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| <?xml version="1.0" encoding="UTF-8"?><!DOCTYPE article PUBLIC "-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD v1.1d1 20130915//EN" "JATS-archivearticle1.dtd"><article article-type="research-article" dtd-version="1.1d1" xmlns:xlink="http://www.w3.org/1999/xlink"><front><journal-meta><journal-id journal-id-type="nlm-ta">elife</journal-id><journal-id journal-id-type="hwp">eLife</journal-id><journal-id journal-id-type="publisher-id">eLife</journal-id><journal-title-group><journal-title>eLife</journal-title></journal-title-group><issn publication-format="electronic">2050-084X</issn><publisher><publisher-name>eLife Sciences Publications, Ltd</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="publisher-id">03077</article-id><article-id pub-id-type="doi">10.7554/eLife.03077</article-id><article-categories><subj-group subj-group-type="display-channel"><subject>Short report</subject></subj-group><subj-group subj-group-type="heading"><subject>Cell biology</subject></subj-group></article-categories><title-group><article-title>The role of IMP dehydrogenase 2 in Inauhzin-induced ribosomal stress</article-title></title-group><contrib-group><contrib contrib-type="author" id="author-13130"><name><surname>Zhang</surname><given-names>Qi</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="aff" rid="aff2"/><xref ref-type="fn" rid="con1"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-13236"><name><surname>Zhou</surname><given-names>Xiang</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="aff" rid="aff2"/><xref ref-type="fn" rid="con2"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-17640"><name><surname>Wu</surname><given-names>RuiZhi</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="aff" rid="aff2"/><xref ref-type="fn" rid="con3"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-13240"><name><surname>Mosley</surname><given-names>Amber</given-names></name><xref ref-type="aff" rid="aff3"/><xref ref-type="fn" rid="con4"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-13235"><name><surname>Zeng</surname><given-names>Shelya X</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="aff" rid="aff2"/><xref ref-type="fn" rid="con5"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-13238"><name><surname>Xing</surname><given-names>Zhen</given-names></name><xref ref-type="aff" rid="aff4"/><xref ref-type="fn" rid="con6"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" corresp="yes" id="author-1470"><name><surname>Lu</surname><given-names>Hua</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="aff" rid="aff2"/><xref ref-type="corresp" rid="cor1">*</xref><xref ref-type="other" rid="par-1"/><xref ref-type="other" rid="par-2"/><xref ref-type="other" rid="par-3"/><xref ref-type="other" rid="par-4"/><xref ref-type="fn" rid="con7"/><xref ref-type="fn" rid="conf1"/></contrib><aff id="aff1"><institution content-type="dept">Department of Biochemistry and Molecular Biology</institution>, <institution>Tulane University School of Medicine</institution>, <addr-line><named-content content-type="city">New Orleans</named-content></addr-line>, <country>United States</country></aff><aff id="aff2"><institution content-type="dept">Tulane Cancer Center</institution>, <institution>Tulane University School of Medicine</institution>, <addr-line><named-content content-type="city">New Orleans</named-content></addr-line>, <country>United States</country></aff><aff id="aff3"><institution content-type="dept">Department of Biochemistry and Molecular Biology</institution>, <institution>Indiana University School of Medicine</institution>, <addr-line><named-content content-type="city">Indianapolis</named-content></addr-line>, <country>United States</country></aff><aff id="aff4"><institution content-type="dept">Department of Molecular and Cellular Oncology</institution>, <institution>University of Texas MD Anderson Cancer Center</institution>, <addr-line><named-content content-type="city">Houston</named-content></addr-line>, <country>United States</country></aff></contrib-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Prives</surname><given-names>Carol</given-names></name><role>Reviewing editor</role><aff><institution>Columbia University</institution>, <country>United States</country></aff></contrib></contrib-group><author-notes><corresp id="cor1"><label>*</label>For correspondence: <email>hlu2@tulane.edu</email></corresp></author-notes><pub-date date-type="pub" publication-format="electronic"><day>27</day><month>10</month><year>2014</year></pub-date><pub-date pub-type="collection"><year>2014</year></pub-date><volume>3</volume><elocation-id>e03077</elocation-id><history><date date-type="received"><day>13</day><month>04</month><year>2014</year></date><date date-type="accepted"><day>06</day><month>10</month><year>2014</year></date></history><permissions><copyright-statement>© 2014, Zhang et al</copyright-statement><copyright-year>2014</copyright-year><copyright-holder>Zhang et al</copyright-holder><license xlink:href="http://creativecommons.org/licenses/by/4.0/"><license-p>This article is distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">Creative Commons Attribution License</ext-link>, which permits unrestricted use and redistribution provided that the original author and source are credited.</license-p></license></permissions><self-uri content-type="pdf" xlink:href="elife03077.pdf"/><abstract><object-id pub-id-type="doi">10.7554/eLife.03077.001</object-id><p>The ‘ribosomal stress (RS)-p53 pathway’ is triggered by any stressor or genetic alteration that disrupts ribosomal biogenesis, and mediated by several ribosomal proteins (RPs), such as RPL11 and RPL5, which inhibit MDM2 and activate p53. Inosine monophosphate (IMP) dehydrogenase 2 (IMPDH2) is a rate-limiting enzyme in de novo guanine nucleotide biosynthesis and crucial for maintaining cellular guanine deoxy- and ribonucleotide pools needed for DNA and RNA synthesis. It is highly expressed in many malignancies. We previously showed that inhibition of IMPDH2 leads to p53 activation by causing RS. Surprisingly, our current study reveals that Inauzhin (INZ), a novel non-genotoxic p53 activator by inhibiting SIRT1, can also inhibit cellular IMPDH2 activity, and reduce the levels of cellular GTP and GTP-binding nucleostemin that is essential for rRNA processing. Consequently, INZ induces RS and the RPL11/RPL5-MDM2 interaction, activating p53. These results support the new notion that INZ suppresses cancer cell growth by dually targeting SIRT1 and IMPDH2.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03077.001">http://dx.doi.org/10.7554/eLife.03077.001</ext-link></p></abstract><abstract abstract-type="executive-summary"><object-id pub-id-type="doi">10.7554/eLife.03077.002</object-id><title>eLife digest</title><p>Cancer develops when cells lose the ability to control their own growth. About half of cancerous tumors carry a dysfunctional version of a protein called p53, while the other half have defects in proteins that are important for p53's production and function. When a healthy cell is exposed to damaging chemicals or agents, the p53 protein triggers responses that are aimed at repairing the damage. However, if these attempts fail, p53 causes the damaged cell to essentially destroy itself.</p><p>As defects in p53-controlled processes cause cells to grow unrestrictedly and can lead to cancer, it is a very attractive target for cancer therapies. Cancer drug developments have focused on both targeting p53 directly and targeting the proteins that work with p53. Two proteins called Mdm2 and SIRT1 are of particular interest. Mdm2 binds to, inactivates, and leads to the degradation of p53. SIRT1 can modify p53 and make it more accessible to Mdm2, and is often found in very high levels in cancer cells.</p><p>In 2012, researchers identified Inauhzin as a small molecule that could potentially be used to treat tumors that still have a functional version of the p53 protein. Inauhzin was thought to work by inhibiting SIRT1, which increases p53 levels—probably through its effects on Mdm2. This restores the cell's ability to control its growth and to die if it is irreparably damaged. However, not all of this small molecule's effects on cells can be explained by its interaction with SIRT1.</p><p>Now Zhang et al., including some of the researchers involved in the 2012 work, have investigated whether Inauhzin also interacts with other proteins in the cell; and Inauhzin was revealed to bind an enzyme called IMPDH2. This enzyme is involved in making GTP—a small molecule that is involved in many important processes in living cells. Zhang et al. demonstrated that Inauhzin's effect on the IMPDH enzyme triggered a response that did not involve the SIRT1 protein, and that ultimately led to a decrease in Mdm2 activity and restored p53 activity.