017). Both are targets Chk2 T68 or H2AX S139 (c-H2AX), nor increases the number sensitive to IMPDH inhibitors, but apparently through different of c-H2AX or 53BP1 foci, indicating that DNA double-strand breaks mechanisms: the first through the inhibition of ribosome biogenesis (DSBs) are not a primary consequence of GMP depletion (Fig 1A (Huang et al, 2018) and the second through DDR (Valvezan et al, and Appendix Fig S1B–D, respectively). Similar findings were 2017). The differences in these findings are not clearly understood, obtained with the non-competitive inhibitor of IMPDH, AVN944 as is the apparent incompatibility between the induction of the (Appendix Fig S1E). In contrast, inhibition of topoisomerase II by 2 of 19 The EMBO Journal 39: e103838 | 2020 ª 2020 The Authors Joffrey Pelletier et al The EMBO Journal etoposide, which generates DNA-double strand breaks, led to an can regenerate nucleotides from degradative intermediates. The increase in Chk2 T68 phosphorylation and c-H2AX (Appendix Fig addition of exogenous guanosine bypassed the effects of MPA on S1F). These results suggest that at lower concentrations of IMPDH guanine nucleotide levels (Fig 1B), completely preventing the induc- inhibitors the IRBC is activated and p53 stabilized, whereas at tion of p53, p21, and Chk1 S345 phosphorylation by 10 lM MPA higher concentrations ATR and p53 phosphorylation are induced. (Fig 1E), with none of the other three nucleosides showing a similar Given the differential effects of IMPDH inhibitors on p53 stabi- effect on p53 (Appendix Fig S1O). These results support the notion lization and phosphorylation, we asked whether these differences that as guanine nucleotide levels decrease distinct responses are were associated with changes in guanine nucleotides pools, as activated, which converge on p53. measured by liquid chromatography-mass spectrometry (LC-MS). Following 24 h treatment with 1 or 10 lM MPA, we observed a The IRBC and ATR contribute distinctly to the regulation of p53 dose-dependent drop in all guanine ribonucleotides, whereas dGTP levels only decreased significantly in 10 lM MPA-treated cells The high demand of ribonucleotides for ribosome biogenesis (Fig 1B). Moreover, IMPDH inhibition leads to the accumulation of (Pelletier et al, 2018) supports the concept that the p53 stabilization, the substrate IMP (Appendix Fig S1G) but has no effect on adenine by low concentrations of IMPDH inhibitors, is mediated by the nucleotide levels (Appendix Fig S1H), pyrimidine nucleotide UTP IRBC. To test this hypothesis, we pretreated cells with a siRNA NT levels (Appendix Fig S1I), or on the NAD+/NADH redox state, or a siRNA against RPL11 (siRPL11), an essential component of the despite NAD+ being an IMPDH coenzyme (Appendix Fig S1J). The IRBC (Pelletier et al, 2018). The results show that the upregulation differential effects of increasing concentrations of MPA on guanine of p53 by 1 or 10 lM MPA is reduced by depletion of RPL11; nucleotide levels appear to be compatible with IMPDH inhibition however, the levels of p53 are significantly higher in cells treated eliciting distinct effects on ribosome biogenesis and replicative with 10 lM MPA (Fig 2A), despite an equivalent decrease in RPL11 stress. mRNA levels (Appendix Fig S2A). To ensure that the second input to p53 is independent of the IRBC, we depleted cells of RPL7a (or The induction of p53 by MPA is selectively mediated by eL8), an essential RP of the 60S ribosomal subunit whose depletion IMPDH inhibition leads to the activation of the IRBC (Fumagalli et al, 2012), or co- depleted RPL7a and RPL11 (Fig 2B and Appendix Fig S2B), followed To ensure that the effects of MPA on p53 were mediated by IMPDH by treatment with 10 lM MPA for 6 h. The results show that deple- inhibition, we depleted cells of IMPDH1 and/or IMPDH2, the domi- tion of RPL11 alone has no effect on p53 or p21 levels, whereas the nant isoform in most mammalian tissues (Carr et al, 1993; Senda & strong induction of both responses following depletion of RPL7a is Natsumeda, 1994), and measured the responses above. We first completely reversed by co-depletion of RPL11 (Fig 2B). However, generated a stable HCT116 cell line expressing a tetracycline (Tet)- p53 upregulation by MPA in control or RPL7a-depleted cells is only inducible miR30 based shRNA (Zuber et al, 2011) against IMPDH2 partially reversed by RPL11 co-depletion (Fig 2B). These findings (IM2iKD). The IM2iKD parental cell line was then transfected with support the role of the IRBC in the primary response to nucleotide either a non-targeting (NT) or IMPDH1 shRNA, to generate the depletion, and that there is a distinct secondary input to p53 when IM2iKD-shNT and IM2iKD–shIM1 stable cell lines. In IM2iKD-shNT the inhibition of GMP synthesis is more severe. cells, tetracycline treatment did not affect IMPDH1 mRNA levels As ATR-mediated p53 S15 phosphorylation (Fig 1A) impairs its (Fig 1C, left panel), whereas IMPDH2 mRNA levels were reduced interaction with HDM2 (Bode & Dong, 2004), the ATR pathway is (Fig 1C, right panel). In IM2iKD–shIM1 cells, IMPDH1 mRNA levels the likely candidate for the induction of the second p53 checkpoint were decreased (Fig 1C, left panel), with tetracycline treatment by MPA. Although selective ATR inhibitors block kinase activity, inducing a reduction in IMPDH2 mRNA levels (Fig 1C, right panel). ATR protects the stability of replicative forks, such that its inhibi- The shRNA targeting of IMPDH1 had no effect on total IMPDH (see tion leads to the collapse of replicative forks and to ATM-mediated Materials and Methods), p53 or p21 protein levels, nor on Chk1 p53 S15 phosphorylation, which is amplified by nucleotide short- S345 phosphorylation (Fig 1D). Despite IMPDH2 depletion decreas- age (Chanoux et al, 2009; Toledo et al, 2013). Consistent with this ing the levels of IMPDH2 and total IMPDH protein, while inducing observation, the ATR inhibitor (VE-821, ApexBio) strongly inhib- p53 and p21 protein, it had no effect on Chk1 S345 phosphorylation ited Chk1 S345 phosphorylation, but induced the phosphorylation (Fig 1D). Similar results were obtained with an independent shRNA of p53 S15, Chk2 T68, and c-H2AX (Appendix Fig S2C), markers sequence against IMPDH2 (Appendix Fig S1K and L). These findings of ATM activation. The increase in p53 S15 and Chk2 T68 phos- argue that IMPDH2 is the predominant isoenzyme in HCT116 cells phorylation was suppressed by the ATM inhibitor (KU-55933, and that its depletion induces p53, but apparently independent of Sigma-Aldrich), whereas that of c-H2AX was unaffected, most replicative stress. likely attributed to the activation of DNA-dependent protein kinase The failure of IMPDH depletion to induce Chk1 phosphorylation (Stiff et al, 2004). Analysis of the combination of ATR and ATM (Fig 1D) suggested that the decrease in IMPDH levels was not inhibitors on p53 activation by 10 lM MPA showed only a partial severe enough to lower nucleotide levels to the level achieved with inhibition of p53 stabilization (Fig 2C and Appendix Fig S2C), MPA (Fig 1B). In agreement with this observation, addition of which was also the case for RPL11 depletion (Fig 2C and 10 lM MPA in cells depleted of IMPDH2 led to an increase in Chk1 Appendix Fig S2D). However, ATR/ATM inhibition in cells S345 phosphorylation (Appendix Fig S1M) and a further decrease in depleted of RPL11 rescued p53 to almost basal levels (Fig 2C), guanine nucleotide levels (Appendix Fig S1N). We also assessed consistent with a milder lesion in guanine nucleotide synthesis potential off-target effects of MPA by supplementing cells with inducing the IRBC, whereas a more severe lesion causes induction exogenous guanosine, through which the purine salvage pathway of ATR and p53 phosphorylation. ª 2020 The Authors The EMBO Journal 39: e103838 | 2020 3 of 19 The EMBO Journal Joffrey Pelletier et al A B C D E Figure 1. 