Undermining ribosomal RNA transcription in both the nucleolus and mitochondrion: an offbeat approach to target MYC-driven cancer

The MYC transcription factor coordinates, via different RNA polymerases, the transcription of both ribosomal RNA (rRNA) and protein genes necessary for nucleolar as well as mitochondrial ribogenesis. In this study we tested if MYC-coordination of rRNA transcription in the nucleolus and in the mitochondrion drives (cancer) cell proliferation. Here we show that the anti-proliferative effect of CX-5461, a Pol I inhibitor of rRNA transcription, in ovarian (cancer) cell contexts characterized by MYC overexpression is enhanced either by 2’-C-Methyl Adenosine (2’-C-MeA), a ribonucleoside that inhibits POLRMT mitochondrial rRNA (mt-rRNA) transcription and doxycycline, a tetracycline known to affect mitochondrial translation. Thus, hindering not only mt-rRNA transcription, but also mitoribosome function in MYC-overexpressing ovarian (cancer) cells, potentiates the antiproliferative effect of CX-5461. Targeting MYC-regulated rRNA transcription and ribogenesis in both the nucleolus and mitochondrion seems to be a novel approach worth of consideration for treating MYC-driven cancer.


INTRODUCTION
The nucleolus is the major site of ribosomal RNA (rRNA) synthesis. Moreover, the mitochondrion is another site of rRNA synthesis. The MYC transcription factor regulates genes encoding proteins necessary for both nucleolar and mitochondrial rRNA transcription. MYC enables RNA Polymerase I (Pol I)-mediated nucleolar rRNA transcription by binding directly the rDNA promoter to induce chromatin modifications that enhance Pol I accessibility and facilitate the assembly of the Pol I transcription machinery [1][2][3][4]. MYC also controls RNA Polymerase II (Pol II)-mediated transcription of "Pol I regulon" genes encoding for basal components of the Pol I transcription machinery, including RRN3, UBTF, the SL1 complex component TAF1C, and the Pol I subunits POLR1B and POLR1E [5,6]. In this way, MYC regulates the availability of nucleolar "Pol I regulon" factors necessary to activate rDNA genes [6]. As far as mitochondrial rRNA transcription is concerned, MYC regulates, via Pol II, the transcription of both the mitochondrial RNA polymerase POLRMT [7] and the mitochondrial transcription factor TFAM [8]. Both POLRMT and TFAM are necessary for transcription of mitochondrial DNA (mtDNA) into mitochondrial 12S and 16S rRNA (mt-rRNA) [9]. Moreover, MYC via Pol II controls the transcription of several proteins required for mitoribogenesis [10]. Hence the hypothesis that targeting both nucleolar and mitochondrial rRNA synthesis in MYC-overexpressing cancer can have a potential antiproliferative value.
In this study we provide evidence that MYCinduced rRNA transcription both in the nucleolus and mitochondrion concur to promote the proliferation of MYC-overexpressing cancer cells. Consistently, MYC overexpression sensitizes cells to the antiproliferative action of drugs targeting on one hand nucleolar Pol I-mediated rRNA transcription (e.g. CX-5461), and on the other hand, either POLRMT-mediated mitochondrial rRNA transcription (e.g. 2'-C-MeA) or mitoribosome function (e.g. doxycycline). Moreover, both 2'-C-MeA and doxycycline, when combined with CX-5461, induce a more effective antiproliferative action on MYCoverexpressing cells. Overall, targeting MYC-regulated rRNA transcription and ribogenesis in two distinct cell sites is an unconventional approach worth of consideration for treating MYC-driven cancer.

Evidence of MYC-induced proliferation consequent to upregulation of both "Pol I regulon" factors and rRNA transcription in human HFF fibroblasts
MYC is known to promote nucleolar rRNA transcription through direct activation of the Pol I transcription machinery [2][3][4] as well as activation of Pol II-mediated transcription of MYC-target genes encoding "Pol I regulon" factors, including RRN3, UBTF, TAF1C, and the Pol I subunits POLR1B and POLR1E [5,6]. To start to assess the proliferation effects of MYC overexpression due to both Pol II-mediated transcription of "Pol I regulon" factors and Pol I-mediated rRNA transcription, we used a well-characterized model of MYC-overexpressing human fibroblasts (HFF-MYC) [3,28,29].
MYC overexpression significantly promoted HFF-MYC cell proliferation, which was evaluated both by both MTT assay ( Figure 1E, left) and incorporation of 5-ethynyl-2′-deoxyuridine (EdU) into nascent DNA of cells in S phase ( Figure 1E, middle and right). To further assess if upregulation of rRNA transcription contributes to MYC-induced cell proliferation, we treated cells with the Pol I inhibitor CX-5461, which prevents the formation of the Pol I pre-initiation complex [17]. Treatment with CX-5461 (100 nM) significantly counteracted MYC-induced rRNA transcription (assessed as pre-rRNA) in HFF-MYC cells ( Figure 1F, red bars). CX-5461 also counteracted HFF-MYC proliferation in a dose-dependent manner ( Figure 1G, left, red bars). To a lesser extent, CX-5461 inhibited rRNA transcription ( Figure 1F, blue bars) and, consequently, proliferation ( Figure 1G, left, blue bars) also in the HFF-Ctrl normal cell context. However, when HFF-MYC and HFF-Ctrl proliferation data were normalized to their respective untreated controls, HFF-MYC showed significantly higher sensitivity to the anti-proliferative action of CX-5461 relative to normal HFF-Ctrl ( Figure 1G, right).
Overall, these preliminary results in a human fibroblast cell context indicate that MYC-induced proliferation can be traced to increased rRNA transcription due to: a) MYC-induced direct activation of Pol I, and b) MYC-induced upregulation of "Pol I regulon" factors. Moreover, MYC overexpression sensitizes HFF cells to the anti-proliferative action of the Pol I inhibitor CX-5461.