</p><p>Cancer treatments often include a combination of drugs that target different proteins with the goal of reducing the likelihood of a tumor becoming resistant to the treatment. Inauhzin's effect on two different proteins that lead to p53 activation not only increases its potency, but also makes it less likely that drug resistance will develop.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03077.002">http://dx.doi.org/10.7554/eLife.03077.002</ext-link></p></abstract><kwd-group kwd-group-type="author-keywords"><title>Author keywords</title><kwd>Inauhzin</kwd><kwd>p53</kwd><kwd>ribosomal stress</kwd><kwd>IMPDH2</kwd><kwd>nucleostemin</kwd><kwd>MDM2</kwd></kwd-group><kwd-group kwd-group-type="research-organism"><title>Research organism</title><kwd>human</kwd></kwd-group><funding-group><award-group id="par-1"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000054</institution-id><institution>National Cancer Institute</institution></institution-wrap></funding-source><award-id>CA 172468-02</award-id><principal-award-recipient><name><surname>Lu</surname><given-names>Hua</given-names></name></principal-award-recipient></award-group><award-group id="par-2"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000054</institution-id><institution>National Cancer Institute</institution></institution-wrap></funding-source><award-id>CA0954412-12</award-id><principal-award-recipient><name><surname>Lu</surname><given-names>Hua</given-names></name></principal-award-recipient></award-group><award-group id="par-3"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000054</institution-id><institution>National Cancer Institute</institution></institution-wrap></funding-source><award-id>CA127724-05</award-id><principal-award-recipient><name><surname>Lu</surname><given-names>Hua</given-names></name></principal-award-recipient></award-group><award-group id="par-4"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000054</institution-id><institution>National Cancer Institute</institution></institution-wrap></funding-source><award-id>CA129828-04</award-id><principal-award-recipient><name><surname>Lu</surname><given-names>Hua</given-names></name></principal-award-recipient></award-group><funding-statement>The funder had no role in study design, data collection and interpretation, or the decision to submit the work for publication.</funding-statement></funding-group><custom-meta-group><custom-meta><meta-name>elife-xml-version</meta-name><meta-value>2</meta-value></custom-meta><custom-meta specific-use="meta-only"><meta-name>Author impact statement</meta-name><meta-value>The small molecule Inauhzin possesses a dual targeting ability, and activates p53 by targeting two enzymes—SIRT1 and IMP dehydrogenase 2 (IMPDH2)—which are highly expressed in human cancers.</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="s1" sec-type="intro"><title>Introduction</title><p>With ∼22 million people living with cancers that are highly associated with alterations of multiple molecules and pathways, it is important to develop a multiple molecules-targeted therapy that can effectively kill cancer cells. The tumor suppressor p53 pathway is one such a target because nearly all cancers show defects in this pathway. Approximately 50% of human cancers have mutations in the TP53 gene itself, while the rest of them harbor functionally inactive p53 proteins, because active p53 can trigger cell growth arrest, apoptosis, autophagy, and/or senescence, which are detrimental to cancer cells (<xref ref-type="bibr" rid="bib33">Vogelstein et al., 2000</xref>; <xref ref-type="bibr" rid="bib34">Vousden and Prives, 2009</xref>), and impede cell migration, metabolism, and/or angiogenesis. A major mechanism for functional inactivation of p53 is through overexpression of two chief p53 suppressors, MDM2 and MDMX, which work together to inactivate p53 by directly interacting with p53, inhibiting its transcriptional activity and mediating its ubiquitin dependent degradation (<xref ref-type="bibr" rid="bib35">Wade et al., 2010</xref>; <xref ref-type="bibr" rid="bib15">Huang et al., 2011</xref>; <xref ref-type="bibr" rid="bib30">Tollini and Zhang, 2012</xref>). This MDM2/MDMX-mediated p53 degradation is also facilitated by SIRT1, a nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase (<xref ref-type="bibr" rid="bib32">Vaziri et al., 2001</xref>; <xref ref-type="bibr" rid="bib6">Cheng et al., 2003</xref>). SIRT1 is highly expressed in human cancers due to down regulation of another p53 target tumor suppressor called hypermethylated in cancer-1 (HIC-1) (<xref ref-type="bibr" rid="bib4">Chen et al., 2005</xref>).</p><p>Our previous study identified a small molecule named Inauhzin (INZ) that effectively inhibits SIRT1 activity and induces p53 acetylation, leading to the increase of p53 level and activity (<xref ref-type="bibr" rid="bib45">Zhang et al., 2012b</xref>). Consequently, INZ induces p53-dependent apoptosis and senescence in various p53-wild type human cancer cells, such as H460, and HCT116 by inducing the expression of p53-dependent transcriptome (<xref ref-type="bibr" rid="bib19">Liao et al., 2012</xref>). INZ markedly inhibits the growth of H460 or HCT116 xenograft tumors, but is not toxic to normal cells and tissues. Also, INZ sensitizes the anti-cancer effect of cisplatin, doxorubicin, or Nutlin-3 (an MDM2 inhibitor) as tested in xenograft cancer models (<xref ref-type="bibr" rid="bib46">Zhang et al., 2012c</xref>; <xref ref-type="bibr" rid="bib47">Zhang et al, 2013</xref>). Thus, this small molecule presents as a promising contender for a molecule-targeted anti-cancer therapy. Since its discovery, we have optimized INZ (<xref ref-type="bibr" rid="bib44">Zhang et al., 2012a</xref>) and determined additional cellular proteins that INZ might target via a set of biochemical, proteomic, and cell-based analyses. As detailed below, our study unveils inosine monophosphate (IMP) dehydrogenase 2 (IMPDH2) as a novel cellular target of INZ.</p></sec><sec id="s2" sec-type="results|discussion"><title>Results and Discussion</title><sec id="s2-1"><title>Identification of IMPDH2 as a cellular target of INZ</title><p>IMPDH is the key metabolic enzyme supplying guanine nucleotides to a cell as the first and rate-limiting enzyme of de novo GTP biosynthesis by catalyzing NAD+-dependent oxidation of IMP to xanthosine monophosphate (XMP) (<xref ref-type="bibr" rid="bib42">Zimmermann et al., 1995</xref>; <xref ref-type="bibr" rid="bib37">Zhang et al., 1999</xref>). IMPDH2 is the predominant isoform among its two isoenzymes, and often highly expressed in proliferating cells and neoplastic tissues (<xref ref-type="bibr" rid="bib17">Ishitsuka et al., 2005</xref>; <xref ref-type="bibr" rid="bib43">Gu et al., 2003</xref>), correlated to drug resistance, and thus has been used as a validated target for immunosuppressive (mycophenolic acid [MPA] [<xref ref-type="bibr" rid="bib27">Sintchak et al., 1996</xref>] and mizoribine [<xref ref-type="bibr" rid="bib11">Gan et al., 2003</xref>]), antiviral (ribavirin [<xref ref-type="bibr" rid="bib25">Prosise et al., 2002</xref>]), and cancer-chemotherapeutic development [tiazofurin] (<xref ref-type="bibr" rid="bib21">Malek et al., 2004</xref>; <xref ref-type="bibr" rid="bib13">Gu et al., 2005</xref>; <xref ref-type="bibr" rid="bib3">Chen and Pankiewicz, 2007</xref>; <xref ref-type="bibr" rid="bib2">Borden and Culjkovic-Kraljacic, 2010</xref>).</p><p>Interestingly, by performing a biotin-INZ avidin affinity purification coupled with mass spectrometry (MS) analysis, we identified IMPDH2 as one of the top candidate proteins that INZ specifically targets in cancer cells. Biotinylated INZ analogs (<xref ref-type="fig" rid="fig1">Figure 1A</xref>) were synthesized for these analyses. Here, Biotin-INZ was as active as INZ (<xref ref-type="bibr" rid="bib45">Zhang et al., 2012b</xref>; <xref ref-type="bibr" rid="bib44">Zhang et al., 2012a</xref>), while Biotin-INZ (O) was inactive and thus used as a negative control. Comparison of the most abundant proteins based on normalized spectral abundance factor (NSAF) in the cells treated Biotin-INZ vs DMSO or Biotin-INZ (O) revealed the high enrichment of IMPDH2 proteins in the former (<xref ref-type="fig" rid="fig1">Figure 1B</xref>) with enriched IMPDH2 peptides shown in <xref ref-type="fig" rid="fig1">Figure 1C</xref>. This result was firmly validated by immunoblot (IB) analysis of the pulled down proteins, as IMPDH2 was specifically brought down with Biotin-INZ, as well as together with our previously identified SIRT1, but not Biotin-INZ (O) or other controls, in both H460 and HCT116 cells (<xref ref-type="fig" rid="fig1">Figure 1C</xref>).<fig id="fig1" position="float"><object-id pub-id-type="doi">10.7554/eLife.03077.003</object-id><label>Figure 1.</label><caption><title>Identification of IMPDH2 as a potential target of INZ.