4 of 19 The EMBO Journal 39: e103838 | 2020 ª 2020 The Authors Joffrey Pelletier et al The EMBO Journal ◀ 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 lM MPA, 10 lM MPA, or the combination of 10 lM MPA with 400 lM 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 lg/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 b-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 lM 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. Severe nucleotide imbalance leads to increased p21 1 lM MPA and ActD (Fig 3C). However, there was no change in protein degradation the amount of RPL5 and RPL11 co-immunoprecipitating with HDM2, in cell extracts cleared of mature ribosomes, after treat- The levels of p21, an important marker of IRBC activation, fall at ment with 1 versus 10 lM MPA (Fig 3D), though there was a higher concentrations of IMPDH inhibitors (Fig 1A, and small reduction in the amount of associated 5S rRNA (Fig 3E). Appendix Fig S1A and E). We hypothesized that decreased Consistent with p21 mRNA levels (Fig 3A), we found higher levels guanine nucleotide synthesis would inhibit transcription of of IRBC complex components bound to HDM2 in the presence of nascent 5S rRNA, IRBC complex formation, and p21 transcription. ActD (Fig 3D and E). Similar results were obtained with the Unexpectedly, the results show that p21 mRNA levels are simi- immunoprecipitation of endogenous RPL5 (Appendix Fig S3E and larly increased after treatment with 1 lM or 10 lM MPA, though F). Thus, the reduction in 5S rRNA synthesis by GMP depletion to a lower extent than induced by ActD treatment (Fig 3A), did not appear to limit formation of the IRBC complex, supporting suggesting that the transcriptional activity of p53 toward p21 is the finding that p21 transcription is not differentially regulated as not differently affected by increasing concentrations of MPA. Like- a function of the dose of MPA. wise, Pol II transcription did not appear to be largely affected, by The results suggested that the effects on p21 were regulated at analyzing a number of mRNA transcripts (Appendix Fig S3A) nor the translational or post-transcriptional level. The half-life of p21 in using a Renilla luciferase reporter (Appendix Fig S3B). is known to be very short (Abbas & Dutta, 2009), raising the However, increasing concentrations of MPA led to a strong dose- possibility that the decrease in p21 protein levels is attributed to a dependent decrease in Pol I transcribed 47S rRNA, as measured decrease in its half-life. To test this possibility, cells were pre-incu- by the analysis of the Internal Transcribed Spacers (ITS) 1 and bated with 1 lM or 10 lM MPA for 24 h, then treated with cyclo- ITS2 (Appendix Fig S3A), and to a reduction in mature 18S and heximide for increasing times. The results show that the half-life 28S rRNA, measured by 3H-uridine pulse-chase labeling of nascent of p21 is dramatically reduced at 10 lM MPA versus 1 lM MPA, rRNA (Fig 3B). These findings are consistent with the induced whereas that of p53 is enhanced, showing differential regulation nucleolar disruption by MPA, evidenced by the redistribution of of p21 versus p53 following guanine nucleotide shortage (Fig 3F). upstream binding factor (UBF) and fibrillarin to adjacent nucleolar Moreover, p21 degradation appears to be due to enhanced protea- cap structures (Appendix Fig S3C), similar to our earlier findings somal degradation, as MG132 had little effect in cells pretreated (Fumagalli et al, 2012) and those of others (Shav-Tal et al, 2005). with 1 lM MPA, but dramatically enhanced p21 levels in cells The inhibition of ribosome biogenesis also agreed with a 30% treated with 10 lM MPA (Fig 3G), whereas the opposite effect decrease in 3H-leucine incorporation into nascent proteins, which was observed for p53 protein levels (Appendix Fig S3G). Taken is further reduced at 48 h (Appendix Fig S3D). In parallel to inhi- together, the results argue that the decrease in p21 levels by bition of Pol I transcription, 10 lM MPA treatment led to a strong severe IMPDH inhibition is due to its enhanced proteasomal inhibition of 5S rRNA, and 5.8S rRNA synthesis, as compared to degradation. Figure 2. The IRBC and ATR distinctly regulate p53 stabilization upon nucleotide depletion. A 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). B HCT116 cells were transfected with the indicated siRNA and 24 h later treated with the vehicle alone or 10 lM 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). C 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 lM of the ATR inhibitor VE-821 (ATRi) and 10 lM the ATM inhibitor KU-55933 (ATMi), followed by addition of the vehicle alone () or MPA 10 lM 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. ª 2020 The Authors The EMBO Journal 39: e103838 | 2020 5 of 19 The EMBO Journal Joffrey Pelletier et al A B C Figure 2. Severe nucleotide imbalance overcomes the IRBC-induced G1/S G0/G1 and G2/M, recapitulating the IRBC-dependent effects of ActD- checkpoint leading to replicative stress mediated Pol I inhibition, a large proportion of cells treated with 10 lM MPA were found in early S phase (Fig 4A and Appendix Fig The downregulation of p21 and the induction of replicative stress by S4A; Fumagalli et al, 2012). Importantly, this event appears to be a higher doses of MPA (Fig 1A) indicate that cells bypass the G1/S direct consequence of a compromised G1/S checkpoint, as cells first checkpoint upon severe nucleotide depletion. To test this possibility, synchronized in G1 by serum deprivation and re-stimulated with we first analyzed cell-cycle profiles of cells treated with either MPA or serum in the presence of 10 lM MPA, also initially accumulate in ActD for 24 h. Unlike cells treated with 1 lM MPA, largely arrested in early S phase (Appendix Fig S4B) and appear to slowly progress 6 of 19 The EMBO Journal 39: e103838 | 2020 ª 2020 The Authors Joffrey Pelletier et al The EMBO Journal A B C D E F G Figure 3. ª 2020 The Authors The EMBO Journal 39: e103838 | 2020 7 of 19 The EMBO Journal Joffrey Pelletier et al ◀ Figure 3. Protein stability