Increased proliferation consequent to upregulation of both "Pol I regulon" genes and rRNA transcription in MYC-overexpressing human ovarian epithelial cells
According to The Cancer Genome Atlas, MYC amplification/upregulation is frequent in cancer, including ovarian cancer, where it is detected in over 30% of cases [30]. For this reason, we tested if MYC overexpression in ovarian cancer induces Pol I-mediated rRNA transcription as well as Pol II-mediated transcription of "Pol I regulon" genes. As shown here, CAOV4 ovarian cancer cells, which overexpress MYC (Figure 2A), display increased levels of rRNA transcription (assessed as pre-rRNA) ( Figure 2B) and upregulation of "Pol I regulon" genes ( Figure 2C) relative to CAOV3 ovarian cancer cells, which do not overexpress MYC.
Next, we set out to test the effects of different levels of MYC overexpression on nucleolar rRNA transcription in IOSE and HOSE human ovarian epithelial cells. Using telomerase-immortalized IOSE cells [31] we developed cells with increasing expression levels of exogenous MYC: IOSE-MYC, with MYC expression higher than control IOSE-Ctrl cells, and IOSE-MYC2, with a MYC overexpression level higher than IOSE-MYC ( Figure 3A). The expression of "Pol I regulon" target genes (RRN3, UBF, TAF1C, POLR1B, POLR1E) was significantly higher in MYC-overexpressing IOSE lines than in IOSE-Ctrl, and correlated with MYC level ( Figure 3B). Moreover, MYC-overexpressing IOSE displayed increased rRNA transcription, which was assessed both by pre-rRNA qRT-PCR ( Figure 3C, left) and nucleolar EU incorporation ( Figure 3C, middle and right). Consistently, we detected a MYC level-dependent increase of cell proliferation in IOSE-MYC and IOSE-MYC2 relative to IOSE-Ctrl (see MTT assay in Figure 3D, left, and EdU incorporation assay in Figure 3D, middle and right). We obtained similar findings also in SV40-immortalized HOSE cells (Supplementary Figure 1A-1D).
Thus, regardless of the cell context, increasing MYC overexpression seems sufficient to differentially foster cell proliferation by inducing Pol II-dependent transcription of "Pol I regulon" genes as well as Pol I-dependent rRNA synthesis.

Additional evidence that upregulation of "Pol I regulon" genes contributes to promote cell proliferation in MYC-overexpressing IOSE cells
To mechanistically prove that increased proliferation of MYC-overexpressing IOSE cells was due, in part, to upregulation of "Pol I regulon" genes and rRNA transcription, we transiently transfected IOSE-MYC2 cells with short interfering RNAs (siRNAs) targeting either MYC, or either one of two key components of the "Pol I regulon": RRN3 and UBF.
As shown in Figure 4A, transient MYC knock down by siMYC in IOSE-MYC2 cells (left) resulted in downregulation of both RRN3 and UBF mRNAs, as well as in decreased pre-rRNA level (right) relative to cells transfected with control siRNA (siCtrl). Importantly, MYC knock down also counteracted MYC-induced proliferation of IOSE-MYC2 cells (see EdU incorporation assay in Figure 4B). In turn, transient knock down of either RRN3 ( Figure 4C, left) or UBF ( Figure 4C, right) in IOSE-MYC2 was sufficient to significantly reduce both rRNA transcription (see pre-rRNA qRT-PCR in Figure 4C, left and right), and cell proliferation (see EdU incorporation assay in Figure 4D).
Based on these findings, the pro-proliferative effects of MYC overexpression in human ovarian epithelial cells rely, at least in part, on MYC transcriptional ability to upregulate "Pol I regulon" genes.