</title><p>(<bold>A</bold>) Structure of INZ analogs conjugated with Biotin (Biotin-INZ) used for INZ target identification, the oxygen-substituent (Biotin-INZ (O)) as a negative control. (<bold>B</bold>–<bold>C</bold>) Cells were treated with indicated compounds individually for 18 hr. Cleared cell lysates were incubated with NeutrAvidin beads and washed. The samples from HCT116 cells were then in-beads digested for MS analysis. NSAF: normalized spectral abundance factor. Samples were also resolved by SDS-PAGE and subjected to IB with indicated antibodies. (<bold>D</bold>–<bold>F</bold>) Knockdown of IMPDH2 alleviates INZ induction of p53. HCT116 cells were transfected with scrambled siRNA or IMPDH2 siRNA. 18 hr prior to harvesting, cells were treated with 2 µM INZ and harvested for IB with indicated antibodies (<bold>D</bold>). HCT116 and HCT116<sup>−/−</sup> cells were exposed to INZ for 72 hr and evaluated by WST cell growth assays (<bold>E</bold> and <bold>F</bold>). The IC<sub>50</sub> values of INZ in the scrambled siRNA and IMPDH2 siRNA transfected cells are 0.87 ± 1.08 µM and 10.24 ± 2.57 µM for HCT116 cells, and 5.28 ± 2.43 µM and 12.34 ± 2.02 µM for HCT116<sup>−/−</sup> cells, respectively (Mean ± SD, n = 3).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03077.003">http://dx.doi.org/10.7554/eLife.03077.003</ext-link></p></caption><graphic xlink:href="elife03077f001"/></fig></p><p>To test if IMPDH2 is required for INZ activation of p53, we knocked down IMPDH2 in HCT116 cells with specific siRNAs in the presence or absence of INZ. As shown in <xref ref-type="fig" rid="fig1">Figure 1D</xref>, knockdown of IMPDH2 impeded INZ-induced p53 activation as indicated by the reduction of INZ-induced p53, p21, MDM2, and Puma levels. Consistently, the growth inhibition by INZ was compromised as the IC<sub>50</sub> value for INZ in cell growth analysis decreased by almost ∼10-fold when IMPDH2 was knocked down (<xref ref-type="fig" rid="fig1">Figure 1E</xref>). Knockdown of IMPDH2 in HCT116 cells also conveyed much more significant effect on compromising the cytotoxicity of INZ compared to p53 null HCT116 (HCT116<sup>−/−</sup>) cells (<xref ref-type="fig" rid="fig1">Figure 1F</xref>), indicating that INZ suppresses cancer cell growth mainly by targeting IMPDH2 in the cells and consequently activating the p53 pathway.</p><p>Although Biotin-INZ was associated with cellular IMPDH2 (<xref ref-type="fig" rid="fig1">Figure 1</xref>), INZ did not appear to affect the activity of the purified enzyme (date not shown). This discrepancy could be due to differences between the recombinant IMPDH2 in vitro and its native form in cells, as the latter could be regulated via post-translational modifications or partner proteins in cells, or INZ might mimic a nucleoside and be phosphorylated by a kinase in cells to target IMPDH2. These results also suggest that INZ might not directly bind to the active site of this enzyme. These possibilities remain to be addressed in the future.</p></sec><sec id="s2-2"><title>INZ causes the depletion of nucleostemin and consequent ribosomal stress</title><p>Our previous study showed that inhibition of IMPDH2 activity by MPA leads to RS and consequent p53 activation by reducing the level of nucleostemin (NS) (<xref ref-type="bibr" rid="bib7">Dai et al., 2008</xref>; <xref ref-type="bibr" rid="bib20">Lo et al., 2012</xref>), a nucleolar GTP-binding protein important for rRNA processing (<xref ref-type="bibr" rid="bib31">Tsai and McKay, 2005</xref>; <xref ref-type="bibr" rid="bib20">Lo et al., 2012</xref>). The association of INZ with IMPDH2 suggested that INZ might have a similar effect. As shown in <xref ref-type="fig" rid="fig2">Figure 2A</xref>, INZ, but not INZ(O), indeed significantly reduced NS protein levels, which was inversely correlated with the INZ induction of p53, p21, MDM2 and cleaved PARP. This result was further confirmed by immunofluorescence staining, as INZ, but not INZ(O), led to apparent decrease of nucleolar NS (<xref ref-type="fig" rid="fig2">Figure 2B</xref>). This decrease was due to the reduction of NS's half-life from >10 hr to <6 hr, as shown in <xref ref-type="fig" rid="fig2">Figure 2C</xref>, but NS mRNA level did not alter (data not shown). This result, also repeated in HCT116 cell lines (data not shown), demonstrates that INZ can destabilize cellular NS.<fig id="fig2" position="float"><object-id pub-id-type="doi">10.7554/eLife.03077.004</object-id><label>Figure 2.</label><caption><title>INZ, not its inactive analog INZ (O), treatment, decreases NS expression and destabilizes NS protein.</title><p>H460 cells were treated with 2 μM INZ or its analogue INZ (O) for 20 hr. Cells were harvested and immunoblotted with p53, NS, MDM2, p21, cleaved PARP and β-actin (<bold>A</bold>), or immunostained with anti-NS (green) and anti-p53 (red) (<bold>B</bold>). (<bold>C</bold>) H460 cells were treated with 2 μM INZ for 9 hr before then 50 μg/ml of CHX was added. Cells were harvested at different time points as indicated and assayed for levels of NS.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03077.004">http://dx.doi.org/10.7554/eLife.03077.004</ext-link></p></caption><graphic xlink:href="elife03077f002"/></fig></p><p>Next, we tested if the depletion of NS by INZ could induce the interaction of the RPL11 and RPL5 with MDM2, because we previously showed that the reduction of NS by MPA could induce RS and activate p53 by enhancing this interaction (<xref ref-type="bibr" rid="bib29">Sun et al., 2008</xref>). As shown in <xref ref-type="fig" rid="fig3">Figure 3A</xref>, INZ, but not INZ(O), indeed enhanced the interaction of MDM2 with RPL5 and RPL11 in H460 cells by immunoprecipitation (IP) using anti-MDM2 antibodies followed by IB (<xref ref-type="fig" rid="fig3">Figure 3A</xref>). The increased binding of MDM2 to L11 was true in a reciprocal co-IP using anti-L11 antibodies (<xref ref-type="fig" rid="fig3">Figure 3A</xref>). This result indicates that INZ-induced p53 activation involves suppression of MDM2 activity by the RPs, further supporting the RS-p53 response of INZ-treated cells.<fig id="fig3" position="float"><object-id pub-id-type="doi">10.7554/eLife.03077.005</object-id><label>Figure 3.</label><caption><title>INZ treatment enhances the interaction of MDM2 with L5 and L11 by inducing ribosome-free form of RPL5 and RPL11.</title><p>(<bold>A</bold>) H460 cells were treated with 2 μM INZ or INZ (O) for 18 hr. Cell lysates were used for IP with anti-MDM2 antibodies or anti-L11 antibodies followed by IB using anti-RPL5, RPL11 or MDM2 antibodies. (<bold>B</bold> and <bold>C</bold>) HCT116 cells were transfected with siRNAs against RPL5 and RPL11, or control, for 48–72 hr and treated with 2 µM INZ 18 hr before harvesting, followed by IB using indicated antibodies or subG1 analysis by flow cytometry. (<bold>D</bold>) Ribosomal profile assay. Cytoplasmic extracts containing ribosomes from H460 cells treated with or without 2 µM INZ for 18 hr were subjected to a 10–50% linear sucrose gradient sedimentation centrifugation. Fractions were collected and subjected to IB with anti-RPL11, anti-RPL5, anti-p53, or anti-MDM2 antibodies. The distribution of ribosomes is indicated.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03077.005">http://dx.doi.org/10.7554/eLife.03077.005</ext-link></p></caption><graphic xlink:href="elife03077f003"/></fig></p><p>To determine if RPL5 and RPL11 are required for INZ activation of p53, we performed a knockdown experiment. As expected, reduction of either RPL5 or RPL11 (data not shown) or both levels by siRNA markedly inhibited INZ-induced p53 level, compared to that in scrambled siRNA-transfected cells (<xref ref-type="fig" rid="fig3">Figure 3B</xref>). Consistently, knocking down RPL5 and RPL11 abrogated INZ-induced p21 and MDM2 levels (<xref ref-type="fig" rid="fig3">Figure 3B</xref>) and apoptosis (<xref ref-type="fig" rid="fig3">Figure 3C</xref>), indicating that RPL5 and RPL11 are required for INZ-induced p53 activation and apoptosis. Also, ribosome profile analysis followed by IB revealed that INZ significantly increases the levels of ribosome-free RPL5 and RPL11 (fractions 1–10), whereas it markedly reduces the level of polysomes (fractions 37–51) (<xref ref-type="fig" rid="fig3">Figure 3D</xref>), suggesting that INZ could suppress ribosome biogenesis and possibly protein translation. All together, these results demonstrate that INZ can activate p53-dependent apoptosis by interfering with ribosome biogenesis through depletion of NS, causing RS, which then induces the release of ribosome-free RPL11 and RPL5 that bind to MDM2 and consequently inhibit its activity toward p53.