of p21, but not formation of the IRBC complex, is reduced by higher concentrations of MPA. A HCT116 cells were treated with either vehicle alone (), 1 lM MPA, 10 lM MPA, or 5 nM ActD for 24 h and mRNA levels of p21 were quantified by real-time qPCR in 2 independent experiments carried out in triplicate and normalized to 28S rRNA. B Autoradiogram (upper panel) and Ethidium Bromide (EtBr)-stained agarose gel (lower panel) of 18S and 28S rRNA from HCT116 cells treated as indicated in the Materials and Methods section. C Autoradiogram (upper panel) and EtBr-stained TBE-urea polyacrylamide gel (lower panel) of 5S, 5.8S rRNA and tRNAs in lysates from (B). D, E HCT116 cells were treated as in (B) for 24 h. Cell lysates were collected, subjected to ultracentrifugation, spiked with firefly luciferase mRNA, and immunoprecipitated with anti-HDM2 rabbit antibody (IP HDM2) or the IgG control (IgG). (D) Levels of HDM2, RPL5, RPL11, and the IP-negative control mTOR in the whole cell extract (WCE), the post-ribosomal lysates (INPUT, left panel) and in the HDM2 immunoprecipitated fraction (right panel) were analyzed on Western blots. The results are representative of two independent experiments. Quantification of band intensity of HDM2, RPL5, and RPL11 of at least two independent experiments is shown (lower right panel). (E) Levels of immunoprecipitated 5S rRNA were determined by real-time qPCR and normalized to the firefly luciferase mRNA. Data are normalized and presented as relative to untreated cells immunoprecipitated with anti-HDM2. The results are representative of three independent experiments carried out in triplicates. F HCT116 cells were pretreated with vehicle alone (), 1 lM, or 10 lM MPA for 24 h, and the levels of p53 and p21 were followed on Western blots in presence of 100 lg/ml cycloheximide (CHX) for the indicated time, in the continued presence of the treatment. GAPDH was used as a loading control. Quantification of band intensities is shown in the lower panels as relative to time 0 of CHX treatment. G HCT116 cells were pretreated as in (F), and 10 lM of the proteasome inhibitor MG132 was added for 4 h when indicated. The levels of p53 and p21 were analyzed on Western blots. GAPDH was used as a loading control. Quantification of p21 band intensity of at least four independent experiments is shown in the lower panel. Data information: In panels (A, E, and G), 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. through the cell cycle (Appendix Fig S4C). The effects of MPA on cell- levels of ATM-dependent phosphorylation of H2AX S139 and Chk2 cycle progression were not ascribed to cellular context as similar T68 increase (Fig 4F). This finding, combined with H2AX phospho- results were observed in a number of CRC cell lines, including RKO, rylation occurring exclusively in DNA replicating cells LoVo, and LS174, harboring oncogenic alterations in K-Ras, and/or (Appendix Fig S4I and J), and the enhancement of cH2AX by ATR B-Raf, and mutations in the APC-Wnt-ß-catenin pathway leading to inhibition in MPA-treated cells (Appendix Fig S2C) indicate that c-Myc upregulation (TCGA, 2012), but all having a functional p53 prolonged nucleotide imbalance leads to persistent fork stalling and (Fig 4B and Appendix Fig S4D). As importantly, the analysis of p53, the generation of DSBs, likely due to the collapse of the replicative p21, and Chk1 S345 phosphorylation levels suggests that the differen- forks (Saintigny et al, 2001; Zhang et al, 2005; Hanada et al, 2006; tial activation of the IRBC and the induction of replicative stress as a Toledo et al, 2013). Moreover, the results further suggest that severe function of MPA concentrations is a conserved molecular response in nucleotide depletion impedes G1-arrest, allowing cells to enter S CRC cell lines (Appendix Fig S4E). phase where they encounter replicative stress, which with time Consistent with cell-cycle profiles (Fig 4A), the analyses of a leads to DNA damage. number of proteins involved in regulating cell-cycle progression show that treatment with 1 lM MPA or ActD decreases the levels of The suppression of p21 drives S phase entry upon cyclin A and the phosphorylation of retinoblastoma (RB) S780, nucleotide imbalance whereas levels of p21 and cyclin D1 increase (Appendix Fig S4F). In contrast, 10 lM MPA-treated cells display reduced levels of p21, To determine whether the loss of a robust p21 response is involved and cyclin D1 similar to untreated cells, while the levels of RB S780 in the failure of the IRBC to preserve cells in G1, we first analyzed the phosphorylation and cyclin A, required for S phase progression, are effects of MPA on the cell-cycle progression, as a function of p21 increased, compatible with a compromised G1/S checkpoint protein levels. We initially set a threshold for p21 analyses based on (Appendix Fig S4F). Moreover, these cells are delayed in completing antibody detection of the protein in isogenic HCT116 p21/ cells S phase (Appendix Fig S4C), indicating a hindrance in DNA replica- and discriminated two cellular populations in the parental cells, p21 tion, potentially ascribed to the reduced levels of dGTP (Fig 1B), a low (p21low) and p21 high (p21high). Cell-cycle analyses of total popu- common cause of replication fork stalling (Awasthi et al, 2015). To lation (Fig 5A, top panel) show similar profiles as described above test this hypothesis, we monitored the speed of DNA replication by (Fig 4A and Appendix Fig S4A). In the absence of treatment, p21high single-molecule DNA fiber analysis, employing two nucleotide cells represented 11.4% of the total population, but this proportion analogues (Ercilla et al, 2016; Appendix Fig S4G), showing that reaches 85.6% of total cells in the presence of 1 lM MPA, and only MPA-treated cells display a greatly reduced rate of DNA fiber elon- 38.0% when treated with 10 lM MPA (Fig 5A). Critically, p21high gation as compared to non-treated cells (Fig 4C and D). Importantly, cells are mostly arrested in G0/G1 and the G2/M regardless of MPA reduced rate of DNA fiber elongation was associated with asymmet- concentration, whereas p21low cells treated with 10 lM MPA are ric replication of the two DNA strands indicative of replicative stress mostly found in early S phase. A parallel comparative analysis of (Fig 4C, right insert). We also detected an increase in ssDNA gener- HCT116 wild-type and p21/ isogenic cells showed that 10 lM MPA ation, by BrdU incorporation under non-denaturing conditions and strongly reduces the proportion of p21/ cells present in G1, while accumulation of chromatin-loaded RPA, in cells treated with MPA, enriching the proportion of cells in early S phase (Appendix Fig S5A), compared with hydroxyurea (HU) which causes rapid depletion of consistent with a key role of p21 in mediating the effects of MPA on dNTPs (Fig 4E and Appendix Fig S4H). This result suggests that cell-cycle regulation, downstream of the IRBC and p53. MPA leads to replicative fork arrest, and to the