</p></sec><sec id="s2-3"><title>INZ-mediated GTP depletion by targeting IMPDH2</title><p>Because Inhibition of IMPDH2 reduces cellular GTP level (<xref ref-type="bibr" rid="bib18">Ji et al., 2006</xref>), and INZ associates with cellular IMPDH2 and reduces nucleolar NS level, consequently causing RS and p53 activation (<xref ref-type="fig" rid="fig1 fig2 fig3">Figures 1–3</xref>), we then tested if this INZ effect on p53 could be suppressed by supplementing culture media with extra GTP or guanosine. As shown in <xref ref-type="fig" rid="fig4">Figure 4A–B</xref>, addition of either GTP or guanosine to cells significantly, though partially, alleviated the INZ induction of p53 level and activity as measured by IB analysis of p53, Puma and cleaved PARP. This result is well correlated with <xref ref-type="fig" rid="fig4">Figure 4C</xref>, showing that INZ markedly reduced the GTP level in H460 cells by 6.3-fold and in HCT116 cells by 3.7-fold, respectively, as measured by HPLC analysis (<xref ref-type="bibr" rid="bib9">Di Pierro et al., 1995</xref>). These results indicate that INZ can reduce cellular GTP level likely by inhibiting IMPDH2 in cells. Indeed, knockdown of IMPDH2 compromised the GTP depletion by INZ treatment in both p53 wild type and p53 null HCT116 cancer cells (<xref ref-type="fig" rid="fig4">Figure 4D</xref>). Since it has been shown that NS is very sensitive to cellular GTP level and low GTP level triggers NS re-localization from the nucleolus to the nucleoplasm, consequently destabilizing it (<xref ref-type="bibr" rid="bib31">Tsai and McKay, 2005</xref>; <xref ref-type="bibr" rid="bib20">Lo et al., 2012</xref>), these results also suggest that it must be by decreasing cellular GTP level that INZ causes NS degradation and consequent RS, leading to p53 activation (<xref ref-type="fig" rid="fig4">Figure 4E</xref>).<fig id="fig4" position="float"><object-id pub-id-type="doi">10.7554/eLife.03077.006</object-id><label>Figure 4.</label><caption><title>GTP or guanosine lessens INZ activation of p53 in cells.</title><p>(<bold>A</bold> and <bold>B</bold>) H460 cells were pretreated with GTP or guanosine for 2 hr before the addition of 2 μM INZ. Cells were harvested and followed by IB with indicated antibodies. (<bold>C</bold>) Effect of INZ on cellular GTP level. The nucleotides were extracted from H460 or HCT116 cells treated with 2 μM INZ by 80% acetonitrile and SPE column. Samples were subjected to GTP analysis by HPLC. Results of quantification of HPLC spectra presented in arbitrary units (AU) were presented in this Table. (<bold>D</bold>) HCT116<sup>−/−</sup> and HCT116 cells transfected IMPDH2 siRNA (SiIMPDH2) or scrambled siRNA (SiControl) were exposed to INZ for 18 hr, and cellular GTP was extracted, measured and quantitated by LC-MS/MS. Values represent means ±SD (n = 2). (<bold>E</bold>) A schematic diagram of the role of IMPDH2 in INZ-induced ribosomal stress (RS) and p53 activation. IMPDH2 is a rate-limiting enzyme in the de novo guanine nucleotide biosynthesis. INZ reduced the levels of cellular GTP and NS by targeting IMPDH2 (or its complex), resulting in RS that leads to the enhancement of the RPL11/RPL5-MDM2 interaction, consequently MDM2 inactivation and p53 activation.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03077.006">http://dx.doi.org/10.7554/eLife.03077.006</ext-link></p></caption><graphic xlink:href="elife03077f004"/></fig></p><p>Cancers are caused by alterations of multiple tumor-associated proteins or genes at the genetic and epigenetic levels (<xref ref-type="bibr" rid="bib14">Hanahan and Weinberg, 2011</xref>), including the p53 pathway (<xref ref-type="bibr" rid="bib34">Vousden and Prives, 2009</xref>). Thus, targeting multiple proteins of one or more signaling pathways in cancers is necessary for developing a more effective cancer therapy. Several individual SIRT1 or IMPDH2 inhibitors have been reported (<xref ref-type="bibr" rid="bib1">Alcain and Villalba, 2009</xref>; <xref ref-type="bibr" rid="bib5">Chen et al., 2010</xref>). However, dual targeting SIRT1 and IMPDH2 by INZ to activate p53 would offer the first paradigm for anti-cancer drug development. Our studies (<xref ref-type="fig" rid="fig1 fig2 fig3 fig4">Figures 1–4</xref>) together with previously published findings strongly suggest that INZ effectively activates p53 and suppresses tumor growth in a p53-dependent fashion by targeting SIRT1 and IMPDH2 (<xref ref-type="fig" rid="fig4">Figure 4E</xref>, (<xref ref-type="bibr" rid="bib45">Zhang et al., 2012b</xref>)). This dual targeting strategy could also explain why INZ can still partially activate p53 in IMPDH2 knockdown or GTP-supplemented cells (<xref ref-type="fig" rid="fig1">Figure 1D</xref> and <xref ref-type="fig" rid="fig4">Figure 4A,B</xref>), although the partial impairment of p53 induction could also be due to the inefficiency of completely knockdown IMPDH2 or the possible non-continuous availability of intracellular GTP throughout the experiment.</p></sec></sec><sec id="s3" sec-type="materials|methods"><title>Materials and methods</title><sec id="s3-1"><title>Cell culture, reagents and antibodies</title><p>Human lung carcinoma H460 and human colon cancer HCT116 were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (PBS), penicillin, and streptomycin. Inauhzin (INZ), Inauhzin inactive analogue INZ (O) (INZ9 in [<xref ref-type="bibr" rid="bib44">Zhang et al., 2012a</xref>]) and Biotinylated INZs were synthesized and characterized by NMR and LC-MS as described (<xref ref-type="bibr" rid="bib45">Zhang et al., 2012b</xref>). The purity of the compounds is higher than 90%. Mycophenolic acid (MPA) was purchased from Sigma-Aldrich (St.Louis, Missouri). Mouse monoclonal anti-p53 (DO-1), rabbit anti-p21 (M19), mouse anti-p21 (F5), rabbit anti-SIRT1 (H300) and goat anti-RPL11 (N17) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, Texas). for immunoblotting. Cleaved PARP, PARP (9542), Puma were from Cell Signaling Technologies. Mouse anti-MDM2 (2A10), rabbit anti-RPL11 and anti-RPL5 antibodies were described previously (<xref ref-type="bibr" rid="bib36">Zeng et al., 1999</xref>; <xref ref-type="bibr" rid="bib29">Sun et al., 2008</xref>). Antibodies for immunostaining were rabbit polyclonal anti-p53 (FL-393; Santa Cruz) and monoclonal nucleostemin antibodies (Chemicon, Billerica, Massachusetts).</p></sec><sec id="s3-2"><title>Biotin-avidin pull down assays</title><p>Cells were plated on 10 cm dishes and treated with compounds at about 60–70% confluence. Cells were harvested and lysed in the PBS buffer with 0.1% (wt/vol) NP40 (freshly adding protease inhibitors and 1 mM DTT). Incubate the cell lysate with 25 μL of NeutrAvidin Agarose beads (Thermo Scientific, Waltham, Massachusetts) (beads volume) in for 2 hr at 4°C with end-over-end mixing. Centrifuge at 13,000 rpm for 10 s at 4°C in a microcentrifuge. The beads were washed three times with 0.5% (wt/vol) NP-40, 0.2% (wt/vol) Tween20/Tris buffered saline and then subjected to on-beads digestion and mass spectrometry as shown below.</p></sec><sec id="s3-3"><title>On-bead Trypsin digestion</title><p>On-bead digestions were performed to release the proteins and resulting tryptic peptides from the NeutrAvidin beads. In brief, beads were resuspended in 100 μl of 50 mM ammonium bicarbonate pH 8.0 followed by the addition of 1 μg of Trypsin Gold (Promega, Madison, Wisconsin). The samples were then incubated at 37°C for 12 hr with shaking. Following digestion, samples were run through spin columns to remove any trace of the residual purification resin. The digestions were then quenched through the addition of 8 μL of formic acid.</p></sec><sec id="s3-4"><title>MudPIT analyses</title><p>Protein samples from cells that were MOCK treated with Biotin-INZ (O) or treated with DMSO or Biotin-INZ were pressure loaded onto three-phase MudPIT columns containing Aqua C18 and Luna SCX resins (Phenomenex, Torrance, California) as previously described (<xref ref-type="bibr" rid="bib22">Mosley et al., 2009</xref>, <xref ref-type="bibr" rid="bib23">2011</xref>). Ten-step MudPIT was performed using increasing concentrations of ammonium acetate to initiate each step followed by a 100-min gradient of 0–80% acetonitrile. All samples were analyzed on a LTQ Velos mass spectrometer (Thermo Scientific) with the dynamic exclusion set to 90 s. The spectra obtained through MudPIT analysis were searched through Proteome Discoverer 1.3 (Thermo Scientific) using SEQUEST as the peptide-spectrum matching algorithm against the Human NCBI 11-22-10 database containing 29,535 protein sequences. In addition to the human proteins, the database also contained ∼140 common contaminant sequences for proteins such as keratins, BSA, and proteolytic enzymes. Using Proteome Discoverer 1.3, all peptides were required to pass a 2% false discovery threshold. The number of spectra obtained for proteins found to interact with Biotin-INZ was compared to the levels of spectra for those same proteins observed in MOCK and DMSO treatments to ensure that the candidate interacting proteins are detected at levels higher than background.</p></sec><sec id="s3-5"><title>Immunoblotting</title><p>Cells were seeded in 6-well plates. All compounds were dissolved in DMSO and diluted directly into the medium to the indicated concentrations, and 0.1% DMSO was used as a control. After incubation with the compounds for the indicated times, cells were harvested and lysed in 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40 supplemented with 2 mM DTT and 1 mM PMSF. An equal amount of protein samples (50 μg) was subjected to SDS-PAGE and transferred to a PVDF membrane (PALL Life Science, Port Washington, New York). The membranes with transferred proteins were probed with primary antibodies followed by horseradish-peroxidase-conjugated secondary antibody (1:10,000; Pierce). The blots were then developed using an enhanced chemiluminescence detection kit (Thermo Scientific), and signals were visualized by Omega 12iC Molecular Image System (UltraLUM, Claremont, California).