uncoupling between To confirm that a reduction in p21 levels is a key determinant of replicative helicase and DNA polymerases. Moreover, after S phase entry upon nucleotide depletion, an empty plasmid (EV) or prolonged nucleotide imbalance, Chk1 is downregulated and the a plasmid encoding a p21wt cDNA under the control of an ectopic 8 of 19 The EMBO Journal 39: e103838 | 2020 ª 2020 The Authors Joffrey Pelletier et al The EMBO Journal A B C E D F Figure 4. ª 2020 The Authors The EMBO Journal 39: e103838 | 2020 9 of 19 The EMBO Journal Joffrey Pelletier et al ◀ Figure 4. S phase entry and inhibition of DNA replication are induced at high concentrations of MPA. A HCT116 were treated as indicated for 24 h, subjected to propidium iodide staining, and analyzed by fluorescence-activated cell sorting (FACS). Data are representative of at least three independent experiments. B Colon adenocarcinoma cell lines HCT116 (circles), LoVo (squares), LS174 (triangles), or RKO (inversed triangles) were treated with the vehicle alone (), 1 lM MPA or 10 lM MPA for 24 h and subjected to propidium iodide staining and FACS analysis. Data are shown as the fold induction of the cell number in G0/G1 phases (blue), S phase (red), and G2/M phases upon treatment with 1 lM MPA (dark) or 10 lM MPA (clear) over vehicle-treated cells. C IdU track length distribution, and distribution of long fork/short fork IdU track length ratios (insert). IdU track length of at least 300 fibers was measured for each condition. For fork symmetry analyses, IdU track length was measure in at least 38 bidirectional forks (see Materials and Methods). ***P < 0.001, ****P < 0.0001 by Kruskal–Wallis test of the means. D Representative images from (C). E HCT116 cells, pre-incubated with 10 lM BrdU for 48 h, were treated with Vehicle (), 10 lM MPA for 6 or 24 h, or 20 lM Hydroxyurea for 4 h before cell permeabilization and fixation (see Materials and Methods). Cells were immunostained for native BrDU (green) or RPA (red). DNA was counterstained with DAPI (blue). One representative z-confocal stack is shown per condition. Scale bars correspond to 5 lm. F HCT116 cells treated with 10 lM MPA for increasing time were analyzed by Western blots for the indicated proteins. a-tubulin was used as a loading control. promoter (p21-OE) was transiently introduced into p21/ cells Likewise if the synthesis of 5S rRNA is first blocked by depleting cells (Appendix Fig S5B) and cells were synchronized in G1 and serum of TFIIIa, required for Pol III-mediated 5S rRNA transcription stimulated as described above (Appendix Fig S4B). Analysis of the (Appendix Fig S6I and J), we also observed the induction of c-H2AX cell-cycle distribution of p21-OE cells was analyzed in p21high versus and the amplification of the response in cells treated with 10 lM p21low populations and showed that the p21high cells were unable to MPA, as well as a reduction in p53 induction by MPA (Appendix Fig bypass the G1/S checkpoint, even in the presence of 10 lM MPA S6K–M). These results show that unexpectedly the inhibition of the (Fig 5B). These results support that at higher doses of MPA the IRBC alone induces DNA damage. Moreover, it amplifies those induction of p21 is hindered, allowing cells to progress into S phase. caused by IMPDH inhibition, potentially due to the favored S phase entry despite nucleotide levels shortage leading to replicative stress The IRBC protects from replicative stress and DNA damage upon and DNA damage (see Discussion and model Fig 7), suggesting that nucleotide imbalance the IRBC serves as barrier against genomic instability caused by limiting pools of GMP. As the IRBC complex triggers p21 transcription, and its loss reduces MPA-induced p21 expression (Fig 2A), we predicted that the inhibi- tion of IRBC complex formation would further enhance the ability of Discussion MPA to drive cells into S phase. Thus, cells were depleted of either RPL11 or p53, prior to G1 synchronization and serum stimulation, as This study demonstrates a hierarchy of p53-dependent checkpoints above. In either condition, serum deprivation leads to the same activated by the inhibition of de novo guanine nucleotide synthesis. extent of G1 arrest (Fig 6A and Appendix Fig S6A). In untreated We have focused on IMPDH inhibitors given their clinical approval cells, p53 depletion does not greatly alter the cell cycle, whereas in other disease settings and the recent exciting pre-clinical findings RPL11 depletion led to an increase in the proportion of cells in S concerning their application in specific cancer types (Valvezan et al, phase, likely due to the slower progression of RPL11-deficient cells 2017; Huang et al, 2018); however, it is most likely that other clini- through the cell cycle, as we previously reported (Teng et al, 2013; cally approved non-genotoxic nucleotide inhibitors operate in a Fig 6A and Appendix Fig S6A). Importantly, the depletion of RPL11 similar manner. We show that the primary response to IMPDH inhi- or p53 potentiates MPA-induced accumulation of cells in S phase, bition is the induction of the IRBC and p21-mediated G1 arrest, particularly in early S phase (Fig 6A, and Appendix Fig S6A and B). consistent with ribosome biogenesis being the most nucleotide As the downregulation of the IRBC allows DNA replication, despite demanding metabolic process in proliferating cells (Pelletier et al, low nucleotide availability, this could favor the occurrence of 2018). However, if guanine nucleotide pools continue to fall, the replicative fork stalling and DNA damage. Unexpectedly, after 72 h, levels of p21 protein are diminished, allowing cells to enter S phase, we found by Western blot analysis that not only MPA, but also the where they encounter replicative stress and DNA damage, an effect depletion of RPL11 alone induced c-H2AX (Fig 6B and Appendix Fig that is strongly enhanced by disruption of the IRBC, and which is S6C). Moreover, RPL11 depletion further increased the effect of MPA known to be a major cause of genomic instability (Gaillard et al, on c-H2AX, while suppressing the induction of p53 (Fig 6B). These 2015). Interestingly, a crosstalk between the DDR and ribosome results were confirmed by quantitative microscopy-based single-cell biogenesis has recently been described, where activation of ATM analyses of c-H2AX or 53BP1, both indicative of DNA-double strand leads to Treacle recruiting Nijmegen Breakage Syndrome protein 1 breaks and the activation of the DDR machinery (Fig 6C–E and (NBS1) to the nucleolus, where this complex suppresses rDNA tran- Appendix Fig S6C). Similar results were obtained with the alkaline scription (Ciccia et al, 2014; Larsen et al, 2014). This regulatory comet assay, indicating global DNA damage, predominantly single- mechanism is an important player in the conservation of the stabil- strand nicks and gaps (Fig 6F and Appendix Fig S6C). To assess ity of the highly repetitive and actively transcribed rDNA genomic whether this response was due to the