</p></sec><sec id="s3-6"><title>Immunofluorescence staining</title><p>H460 cells at 50–70% confluence were treated with 2 µM of Inauhzin (INZ) or Inauhzin (O) (INZ (O)) for 16 hr. Cells were fixed in 4% formaldehyde/PBS for 10 min, permeabilized and blocked with 0.3% Triton-100, 8%BSA/PBS. The primary antibodies used were monoclonal nucleostemin antibodies in 1:250 dilution and polyclonal p53 antibodies in 1: 500 dilution according to the manufactural instruction. Images were taken with a Zeiss Axiovert 200M fluorescent microscope (Germany).</p></sec><sec id="s3-7"><title>RNA interference</title><p>Control scrambled siRNA (Santa Cruz), or siRNA specific to IMPDH2 (Santa Cruz and Ambion, Grand Island, New York) were commercially purchased. These siRNAs (60 nM) were introduced into cells using METAFECTENE SI following the manufacturer's protocol (Biontex, Germany). Cells were treated with INZ for IB, cell viability assays and FACS analysis.</p></sec><sec id="s3-8"><title>Cell viability assay</title><p>To assess cell growth, the cell counting kit (Dojindo Molecular Technologies Inc., Rockville, Maryland) was used according to manufacturer's instructions. Cell suspensions were seeded at 5000 cells per well in 96-well culture plates and incubated overnight at 37°C. Compounds were added into the plates and incubated at 37°C for 72 hr. Cell growth inhibition was determined by adding WST-8 at a final concentration of 10% to each well, and the absorbance of the samples was measured at 450 nm using a Microplate Reader (Molecular Device, SpectraMax M5e (Sunnyvale, California)).</p></sec><sec id="s3-9"><title>FACS analysis</title><p>Cells were harvested, fixed in 70% ethanol overnight and analyzed by propidium iodide (PI) staining and flow cytometry (FACS Calibur, Becton Dickinson, Washington, DC) as previously described (<xref ref-type="bibr" rid="bib26">Riccardi and Nicoletti, 2006</xref>).</p></sec><sec id="s3-10"><title>Ribosomal profiling analysis</title><p>Cytosolic extractions, sucrose gradient sedimentation of polysomes, and analysis of the polysomes/mRNPs distribution of proteins were carried out as previously described (<xref ref-type="bibr" rid="bib28">Sun et al., 2007</xref>; <xref ref-type="bibr" rid="bib8">Dai et al., 2012</xref>; <xref ref-type="bibr" rid="bib16">Ingolia et al., 2012</xref>). Briefly, cells were incubated with 100 μg/ml of cycloheximide for 15 min. Cells were homogenized in polysome lysis buffer containing 30 mM Tris–HCl (pH 7.4), 10 mM MgCl<sub>2</sub>, 100 mM KCl, 0.3% NP-40, 100 μg/ml of cycloheximide, 30 units/ml RNasin inhibitor, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 0.25 μg/ml pepstatin A. After incubation on ice for 5 min, cell lysates were centrifuged at 1300×g at 4°C for 10 min. Supernatants were subjected to sedimentation centrifugation in a 10–50% sucrose gradient solution containing 30 mM Tris–HCl (pH 7.4), 10 mM MgCl<sub>2</sub>, 100 mM KCl in a Beckman SW41 rotor at 37,000 rpm at 4°C for 2 hr. Fractions were collected and absorbance of RNA at 254 nm was recorded using BR-188 Density Gradient Fractionation System (Brandel, Gaithersburg, Maryland) to analyze the distribution of polysomes and monosomes as described (<xref ref-type="bibr" rid="bib10">Esposito et al., 2010</xref>).</p></sec><sec id="s3-11"><title>Extraction and determination of cellular GTP</title><p>We adapted previously established whole cell assays using HPLC to determine cellular GTP level (<xref ref-type="bibr" rid="bib9">Di Pierro et al., 1995</xref>; <xref ref-type="bibr" rid="bib24">Nakajima et al., 2010</xref>). After 48 hr of growth, cells were treated with 2 μM INZ for 18 hr. Cellular nucleotides were extracted from cell monolayers by addition of ice-cold 80% acetonitrile for 1 hr. The extracts were centrifuged to pellet the cellular debris and the cleared supernatant was loaded to the SPE column (SAX column, Sigma–Aldrich). The elutes were analyzed by Agilent 1100 series liquid chromatograph system with a C18 reversed-phase column (Agilent Zorbax Extend-C18, 5 µM, 4.6 × 150 mm). A gradient elution from 0%B to 50%B in 70 min was used at a flow rate of 1 ml/min (solvent A: 0.05M KH<sub>2</sub>PO<sub>4</sub>, 0.005M tetrabutylammonium, pH5.5; B: 50% acetonitrile in 0.05M KH<sub>2</sub>PO<sub>4</sub>, 0.005M tetrabutylammonium, pH 7.0). The GTP level was also analyzed by a nanoACQUITY UPLC/Synapt HDMS mass spectrometer (Waters, Milford, Massachusetts) using acetonitrile/water (0.05M NH<sub>4</sub>Ac buffer solution at pH = 5.5) as the mobile phase with a flow rate of 0.5 μl/min.</p></sec></sec></body><back><sec sec-type="additional-information"><title>Additional information</title><fn-group content-type="competing-interest"><title>Competing interests</title><fn fn-type="conflict" id="conf1"><p>The authors declare that no competing interests exist.</p></fn></fn-group><fn-group content-type="author-contribution"><title>Author contributions</title><fn fn-type="con" id="con1"><p>QZ, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con2"><p>XZ, Conception and design, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con3"><p>RZW, Acquisition of data, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con4"><p>AM, Conception and design, Acquisition of data, Analysis and interpretation of data, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con5"><p>SXZ, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con6"><p>ZX, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con7"><p>HL, Conception and design, Analysis and interpretation of data, Drafting or revising the article</p></fn></fn-group></sec><ref-list><title>References</title><ref id="bib1"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Alcain</surname><given-names>FJ</given-names></name><name><surname>Villalba</surname><given-names>JM</given-names></name></person-group><year>2009</year><article-title>Sirtuin inhibitors</article-title><source>Expert Opinion on Therapeutic 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inosine-5'-monophosphate dehydrogenase type II gene</article-title><source>The Journal of Biological Chemistry</source><volume>270</volume><fpage>6808</fpage><lpage>6814</lpage><pub-id pub-id-type="doi">10.1074/jbc.270.41.23966</pub-id></element-citation></ref></ref-list></back><sub-article article-type="article-commentary" id="SA1"><front-stub><article-id pub-id-type="doi">10.7554/eLife.03077.007</article-id><title-group><article-title>Decision letter</article-title></title-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Prives</surname><given-names>Carol</given-names></name><role>Reviewing editor</role><aff><institution>Columbia University</institution>, <country>United States</country></aff></contrib></contrib-group></front-stub><body><boxed-text><p>eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see <ext-link ext-link-type="uri" xlink:href="http://elifesciences.org/review-process">review process</ext-link>). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.</p></boxed-text><p>Thank you for sending your work entitled “The Role of IMPDH2 in Inauhzin-induced Ribosomal Stress” for consideration at <italic>eLife</italic>. Your article has been favorably evaluated by James Manley (Senior editor) and 3 reviewers, one of whom is a member of our Board of Reviewing Editors.</p><p>The Reviewing editor and the other reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.</p><p>1) The authors previously showed that INZ inhibits SIRT1 and thereby activates p53. Now they provide a different mode by which this compound activates p53 and the authors do not try to consolidate these observations with their previous work showing INZ inhibits SIRT1, so we cannot assess the contribution of each activity to the end effect. When does INZ work via SIRT1 and when by inhibiting IMPDH2? Since both SIRT1 and IMPDH2 catalyze NAD+ dependent reactions is there perhaps a common mechanism by which this compound works in cells? The consequences of each function of INZ in terms of p53-independent effects are also not clear. It would be interesting to know whether INZ(O) has also lost SIRT1 inhibitory activity: could the two activities be separated using this compound?</p><p>2) What are the consequences of INZ interaction with IMPDH2? Where on IMPDH2 does INZ bind? Does it inhibit its enzymatic activity? Is that why the levels of GTP are so dramatically reduced in these cells? Are they similarly reduced in p53 null cell lines? What about normal non-transformed cells? The nature of the interaction between INZ and IMPDH2 remains unclear. Although INZ is implied in the manuscript to inhibit IMPDH2 activity, the RNAi mediated knockdown of IMPDH2 does not replicate the same effects on p53 as INZ treatment. The authors should address this uncertainty and extend the findings on the interaction between the inhibitor and IMPDH2.