loss of the IRBC or to the regions (Pelletier et al, 2018). The ATM/NBS1/Treacle response is impairment of ribosome biogenesis, we depleted cells of RPL5 or particularly critical during rDNA replication, where such conflicts RPS6 (or eS6). While the depletion of RPL5 induced c-H2AX and lead to the formation of R-loops, rRNA:DNA hybrids, giving rise to amplified MPA-induced response, the depletion of RPS6 had no such DNA-double strand breaks (Pelletier et al, 2018). Thus, the impor- effect, but reduced the effect of MPA (Appendix Fig S6C–H). tance of ribosome biogenesis in replicative stress is underscored by 10 of 19 The EMBO Journal 39: e103838 | 2020 ª 2020 The Authors Joffrey Pelletier et al The EMBO Journal A B Figure 5. The downregulation of p21 facilitates S phase entry at high concentrations of MPA. A HCT116 cells were treated with either the vehicle alone (), 1 lM MPA, or 10 lM MPA for 24 h, before p21 immunolabeling, propidium iodide staining, and FACS analysis. Cell-cycle profiles of the total cell population (upper panel, left), the total intensity of p21, and propidium iodide were determined in 20,000 cells and plotted in a scatter diagram (lower panel, left). The specific cell-cycle profiles are shown of p21high (upper panel, right) and p21low (lower panel, right) cell populations. Mean  SD of the percentage of cells in each cell-cycle phase of at least two independent experiments is shown in the right panels. B HCT116 p21/ isogenic cell lines were transfected for 24 h with an empty plasmid (EV) or a plasmid encoding a p21wt cDNA (p21-OE), and then, cells were serum- deprived for 16 h before the re-addition of serum for additional 24 h in the absence or presence of 10 lM MPA. Cells were subjected to p21 immunolabeling, propidium iodide staining, and FACS analysis. The results are representative of two independent experiments. both the IRBC-p21 response and the NBS1/Treacle signaling path- expression of p21 in MPA-treated HCT116 p21/ cells is sufficient to way, each acting at a distinct level to protect from DNA damage. maintain cells in G1. The downregulation of p21 appears to be inde- The loss of p21 with increasing severity of IMPDH inhibition pendent of its transcription, but caused instead by its selective degra- seems responsible for the entry of cells in S phase, as ectopic dation. It has been recently reported in a context of doxorubicin- ª 2020 The Authors The EMBO Journal 39: e103838 | 2020 11 of 19 The EMBO Journal Joffrey Pelletier et al A B C D E F Figure 6. 12 of 19 The EMBO Journal 39: e103838 | 2020 ª 2020 The Authors Joffrey Pelletier et al The EMBO Journal ◀ Figure 6. Disruption of the IRBC enhances MPA-mediated DNA damage. A HCT116 cells were transfected for 8 h with the indicated siRNA then treated and analyzed as in Fig 5B. Here are shown the cell-cycle profiles after 16 h of serum deprivation (top panels), or after an additional 24 h of serum re-addition in the absence (middle panels), or presence of 10 lM MPA (bottom panels). The results are representative of at least three independent experiments. B HCT116 cells transfected with a NT siRNA or a siRNA against RPL11 for 24 h were treated with 10 lM MPA for additional 72 h. The levels of c-H2AX were analyzed on Western blots. GAPDH was used as a loading control. C, D Statistical analysis of the immunostaining of c-H2AX (C) or 53BP1 (D) in HCT116 cells treated as in (B) (see Materials and Methods). Total nuclear intensities were obtained on > 6,000 (c-H2AX) and > 1,000 (53BP1) individual cells for each condition from four or two independent biological replicates, respectively. **P < 0.01, ****P < 0.0001 by Kruskal–Wallis test. A.U. Arbitrary Units. E Representative images from (C, D) of cells labeled with c-H2AX (green), 53BP1 (red), and DAPI (blue). Scale bars correspond to 10 lm. F HCT116 cells treated as in (B) were analyzed by the comet assay. The percentage of tail DNA/total DNA was analyzed in a total of 300 cells for each condition from three independent biological replicates. ****P < 0.0001 by Kruskal–Wallis test. induced DNA double-strand breaks that the proteasomal degradation translation, as we reported earlier (Teng et al, 2013). It is likely that of p21 during S phase is prevented by Chk1 inhibition, suggesting a under such conditions the translation of specific factors is required role for Chk1 signaling in p21 degradation (Hsu et al, 2019). However to sustain the correct replication of DNA, or to sense and resolve in the case of IMPDH inhibitors, replicative stress and Chk1 activation replicative stress to limit DNA damage and potentially genomic would require S phase entry, restricted by p21. In normal cycling instability. cells, the degradation of p21 takes place prior to the G1/S transition Activation of the IRBC would appear to have an important role by the ubiquitin ligases CRL4Cdt2 and SCFSkp2 suggesting a potential in cancer therapy. Many approved chemotherapeutic agents are role of these proteins in the degradation of p21 by IMPDH inhibitors known to interfere with ribosome biogenesis (Burger et al, 2010). (Barr et al, 2017; Sheng et al, 2019). However, it has also been It has been long established that the major effects of 5-FU on p53- reported that p21 binds to the central domain of HDM2 (Jin et al, induced apoptosis are not due to the inhibition of thymidylate 2003; Zhang et al, 2004), where RPL11 also binds (Zheng et al, synthase, but to the inhibition of rRNA synthesis (Pritchard et al, 2015), and that HDM2 promotes the degradation of p21 in tumor cells 1997; Sun et al, 2007). Likewise recent studies showed that oxali- which are either deficient or mutated in p53 (Jin et al, 2003; Zhang platin does not induce p53 by causing DNA damage, but instead et al, 2004). Given that higher doses of MPA lead to p53 S15 phos- through impairing ribosome biogenesis and the apparent activa- phorylation, which is reported to disrupt the HDM2/p53 interaction, tion of the IRBC (Bruno et al, 2017). These insights have led to this would liberate HDM2 to degrade p21. It should be noted that the the development of new drugs specifically designed to attack ribo- effects of HDM2 on p21 are independent of the HDM2 E3 ligase func- some biogenesis, particularly CX-5461 (Drygin et al, 2011; Bywa- tion, probably acting through the binding of both p21 and the C8 ter et al, 2012), with the first results published of a phase 1 dose subunit of the 20S proteasome (Jin et al, 2003; Zhang et al, 2004). It escalation clinical trial in patients with advanced hematologic will be of interest to determine the underlying mechanism(s) by cancers (Khot et al, 2019). Importantly, a recent report showed which severe IMPDH inhibition leads to the degradation of p21 that the IMPDH inhibitor mizoribine, selectively targets tumors protein. developing in TSC2-deficient mice, which display constitutive During early tumorigenesis, oncogenic stresses lead to acceler- mTORC1 activation (Valvezan et al, 2017). mTORC1 coordinates ated rates of rRNA synthesis despite nucleotide deficiencies (Bester the upregulation of ribosome biogenesis by increasing Pol I- and et al, 2011), which may impair ribosome biogenesis, and lead to the Pol III-dependent transcription, and controlling the translation of IRBC-dependent senescence phenotype (Nishimura et al, 2015). The RP mRNAs (Pelletier et al, 2018). In this setting, nucleotide pools acute sensitivity of ribosome biogenesis impairment to nucleotide are apparently diverted to rRNA synthesis to sustain the hyperacti- depletion as compared to that of replicative stress supports the vation of ribosome biogenesis, which appears to sensitize TSC2- notion that the IRBC acts as a first-line barrier against oncogenes. deficient cells to replicative stress caused by guanine nucleotide Consistent with this model, earlier studies conducted in El-Myc depletion. Although Valvezan et al show that IMPDH inhibition transgenic mice harboring the C305F mutation in MDM2, which led to increased Chk1 phosphorylation and DNA damage in TSC2- disrupts its interaction with RPL11 and the inhibitory effect of the deficient models, there was no apparent effect on rRNA synthesis IRBC complex, demonstrated that mutant mice succumb earlier to in either TSC2/or TSC2+/+ cells (Valvezan et al, 2017). It is dif- lymphoma with respect to their WT counterparts (Macias et al, ficult to rationalize this observation with earlier reports (Huang 2010). As the loss of the IRBC enhances MPA-induced DNA damage et al, 2008; Sun et al, 2008), the recent findings of Huang et al, in in cell culture, one might expect the MDM2-mutant tumors, which c-Myc overexpressing SCLCs (Huang et al, 2018), and those are IRBC-defective, to have an increased rate of genomic instability. presented here, particularly with the high demand of nucleotides The mechanism by which the disruption of the IRBC leads to DNA in ribosome biogenesis versus DNA synthesis. Given the potential damage is not known. An interpretation for the amplification of importance in developing inhibitors of ribosome biogenesis to DNA damage may come from studies showing that in RPL11 deleted treat aggressive cancers, such as those driven by c-Myc, it will be mouse embryo fibroblasts, the expression of genes involved in DNA critical to understand whether TSC is unique in its response to repair are downregulated (Morgado-Palacin et al, 2015). On the IMPDH inhibitors or whether it can be more broadly applied to other hand, cells harboring reductions or mutations in components other cancer types. Moreover, given the role of the IRBC as a of the IRBC continue to cycle, but with reduced ribosome content tumor suppressor, a deeper knowledge of the biological processes and translational capacity, leading to alterations in the pattern of controlling the hierarchy of intrinsic tumor checkpoints will be ª 2020 The Authors The EMBO Journal 39: e103838 | 2020 13 of 19 The EMBO Journal Joffrey Pelletier et al critical for the development of novel strategies exploiting anti- hexamers and MMLV Reverse transcriptase were from Invitrogen cancer agents in the clinic. (Carlsbad, CA, USA). The SYBR Green kit was purchased from Roche (Basel, Switzerland). The Dual-Luciferase kit was purchased from Promega (Madison, WI, USA). The sequences of siRNAs used Materials and Methods in the experiments, siRPL5, siRPL11, siRPL7a, sip53, and siNT are reported in Appendix Table S2. For each treatment, the amount of Cell culture siRNA transfected was maintained constant between samples, by using siNT where required. pcDNA 3.1 (Addgene plasmid V790-20), HCT116, LoVo, LS174, and RKO human colorectal carcinoma cell PG13-luc plasmids (wt p53 binding sites, Addgene plasmid #16442), lines were obtained from the American Type Culture Collection and and MG15-luc plasmids (mut p53 binding sites, Addgene plasmid maintained in Dulbecco’s modified Eagle medium (DMEM) supple- #16443) were obtained from Addgene (Cambridge, MA, USA). The mented with 10% heat-inactivated fetal bovine serum (Sigma- pcDNA3.1-p21 plasmid is a kind gift from Dr. Sawaya. Aldrich, St Louis, MO, USA). Isogenic HCT116 cells disrupted for p53 or p21 expression were kindly provided by Dr. Vogelstein Protein analysis (Johns Hopkins University of Medicine, Baltimore, MD, USA). Cell protein extracts and protein concentrations for Western blot Stable cell lines generation analysis were carried out with the indicated antibodies (Appendix Table S1) as recently described (Gentilella et al, 2017). The shRNA sequences for IMPDH1 and IMPDH2 were selected using Immunoblots were developed using secondary horseradish peroxi- the Designer of Small Interfering RNA (http://biodev.cea.fr/DSIR/ dase-coupled antibodies (Polyclonal swine anti-rabbit and poly- DSIR.html). shRNA targeting IMPDH2 were cloned into the retrovi- clonal rabbit anti-mouse, Agilent, CA, USA) and an enhanced ral TRMPVIR vector as previously described (Zuber et al, 2011). chemiluminescence kit (GE Healthcare). Signal was detected using Five shRNA sequences were screened, and two were chosen for iBright CL1000 (Thermo Fisher Scientific, PA, USA), and quan- virus production (see Appendix Table S2). The generation of shRNA tification of band intensities by densitometry was carried out using encoding retrovirus particles and the transduction of HCT116 cells the ImageJ software. were performed as previously described (Zuber et al, 2011). After 24 h of transduction, the cells were sorted for Venus-positive cells Real-time PCR by FACS (MoFlo Astrios cell sorter). Individual clones were grown, treated with doxycycline (2 lg/ml), and analyzed for mRNA and Total cell RNA was isolated using TRIzol reagent (Invitrogen) protein expression. Separately, control non-targeting (shNT) or a according to the manufacturer’s instructions. Reverse transcription shRNA against IMPDH1 (shIM1) were cloned into pLKO.1 plasmid and quantitative real-time PCR were performed as previously and lentiviral particles were generated as described previously (Fon- described (Gentilella et al, 2017). The sequences of primers utilized seca et al, 2015; see Appendix Table S2). HCT116 cells stably are reported in Appendix Table S2. For the quantification of RNA expressing IMPDH2 shRNA were transduced with control shRNA or normalized to the DNA content, cells were harvested in an equal IMPDH1 shRNA followed by 3 weeks of puromycin selection lead- volume of cold PBS and 50 ll of the cell suspension was lysed in ing to the generation of IM2iKD-shNT or IM2iKD-shIM1, respectively. 