</p><p>More minor comments:</p><p><italic>Reviewer #1</italic>:</p><p><xref ref-type="fig" rid="fig1">Figure 1D</xref>: IMZ dramatically induces p53 and Mdm2 but only very modestly increases p21 expression in <xref ref-type="fig" rid="fig1">Figure 1</xref>. Is this because the massively increased Mdm2 is degrading p21 (as the authors have previously showed), or because the impact of INZ on p53 transcriptional program is selective? Have the authors performed a kinetic analysis to show the relative peak times for p53 proteins vs p21, Mdm2 and Puma RNA and protein induction?</p><p><xref ref-type="fig" rid="fig3">Figure 3B</xref>: Can the authors explain why there is more p21 upon siRNA knockdown of RPL11 and RPL5 in DMSO treated cells than in INZ treated cells with these siRNAs while the opposite is seen with Mdm2 protein under the same conditions?</p><p><xref ref-type="fig" rid="fig4">Figure 4</xref> A: The data with GTP and guanosine are a bit strange in that phosphorylated nucleotides are generally not taken up by intact cells. What were the concentrations of GTP and guanosine these experiments and can authors show that adding GTP to cells increased the intracellular levels of GTP?</p><p>Only 1 siRNA is used for each knockdown in all the experiments. They need to show more siRNAs to rule out off-target effects.</p><p><xref ref-type="fig" rid="fig3">Figure 3</xref>: I assume <xref ref-type="fig" rid="fig3">Figure 3E</xref> should be labeled <xref ref-type="fig" rid="fig3">Figure 3D</xref>? I would recommend showing Figure E (D?) as the first panel in this experiment to set the stage for the IP shown in 3A and B.</p><p><italic>Reviewer #2</italic>:</p><p>Citations should not be used in the Abstract.</p><p>Consistent denotation is needed for genes/proteins. For example TP53/p53.</p><p><xref ref-type="fig" rid="fig1">Figure 1</xref>:</p><p>Legend: GRP78 is mentioned in the Figure Legend but the experiment described is actually a knockdown of IMPDH2.</p><p>1C: Treatment of H460 cells with INZ appears to decrease IMPDH2 expression whereas treatment of HCT116 cells with INZ appears to have no effect on IMPDH2 expression in <xref ref-type="fig" rid="fig1">Figures 1C and D</xref>. According to the model in <xref ref-type="fig" rid="fig4">Figure 4</xref>, p53 is activated by a decrease in GTP mediated by decreased IMPDH2 activity; however, a decrease in IMPDH2 expression and correspondingly its activity has no observable effect on p53 activation in the absence of INZ.</p><p>1E: The Introduction states that IMPDH2 is often highly expressed in rapidly proliferating cell populations; yet, siRNA mediated knockdown of IMPDH2 causes an increase in cell proliferation.</p><p><xref ref-type="fig" rid="fig2">Figure 2</xref>:</p><p>2C: The graphical representation of the half-life assay should be presented in log form so that the decrease in expression is linear and the slope can be more easily compared. Furthermore, the x-axis should be labeled in a manner that is proportional to the time between each point; for example, the distance between 2 and 4 should not be equal to that between 9 and 24.</p><p><xref ref-type="fig" rid="fig3">Figure 3</xref>:</p><p>3C: Statistical significance should be added to the error bars if possible.</p><p><xref ref-type="fig" rid="fig4">Figure 4</xref>:</p><p>4B: Again, if statistically significant the rescue by GTP/Guanosine treatment from vehicle should be denoted as such to strengthen the data.</p><p>4C: As a possible explanation for the lack of p53 activation in response to siIMPDH2 treatment this could be added to the GTP concentration assay to determine whether partial IMPDH2 knockdown has a comparable effect on GTP levels as INZ treatment.</p><p><italic>Reviewer #3</italic>:</p><p>1) What is the effect if IMPDH2 depletion on the growth of cells without INZ treatment (<xref ref-type="fig" rid="fig1">Figure 1E</xref>)? Are all the growth effects p53 dependent?</p><p>2) It is difficult to conclude anything about the effect of INZ on Mdm2-L11 binding in <xref ref-type="fig" rid="fig3">Figure 3A</xref> since the input levels of Mdm2 are so different.</p></body></sub-article><sub-article article-type="reply" id="SA2"><front-stub><article-id pub-id-type="doi">10.7554/eLife.03077.008</article-id><title-group><article-title>Author response</article-title></title-group></front-stub><body><p>In response to the comments and suggestions by the reviewers, we have performed a substantial amount of experiments and added 2 new panels (<xref ref-type="fig" rid="fig1 fig4">Figures 1F and 4D</xref>) and 7 modified panel (<xref ref-type="fig" rid="fig1 fig2 fig3 fig4">Figures 1C, 1D, 1E, 2C, 3A, 4B, 4E</xref>) as well as corresponding modified main text. More new results have also been obtained to address each of the reviewers’ comments. Of note, because of his help in detecting cellular GTP levels, Ruizhi Wu was added into our manuscript as a co-author.</p><p><italic>1) The authors previously showed that INZ inhibits SIRT1 and thereby activates p53. Now they provide a different mode by which this compound activates p53 and the authors do not try to consolidate these observations with their previous work showing INZ inhibits SIRT1, so we cannot assess the contribution of each activity to the end effect. When does INZ work via SIRT1 and when by inhibiting IMPDH2? Since both SIRT1 and IMPDH2 catalyze NAD+ dependent reactions is there perhaps a common mechanism by which this compound works in cells? The consequences of each function of INZ in terms of p53-independent effects are also not clear</italic>. <italic>It would be interesting to know whether INZ(O) has also lost SIRT1 inhibitory activity: could the two activities be separated using this compound?</italic></p><p>A) Our previous studies strongly demonstrate that INZ targets the SIRT1-p53 pathway (<xref ref-type="bibr" rid="bib45">Zhang et al 2012b</xref>). In this current study, we mainly focus on a new target of INZ, which is the ribosomal stress (RS)-p53 pathway by inhibiting IMPDH2 in cancer cells. It is possible that INZ inhibits SIRT1 and IMPDH2 in a similar fashion, since both of the enzymes utilize NAD as a cofactor. To prove that INZ can target both pathways by inhibiting SIRT1 and IMPDH2 to achieve maximum cell growth inhibition, we performed INZ dose response experiments in HCT116 cells with double knockdown of SIRT1 and IMPDH2. As shown in <xref ref-type="fig" rid="fig5">Author response image 1</xref>, co-depletion of SIRT1 and IMPDH2 displayed an obvious combination effect on suppression of INZ-induced cytotoxicity, compared to single knockdown of either SIRT1 or IMPDH2, as their IC<sub>50</sub> values increased at least 2 fold, from 3.77 to 10.57μM, and 5.20 to 10.57μM, respectively.<fig id="fig5" position="float"><label>Author response image 1.</label><caption><p>Combination effect of co-depletion of IMPDH2 and SIRT1 on INZ induced cell death. HCT116 cells, transfected with scrambled siRNA (SiControl), IMPDH2 siRNA (SiIMPDH2), SIRT1 siRNA (SiSIRT1) or co-transfected with IMPDH2 and SIRT1 siRNA, were treated with different doses of INZ and cell viability were assessed by WST cell growth assays. IC<sub>50</sub> values are represented as mean ± standard deviation (n=3).</p></caption><graphic xlink:href="elife03077f005"/></fig></p><p>B) It had been suggested that INZ might possess p53 independent effects, for example, INZ promotes cell death in the absence of p53 at high concentrations ((<xref ref-type="bibr" rid="bib19">Liao et al 2012</xref>, <xref ref-type="bibr" rid="bib44">Zhang et al 2012a</xref>), and data not shown). This could be due to other SIRT1's substrates like p73 as we showed in our previous studies. Although there is the existence of other potential protein targets (that are associated with SIRT1 and IMPDH2) for INZ, our results clearly show that this compound at lower doses specifically triggers p53-dependent apoptosis and suppression of cell proliferation in both cultured and xenograft tumors (<xref ref-type="bibr" rid="bib44">Zhang et al 2012a</xref>, <xref ref-type="bibr" rid="bib45">Zhang et al 2012b</xref>, <xref ref-type="bibr" rid="bib46">Zhang et al 2012c</xref>, <xref ref-type="bibr" rid="bib47">Zhang et al 2013</xref>) and data not shown). Indeed, knockdown of IMPDH2 by siRNA conveyed a much more significant effect on compromising the cytotoxicity of INZ in p53 wild type than in p53 null cancer cells (<xref ref-type="fig" rid="fig1">Figure 1E and 1F</xref>), indicating that the suppression of cell growth by INZ is mainly through the inhibition of IMPDH2 and activation of the p53 pathway.