50 ll of 0.1 N NaOH and DNA was quantified using a NanoDrop HCT116 cells stably expressing IMPDH2 shRNA seq#2 (see 1000 spectrophotometer (Thermo Scientific). The remaining cells Appendix Table S2) were transduced with control shRNA leading to were pelleted, lysed in TRIzol, a spike of Firefly luciferase mRNA the generation of IM2iKD#2-shNT. proportional to the amount of DNA was added, and the samples were processed as described above and the RNA amount normalized Reagents and plasmids to luciferase mRNA. MPA, AVN944, actinomycin D, etoposide, hydroxyurea, KU55933, Immunoprecipitation guanosine, doxycyclin and Cycloheximide, CldU and IdU were purchased from Sigma (Sigma-Aldrich, St Louis, MO, USA). VE-821 Cells grown in 15-cm dishes were lysed and subjected to immuno- (ATRi) was purchased from ApexBio. The transfection reagents, precipitation largely as previously described (Donati et al, 2013). lipofectamine RNAiMAX (for siRNA transfection), lipofectamine After lysis, mature ribosomes were pelleted by ultracentrifugation at 2000 (for plasmid transfection), and TRIzol RNA extraction reagent, 200,000 g for 2 h at 4°C and an equivalent amount of protein were purchased from Invitrogen (Carlsbad, CA, USA). Transfections (1 mg) was incubated at 4°C overnight with rotation with anti- were performed according to manufacturer’s instructions. RPL5, anti-HDM2, or anti IgG to a ratio antibody/sample of 4 EN3HANCE autoradiography enhancer, 3H-Leucine, and 3H-Uridine lg/mg. Magna ChIP Protein A/G magnetic beads (Millipore) were were purchased from PerkinElmer. The protein assay kit was added to the extracts for an additional 2 h at 4°C with rotation and purchased from Pierce (Rockford, IL, USA). The antibodies used are washed following manufacturer’s instructions. Beads-containing listed in Appendix Table S1. It should be noted that the anti-IMPDH pellets were resuspended either in protein loading buffer for antibody (pan antibody) was argued to be a specific antibody Western blot analysis or TRIzol reagent, together with a spike of against IMPDH1, but our findings indicate that it recognizes both firefly luciferase mRNA (5 ng/mg of precipitated proteins) before IMPDH1 and IMPDH2. The Magna ChIP Protein A/G magnetic beads RNA purification, to recover immunoprecipitated RNA for 5S rRNA mix was purchased from Millipore (Billerica, MA, USA). Random qRT-PCR analysis. 14 of 19 The EMBO Journal 39: e103838 | 2020 ª 2020 The Authors Joffrey Pelletier et al The EMBO Journal Figure 7. Model of the hierarchical organization of the IRBC and the DDR response upon ribonucleotides depletion. Under normal cell growth conditions, the pre-ribosomal complex formed by RPL5, RPL11, and 5S rRNA is incorporated into nascent ribosomes (Left panel). When nucleotide levels become limiting for 47S rRNA synthesis, ribosome biogenesis is impaired, which converts the RPL5/RPL11/5S rRNA complex into the IRBC complex, which is able to bind and inhibit MDM2, releasing p53, leading to p21 induction and G1 arrest (Middle panel). However, a more severe drop in nucleotide levels leads to the degradation of p21 by the proteasome, impairing IRBC-dependent G1 arrest and allowing S phase entry despite reduced nucleotide pools. Cells are therefore exposed to replicative stress, sensed by the ATR-Chk1 pathway responsible for p53 S15 phosphorylation. Chronic replicative stress leads to DNA damage and eventually genomic instability, key events in tumor progression (Right panel). Autoradiographic analysis of rRNA synthesis After ultraviolet cross-linking, the membranes were sprayed with EN3HANCE (PerkinElmer) and exposed to Kodak BioMax MS film To analyze newly synthesized RNA, cells were pulse-labeled for 2 h (Kodak) at 80°C for 1 week. with 1.2 lCi/ml of [3H]-uridine (PerkinElmer) and then chased in non-radioactive media for 4 h before TRIzol RNA extraction as Measurement of de novo protein synthesis by [3H] described above. 2 lg of total RNA were resolved either on a leucine incorporation formaldehyde-containing 1.2% agarose gel for 18S and 28S rRNAs or on a TBE-urea 10% polyacrylamide gel for 5S rRNA, 5.8S rRNA, After treatment, cells were pulse-labeled for 30 min with 10 lCi/ml or tRNAs and transferred to Hybond N+ membrane (GE Healthcare). of [3H] leucine as previously described (Gentilella et al, 2017). ª 2020 The Authors The EMBO Journal 39: e103838 | 2020 15 of 19 The EMBO Journal Joffrey Pelletier et al Cycloheximide (CHX), at a concentration of 100 lg/ml for 2 h, was 1 min on ice prior to 4% formaldehyde fixation. We then used as a control of protein synthesis inhibition. proceeded as above using a blocking solution containing 3% BSA and 0.01% Triton X-100. Luciferase assay DNA fiber assays Cells plated in 6-well plates were transfected with 2 lg of reporter plasmid (PG13-luc or MG-15-luc, see Reagents and Plasmids) and DNA fiber assays were largely performed as previously described 250 ng of Renilla luciferase control plasmid (Promega, Madison, (Ercilla et al, 2016). Cells were treated and pulse-labeled as indi- WI, USA) for 24 h. Cell extracts were prepared and assayed using cated with CldU (25 lM)/IdU (250 lM). Labeled cells were the Dual Luciferase kit (Promega) according to the manufacturer’s harvested by trypsinization and resuspended in ice-cold PBS at instructions. Firefly Luciferase activity was normalized to Renilla 5 × 105 cells/ml. After DNA spreading, fixation in methanol/acetic luciferase activity. acid (3:1) solution, denaturation, and neutralization steps, CldU and IdU were detected by immunofluorescence with anti-BrdU mono- Cell-cycle analysis clonal antibody (Abcam, ab6326; 1/1,000 for CldU labeling and Becton Dickinson, 347580; 1/200 for IdU labeling) for 1.5 h at 37°C. After treatment, cells were trypsinized, counted, washed, and fixed After incubation with primary antibodies, fiber spreads were fixed in cold 70% ethanol solution overnight. For p21 or c-H2AX with 4% PFA-PBS for 10 min at RT and finally incubated with Alex- immunostaining, cells were permeabilized with 0.1% TritonX-100 a488- and Alexa555-conjugated secondary antibodies (Invitrogen, for 20 min at room temperature (RT), washed with PBS-Tween20 1/500) for 1.5 h at 37°C. Images were obtained using Zeiss LSM880 (0.5%), incubated in 4% FBS–containing PBS-T Blocking Solution confocal microscope with a 63× oil immersion objective and then for 30 min at RT, and incubated with p21 antibody (1/250, Abcam) analyzed using Fiji software. The length of at least 300 IdU tracks or c-H2AX (Abcam, 1/200) in the blocking solution for 1 h at RT. per condition was measured. We only measured the fibers that were Cells were then extensively washed in PBS-T, before alexa647- doubly labeled (by CldU and IdU), to exclude from analysis the conjugated secondary antibody (Invitrogen, 1/500) incubation for fibers that were initiated during the second labeling period. To 30 min at RT in the dark. After washing, cells were stained with assess fork symmetry, the length of IdU tracks was measured in at propidium iodide as previously described (Morcelle et al, 2019) least 38 bidirectional forks (labeled by CldU, and IdU in the two before FACS analysis, using FACSCanto II (BD biosciences, CA, directions), and the ratio of length between the longer and the USA). In all experiments, 20,000–50,000 gated events were shorter track was analyzed. collected. Experiments were analyzed using ModFit LT software (Verity Software House, ME, USA) and BD FACSDiva software (BD Comet assay Biosciences, CA, USA). After treatment, cells were harvested by trypsinization, resuspended Immunofluorescence microscopy in ice-cold PBS at 1 × 106 cells/ml and centrifuged at 1,200 g for 8 min. The cell suspension was mixed 1:10 with 0.75% low melting Immunofluorescence microscopy was performed largely as previ- point agarose at 37°C, dropped on GelBond Films (GBF) (Life ously described (Pelletier et al, 2012). Briefly, after 4% formalde- Sciences, Lithuania) in triplicates and lysed in cold lysis buffer over- hyde fixation for 20 min at RT, and permeabilization with 0.1% night at 4°C. GBF were then incubated in electrophoresis buffer, to Triton X-100 for 10 min at RT, cells were incubated in the block- allow DNA denaturation and expression of alkali-labile sites, for ing solution containing 1% BSA and 0.01% Triton X-100 for 1 h 35 min at 4°C. Subsequently, the electrophoresis step was carried at RT. Cells were then incubated with the indicated primary anti- out at 20 V and 300 mA for 20 min at 4°C. GBF were washed twice bodies (Appendix Table S1) in blocking solution overnight at with cold PBS 1× fixed in absolute ethanol for 1 h, then air-dried 4°C. After extensive washes with PBS-T (0.01%), cells were overnight at room temperature. Cells were stained with 1:10,000 stained with Alexa488- or Alexa555-conjugated secondary anti- SYBR Gold in TE buffer for 20 min at room temperature, mounted, body (Invitrogen, 1/500 in blocking solution) for 45 min at RT in and visualized with an epifluorescence microscope (Olympus BX50) the dark. After additional washes, cells were mounted with with a 20× magnifications. DNA damage was quantified with the Vectashield mounting solution containing DAPI. Images were Komet 5.5 Image analysis system (Kinetic Imaging Ltd, Liverpool, generated either with the Leica spectral confocal microscope TCS UK) as the percentage of DNA in the tail. One hundred randomly SP5 with a 63×/1.4 NA objective or with the Zeiss Axio Observer selected comet images were analyzed per sample (33/34 per tripli- inverted microscope with a 10×/0.25 NA objective. Acquisition cate). Results from three independent biological replicates were settings for the different channels were adjusted and maintained analyzed. to obtain images in non-saturating conditions. For c-H2AX and 53BP1 immunostaining quantification, depending on cell conflu- Targeted liquid chromatography-mass spectrometry ence, 8–15 images were acquired containing n > 1,000 cells per (LC-MS) analyses condition. Images were processed and analyzed using ImageJ software (NIH USA) (Toledo et al, 2013). The analysis of positive Metabolites were extracted from snap-frozen cell pellets by adding foci for native BrDU and RPA required a pre-extraction protocol. methanol: H2O (1:1 v/v) solution and vortexing samples for 30 s. Briefly, after treatment, cells were washed once with PBS and Samples were immersed in liquid N2 to disrupt cell membranes incubated with ice-cold PBS containing Triton X-100 (0.5%) for followed by 30 s of ultrasonication. These two steps were repeated 16 of 19 The EMBO Journal 39: e103838 | 2020 ª 2020 The Authors Joffrey Pelletier et al The EMBO Journal three times. Then, samples were incubated at 20°C for 2 h, centri- Author contributions fuged at 17,000 × g for 15 min, and the supernatant was collected JP, FR-C and GT conceived and designed the study. JP and FR-G performed into a LC-MS vial. Samples were injected in a UHPLC system (1290 most of the experiments, and EA, CM, SS, SF, SM, AD, MG-C, CC and AG contrib- Agilent) coupled to a triple quadrupole (QqQ) MS (6490 Agilent uted to individual experiments. JP, OY, NA, RM, RS, AT, SCK, AG, and GT Technologies) operated in multiple reaction monitoring (MRM) and analyzed the data, and all authors provided intellectual support in the discus- positive (POS) or negative (NEG) electrospray ionization (ESI) sion of the results. JP and GT wrote the manuscript. mode. Metabolites were separated using C18-RP (ACQUITY UPLC BEH C18 1.7 lM, Waters) chromatography at flow rate of 0.3 Conflict of interest ml/min. The solvent system was A = 20 mM ammonium The authors declare that they have no conflict of interest. acetate + 15 mM NH3 in water and B = acetonitrile: water (95:5). The linear gradient elution started at 100% A (time 0–2 min), 65% A (time 2–5 min) and finished at 100% B (time 5.5 min). MRM References transitions are shown in Appendix Table S3. Abbas T, Dutta A (2009) p21 in cancer: intricate networks and multiple Statistical analysis activities. Nat Rev Cancer 9: 400 – 414 Aird KM, Zhang G, Li H, Tu Z, Bitler BG, Garipov A, Wu H, Wei Z, Wagner SN, Statistical analysis was performed using GraphPad Prism version 7.00 Herlyn M et al (2013) Suppression of nucleotide metabolism underlies the (GraphPad Software, San Diego, CA, USA). Data are presented as establishment and maintenance of oncogene-induced senescence. Cell Rep mean  SD. 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Nat Rev Cancer 4: 793 – 805 Cincinnati for sharing ideas and reagents as well as their encouragement Bruno PM, Liu Y, Park GY, Murai J, Koch CE, Eisen TJ, Pritchard JR, Pommier Y, throughout the study, and the CERCA Programme/Generalitat de Catalunya for Lippard SJ, Hemann MT (2017) A subset of platinum-containing institutional support. We thank Dr. J. Bartek for his critical comments of earlier chemotherapeutic agents kills cells by inducing ribosome biogenesis drafts of this manuscript. We also thank Drs. S. Fumagalli and S. Volarevic for stress. Nat Med 23: 461 – 471 their advice during these studies, Drs. B. Vogelstein and K. Vousden that kindly Burger K, Muhl B, Harasim T, Rohrmoser M, Malamoussi A, Orban M, Kellner provided isogenic HCT116 p21/ cells, and Dr B.E. Sawaya that kindly M, Gruber-Eber A, Kremmer E, Holzel M et al (2010) Chemotherapeutic provided the pcDNA3.1-p21 plasmid. These studies were supported by grants drugs inhibit ribosome biogenesis at various levels. J Biol Chem 285: to G. T. from the Spanish Ministry of Economy and Competitiveness (SAF2014- 12416 – 12425 52162-P), the CIG European Commission (PCIG10-GA-2011-304160), the NIH/ Bywater MJ, Poortinga G, Sanij E, Hein N, Peck A, Cullinane C, Wall M, Cluse NCI National Cancer Institute (R01-CA191814), the ISCIII-RTICC (RD12/0036/ L, Drygin D, Anderes K et al (2012) Inhibition of RNA polymerase I as a 0049), the AGAUR (2014SGR-870) and by shared funding from the IDIBELL and therapeutic strategy to promote cancer-specific activation of p53. Cancer the Vall d’Hebron Institute of Oncology (VHIO). A. G. and G. T. also were Cell 22: 51 – 65 supported by a grant from the Spanish Ministry of Economy and Competitive- Carr SF, Papp E, Wu JC, Natsumeda Y (1993) Characterization of human type ness (SAF2017-84301-P) and by the European Social Fund (ESF) “Investing in I and type II IMP dehydrogenases. J Biol Chem 268: 27286 – 27290 your future” (AGAUR—2017 SGR 01743). R. 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