</p><p>C) We used Biotin-INZ (O) for in vitro pull down assay as a negative control to identify additional potential proteins that INZ specifically targets in cancer cells. As compared to the cells treated with Biotin-INZ (O), some specific proteins were pulled down with Biotin-INZ. MS analysis revealed one of them as our previously identified SIRT1 (<xref ref-type="bibr" rid="bib45">Zhang et al 2012b</xref>) and another as IMPDH2 (<xref ref-type="fig" rid="fig2">Figure 2B</xref>), which indicated SIRT1 and IMPDH2 are associated with INZ but not with INZ (O). This was confirmed by immunoblot analysis from both H460 and HCT116 cells (<xref ref-type="fig" rid="fig1">Figure 1</xref>; data not shown). We have modified the text and updated <xref ref-type="fig" rid="fig1">Figure 1C</xref> accordingly.</p><p><italic>2) What are the consequences of INZ interaction with IMPDH2? Where on IMPDH2 does INZ bind? Does it inhibit its enzymatic activity? Is that why the levels of GTP are so dramatically reduced in these cells? Are they similarly reduced in p53 null cell lines? What about normal non-transformed cells? The nature of the interaction between INZ and IMPDH2 remains unclear. Although INZ is implied in the manuscript to inhibit IMPDH2 activity, the RNAi mediated knockdown of IMPDH2 does not replicate the same effects on p53 as INZ treatment. The authors should address this uncertainty and extend the findings on the interaction between the inhibitor and IMPDH2</italic>.</p><p>A) We performed a set of experiments to determine the effect of INZ on IMPDH2 in vitro (<xref ref-type="fig" rid="fig6">Author response image 2</xref>). Indeed, INZ did directly bind to purified IMPDH2 in biotin-avidin pull down assays (<xref ref-type="fig" rid="fig6">Author response image 2C-D</xref>). Although we could pull-down IMPDH2 from cell extracts and observed the direct binding between IMPDH2 and INZ using purified proteins, INZ did not affect IMPDH2 activity using the purified enzyme (<xref ref-type="fig" rid="fig7">Author response image 3B</xref>). This discrepancy could be due to differences between the native state of IMPDH2 inside cells and the purified recombinant protein, as the former could include post-translational modifications and/or partner proteins. Another possibility is that as the chemical structure of INZ mimics a nucleoside, it could be phosphorylated by a kinase and the phosphorylated form directly inhibits the activity of IMPDH2 in vivo, as a number of nucleosideanalogues (e.g. ribavirin, mizoribine) are known to inhibit IMPDH2 after being monophosphorylated by cellular kinases (Leyssen et al 2005, Stuyver et al 2002a, Stuyver et al 2002b). Our results also suggest that INZ might not directly bind to the active site of the enzyme. Currently, we have found some interesting proteins pulled down by INZ are associated with IMPDH2 via proteomics analyses and biochemical and cell-based assays, and we have also identified the metabolites of INZ by LC-MS/MS. It is possible these metabolites of INZ and newly identified IMPDH2-binding proteins might play a role in the regulation of IMPDH2 activity by INZ. Apparently, the mechanism underlying INZ inhibition of IMPDH2 is more complex than that for INZ inhibition of SIRT1. However, studying this complex possibility would take us much longer time and generate substantial amounts of data that could be useful for another independent manuscript. Therefore, we hope that this reviewer would allow us to put these studies into our future manuscripts.<fig id="fig6" position="float"><label>Author response image 2.</label><caption><p>INZ binds to IMPDH2, but does not inhibit enzyme activity <italic>in vitro</italic>. (A) Recombinant IMPDH2 produced in E. coli BL21-CodonPlus (DE3)-RIPL, and purified through Ni-His columns (Lane 1) followed by TEV cleavage (Lane 2) as described in the experimental procedure. The purity of IMPDH2 enzyme (Lane 3) is confirmed before every assay by SDS-PAGE. (B) <italic>In vitro</italic> enzyme activity assays were conducted as described in the Supplementary Information. Mycophenolic Acid (MPA) is used as a positive control for IMPDH2 inhibition. The curve fitting and IC<sub>50</sub> determination of INZ were performed using Igor Pro 4.01A. (C) Purified IMPDH2 was incubated at indicated concentrations with Biotin or Biotin-INZ that was conjugated with avidin beads. The bound IMPDH2 to Biotin-INZ was analyzed using IB with NeutrAvidin Protein-Horseradish Peroxidase Conjugated (Avidin-HRP, 1:1000; Pierce) and anti-IMPDH2 antibodies. (D) Purified IMPDH2 was incubated at indicated concentrations with Biotin-INZ or Biotin overnight at 4°C. After incubation, each mixture was subjected to Native-PAGE analysis, followed by blotting to PVDF. The blot was probed with Avidin-HRP and anti-IMPDH2 antibodies.</p></caption><graphic xlink:href="elife03077f006"/></fig></p><p>B) The cellular GTP level is also depleted by 60% in p53 null cells with INZ treatment (<xref ref-type="fig" rid="fig4">Figure 4D</xref>); however, the decrease is modest compared to the over 90% depletion in the p53 wild type cells with INZ treatment. Knockdown of IMPDH2 could compromise INZ effect on the GTP levels in both p53 wild type and null cancer cells. We did not test INZ in normal cells because INZ is much less toxic to normal cells even though they contain WT p53 (<xref ref-type="bibr" rid="bib44">Zhang et al 2012a</xref>, <xref ref-type="bibr" rid="bib45">Zhang et al 2012b</xref>)</p><p>C) The partial knockdown of IMPDH2 by siRNA and the multiple protein targets of INZ, such as SIRT1 and IMPDH2, could explain why knockdown of only IMPDH2 by siRNA does not replicate the same effects on p53 as with INZ treatment. We have repeated siRNA knockdown experiments using another specific siRNA against IMPDH2. The new immunoblots are included in the revised version (<xref ref-type="fig" rid="fig1">Figure 1D</xref>). Although the efficiency of knockdown of IMPDH2 is better, the effect on p53 activation by knockdown of IMPDH2 is still not comparable to INZ treatment, indicating INZ targets more than one protein besides IMPDH2.</p><p><italic>More minor comments</italic>:</p><p>Reviewer #1:</p><p><xref ref-type="fig" rid="fig1"><italic>Figure 1D</italic></xref><italic>: IMZ dramatically induces p53 and Mdm2 but only very modestly increases p21 expression in</italic> <xref ref-type="fig" rid="fig1"><italic>Figure 1</italic></xref><italic>. Is this because the massively increased Mdm2 is degrading p21 (as the authors have previously showed), or because the impact of INZ on p53 transcriptional program is selective? Have the authors performed a kinetic analysis to show the relative peak times for p53 proteins vs p21</italic>, <italic>Mdm2 and Puma RNA and protein induction?</italic></p><p>In our previous studies, we had shown INZ induced p53 level and transcriptional activity, in a dose and time-dependent fashion (<xref ref-type="bibr" rid="bib45">Zhang et al 2012b</xref>). The p21 expression rises significantly within 3 h, while p53 expression is noticed only after 6h from the treatment in H460 cell line. This might partially be due to p73 activation by INZ inhibition of SIRT1. Another possibility would be that p53 activity might be activated earlier than the increase of p53 steady state level in response to INZ treatment, leading to the earlier expression of p21. The time point for <xref ref-type="fig" rid="fig1">Figure 1D</xref> is 18h, and the MDM2 expression level is very high at that time point, and consequently, MDM2 mediates p21 degradation decreasing the level of p21. Also, please see our response to point 2 as indicated below.</p><p><xref ref-type="fig" rid="fig3"><italic>Figure 3B</italic></xref><italic>: Can the authors explain why there is more p21 upon siRNA knockdown of RPL11 and RPL5 in DMSO treated cells than in INZ treated cells with these siRNAs while the opposite is seen with Mdm2 protein under the same conditions</italic>?</p><p>In DMSO treated cells, the elevation of p21 level is not through transcriptional activation, because the p53 level is reduced upon RP knockdown. Therefore, the p21 expression in these cells may be regulated at the post-translational level. Many mechanisms are responsible for the regulation of p21 protein level; for example, our group and others found that MDM2 could degrade p21 independently of p53 (Jin et al 2003, Zhang et al 2004), which may partially explain the reason why p21 level in lane 2 is lower than lane 1. However, we cannot rule out other possibilities, as RPs are essential proteins, and knocking down any of them is potentially able to cause other defects. These experiments are basically designed to test whether RPs are required for INZ-induced activation of the p53 pathway. Thus we assessed the expression of two typical p53 target genes, p21 and MDM2, which indeed demonstrates that RPs are required for INZ-induced p53 activation.</p><p><xref ref-type="fig" rid="fig4"><italic>Figure 4</italic></xref> <italic>A: The data with GTP and guanosine are a bit strange in that phosphorylated nucleotides are generally not taken up by intact cells. What were the concentrations of GTP and guanosine these experiments and can authors show that adding GTP to cells increased the intracellular levels of GTP?</italic> We appreciate the reviewer’s comment.</p><p>The addition of exogenous GTP or guanosine has been used to increase intracellular GTP level (<xref ref-type="bibr" rid="bib7">Dai et al 2008</xref>, <xref ref-type="bibr" rid="bib20">Lo et al 2012</xref>, Meshkini et al 2011). The evidence that exogenous phosphorylated nucleotides can be actively transported across the cell membrane of intact cells is indicated in the formation of phosphoproteins inside the cells (Amir-Zaltsman and Salomon 1989, Guo et al 1999, Piacentini and Niroomand 1996). For example, in labeling experiments, cells are initially placed in a specific medium designed to deplete the nucleotide pools, followed by addition of radioactive labeled 32P-ATP or GTP (or other nucleotide), leading to labeling of intracellular DNA or proteins. The mechanism is associated with the active transport by transporters of ATP or GTP located on the cell membranes (Amir-Zaltsman and Salomon 1989, Lelong et al 1992, Lelong et al 1994, Piacentini and Niroomand 1996, Wieland et al 1993).</p><p>To demonstrate that this is true to this reviewer, we also confirmed the uptake of GTP by cells using LC-MS. Cells were incubated with 100 μM GTP, and at the time point of 2hr and 4hr, the cells were thoroughly washed, and GTP was extracted, identified and quantitated by LC-MS. As shown in the figure below (<xref ref-type="fig" rid="fig7">Author response image 3</xref>), the concentration of cellular GTP level reached the level of greater than 80 μM 4 hrs after incubation with 100 μM of GTP.<fig id="fig7" position="float"><label>Author response image 3.</label><graphic xlink:href="elife03077f007"/></fig></p><p><italic>Only 1 siRNA is used for each knockdown in all the experiments. They need to show more siRNAs to rule out off-target effects</italic>.</p><p>We appreciate the reviewer’s suggestion.</p><p>Additionally, we have purchased two different siRNA of IMPDH2 from Ambion, and repeated the knockdown experiment. The new immunoblots are included in the revised version (<xref ref-type="fig" rid="fig1">Figure 1D</xref> and data not shown) indicating consistent results.</p><p>Based on our experience, the siRNAs used against RPL5 and RPL11 in the current study are specific for the knockdown of these two RPs. Please also refer to our previous publications (Jin et al 2003, <xref ref-type="bibr" rid="bib19">Liao et al 2013</xref>, Zhou et al 2013).</p><p><xref ref-type="fig" rid="fig3"><italic>Figure 3</italic></xref><italic>: I assume</italic> <xref ref-type="fig" rid="fig3"><italic>Figure 3E</italic></xref> <italic>should be labeled</italic> <xref ref-type="fig" rid="fig3"><italic>Figure 3D</italic></xref><italic>? I would recommend showing</italic> <italic>Figure E</italic> <italic>(D?) as the first panel in this experiment to set the stage for the IP shown in 3A and B</italic>.</p><p>We appreciate the suggestion. We have reorganized the figure panels in <xref ref-type="fig" rid="fig3">Figure 3</xref>.</p><p>Reviewer #2:</p><p><italic>Citations should not be used in the Abstract</italic>.</p><p>Thanks, we have corrected it.</p><p><italic>Consistent denotation is needed for genes/proteins. For example TP53/p53</italic>.</p><p>Thanks, we have corrected it.</p><p><xref ref-type="fig" rid="fig1"><italic>Figure 1</italic></xref><italic>:</italic></p><p><italic>Legend: GRP78 is mentioned in the Figure Legend but the experiment described is actually a knockdown of IMPDH2</italic>.</p><p>Thanks, we have corrected it.</p><p><italic>1C: Treatment of H460 cells with INZ appears to decrease IMPDH2 expression whereas treatment of HCT116 cells with INZ appears to have no effect on IMPDH2 expression in</italic> <xref ref-type="fig" rid="fig1"><italic>Figures 1C and D</italic></xref><italic>. According to the model in</italic> <xref ref-type="fig" rid="fig4"><italic>Figure 4</italic></xref><italic>, p53 is activated by a decrease in GTP mediated by decreased IMPDH2 activity; however, a decrease in IMPDH2 expression and correspondingly its activity has no observable effect on p53 activation in the absence of INZ</italic>.</p><p>We also observed the decrease of IMPDH2 level by INZ treatment in H460 cells but not in HCT116 cells. This is probably a cell-specific event. The depletion of IMPDH2 by siRNA is not as great as in the INZ treatment. One reason might be the low efficiency of siRNA, with the siRNA able to partially knockdown IMPDH2. Another reason might be the multiple targets of INZ besides IMPDH2. We also repeated the experiment using two additional siRNA against IMPDH2 from different vendors. Our results clearly showed that the knockdown of IMPDH2 activates p53 in HCT116 cells, although not as great as in the INZ treatment. We also determined the GTP level in IMPDH2 knockdown cells. It is obvious that the knockdown of IMPDH2 reduced GTP levels significantly in both wild type and null p53 cells. However, it was not comparable to INZ treatment, probably because depletion of IMDPH2 expression cannot activate p53 as much as INZ.</p><p><italic>1E: The Introduction states that IMPDH2 is often highly expressed in rapidly proliferating cell populations; yet, siRNA mediated knockdown of IMPDH2 causes an increase in cell proliferation</italic>.</p><p>Thank you for the comment. It is possible that the scramble siRNA had side effects on cell growth somehow. We repeated the experiments using a different scramble siRNA as control. Indeed, IMPDH2 knockdown inhibited cell growth of cancer cells, as shown in the new figure panel <xref ref-type="fig" rid="fig1">Figure 1E</xref> (the first point indicates the absence of INZ treatment).</p><p><xref ref-type="fig" rid="fig2"><italic>Figure 2</italic></xref></p><p><italic>2C: The graphical representation of the half-life assay should be presented in log form so that the decrease in expression is linear and the slope can be more easily compared. Furthermore, the x-axis should be labeled in a manner that is proportional to the time between each point; for example, the distance between 2 and 4 should not be equal to that between 9 and 24</italic>.</p><p>Thanks for the suggestion. We have done so as suggested.</p><p><xref ref-type="fig" rid="fig3"><italic>Figure 3</italic></xref><italic>:</italic></p><p><italic>3C: Statistical significance should be added to the error bars if possible</italic>.</p><p>Thanks for the suggestion. We have added P values and connecting lines for the relevant comparisons in figures throughout the paper.</p><p><xref ref-type="fig" rid="fig4"><italic>Figure 4</italic></xref><italic>:</italic></p><p><italic>4B: Again, if statistically significant the rescue by GTP/Guanosine treatment from vehicle should be denoted as such to strengthen the data</italic>.</p><p>Thanks. We have done so as suggested.</p><p><italic>4C As a possible explanation for the lack of p53 activation in response to siIMPDH2 treatment this could be added to the GTP concentration assay to determine whether partial IMPDH2 knockdown has a comparable effect on GTP levels as INZ treatment</italic>.</p><p>We appreciate the reviewer’s suggestion. We performed the new experiment as suggested. Knockdown of IMPDH2 significantly reduced the level of cellular GTP, however, not as strong as INZ treatment (<xref ref-type="fig" rid="fig4">Figure 4D</xref>). This is probably one of the reasons that IMPDH2 siRNA could not reach the same effect as INZ in p53 activation.</p><p>Reviewer #3:</p><p><italic>1) What is the effect if IMPDH2 depletion on the growth of cells without INZ treatment (</italic><xref ref-type="fig" rid="fig1"><italic>Figure 1E</italic></xref><italic>)? Are all the growth effects p53 dependent</italic>?</p><p>The depletion of IMPDH2 mediated by siRNA decreased GTP level and inhibited cell growth (<xref ref-type="fig" rid="fig1">Figure 1E</xref>; of note, the first point indicates the absence of INZ treatment and <xref ref-type="fig" rid="fig4">Figure 4D</xref>). The depletion of cellular GTP level and the growth inhibition by IMPDH2 depletion is much more modest in p53 null cells compared to p53 wild type cells. Please also see the above points for more details.</p><p><italic>2) It is difficult to conclude anything about the effect of INZ on Mdm2-L11 binding in</italic> <xref ref-type="fig" rid="fig3"><italic>Figure 3A</italic></xref> <italic>since the input levels of Mdm2 are so different</italic>.</p><p>Thanks for the comment. Unfortunately, we failed to equalize the amount of MDM2 level with the addition of MG132. We then performed a reciprocal co-immunoprecipitation experiment using L11 antibody. Anti-L11 antibodies pulled down same amount of L11 in all samples, but much more MDM2 was co-immunoprecipitated with L11 antibodies in INZ treated samples (<xref ref-type="fig" rid="fig3">Figure 3A</xref>).</p></body></sub-article></article> |