MYC overexpression sensitizes human ovarian epithelial cells to the anti-proliferative action of the Pol I inhibitor CX-5461
Selective Pol I inhibitors have been rapidly emerging as drugs capable of targeting increased rRNA synthesis [19,32]. Based on preliminary evidence of an antiproliferative effect of the Pol I inhibitor CX-5461 in MYC-overexpressing HFF (see Figure 1G), we did test if MYC-induced rRNA transcription was also capable of sensitizing IOSE cells to the anti-proliferative action of CX-5461.
As shown in Figure 5A, CX-5461 (100 nM) reduced rRNA transcription (assessed by pre-rRNA quantitative RT-PCR) significantly more in IOSE-MYC2 relative to  (B) and increased expression of "Pol I regulon" genes (assessed by qRT-PCR) (C) relative to CAOV3 ovarian cancer cells, with no MYC overexpression. All differences between CAOV4 and CAOV3 are significant (p<0.05). IOSE-Ctrl. Consistently, IOSE-MYC2 cells also displayed increased sensitivity to the anti-proliferative action of CX-5461 even at the lowest concentration ( Figure 5B). Indeed by EdU incorporation we found that at 10 nM, CX-5461 reduced the number of cells in S phase significantly more in IOSE-MYC2 than in IOSE-Ctrl cells ( Figure 5C). A similar antiproliferative response to CX-5461 was also found in HOSE cells (Supplementary Figure 1E).
Overall, these findings indicated that a) upregulation of Pol I-mediated rRNA transcription contributes to MYCinduced proliferation in ovarian epithelial cells, and b) MYC-overexpressing human ovarian epithelial cells are more sensitive to the anti-proliferative action of the Pol I inhibitor CX-5461.

Upregulation of genes involved in both mitochondrial rRNA transcription and mitoribogenesis contributes to MYCinduced HFF proliferation
Recent studies in different cell contexts indicate that MYC can promote mitochondrial ribogenesis by activating Pol II-dependent transcription of the mitochondrial RNA polymerase POLRMT and the mitochondrial transcription factor TFAM [7,8]. Further, MYC can induce Pol IIdependent transcription of several mitoribosome proteins (e.g. PTCD3, MRPS5, and MRPS27) [10].
We used the HFF model to assess whether the effects of MYC overexpression on proliferation can    (B) and analysis of EdU incorporation by immunofluorescence (C). * p<0.05, ** p<0.01, *** p<0.001. be traced to either increased mt-rRNA transcription or upregulation of mitoribosome proteins. Relative to HFF-Ctrl, HFF-MYC cells displayed increased levels of both POLRMT and TFAM mRNAs ( Figure 6A, left), as well as increased levels of three pre-mtRNA regions ( Figure 6A, middle and right): i) a region within the 12S mitochondrial RNA, ii) a region within the 16S mtRNA, and iii) a region across the 12S and 16S mt-rRNA. Since the 12S and 16S RNAs are subsequently spliced out to form mature mitochondrial rRNAs, this last region is present only in mt-rRNA precursors, thus indirectly reflecting the rate of mt-rRNA transcription. In addition, HFF-MYC also displayed increased transcript levels of PTCD3, MRPS5, and MRPS27 mitoribosome proteins relative to HFF-Ctrl ( Figure 6B), thus confirming that, also in this cell context, MYC overexpression can promote mitoribosome formation by concomitantly enhancing both mt-rRNA transcription and expression of mitochondrial ribosomal proteins.
Second, we treated both HFF-Ctrl and HFF-MYC cells with doxycycline, a tetracycline that targets the 28S subunit of mitochondrial ribosomes, which is homologous to the 30S subunit of bacterial ribosomes. Doxycycline inhibited HFF-MYC proliferation in a dose-dependent fashion, and its anti-proliferative effects were significantly greater in the HFF-MYC cell context than in the HFF-Ctrl cell context ( Figure 6E).
These preliminary findings in the HFF model showed that: i) MYC-induced mt-rRNA transcription and mitoribogenesis contribute to promote HFF-MYC proliferation; ii) MYC overexpression sensitizes HFF-MYC cells to the anti-proliferative action of drugs inhibiting either mt-rRNA transcription (2'-C-MeA) or mitoribosome function (doxycycline).

MYC-induced upregulation of either mt-rRNA transcription or mitoribogenesis in human ovarian epithelial cell contexts contributes to promote proliferation
We found evidence that, relative to CAOV3 ovarian cancer cells, which do not overexpress MYC, CAOV4 ovarian cancer cells, which overexpress MYC (see Figure  2A), display a) upregulation of both MYC-target genes involved in mt-rRNA transcription (POLRMT and TFAM) and MYC-target genes encoding mitoribosome proteins (PTCD3, MRPS5, MRPS27) ( Figure 7A) as well as b) increased mitochondrial rRNA synthesis ( Figure 7B). For this reason, we set out to test whether MYC ectopic overexpression in human ovarian epithelial cell contexts (IOSE and HOSE) leads to increased mt-rRNA synthesis and affects mitoribogenesis.
Apparently, MYC ectopic overexpression in different human ovarian epithelial cell contexts promotes mt-rRNA transcription and mitoribogenesis by upregulating MYC-target genes that are involved in these two processes. Moreover, MYC-induced mt-rRNA transcription and mitoribogenesis contribute to MYCinduced cell proliferation.

Antiproliferative effect of 2'-C-MeA and doxycycline, alone or in combination with CX-5461, on MYC overexpressing ovarian epithelial cells
Recently, it was reported that selective inhibition of either mt-rRNA transcription or mitoribosome function exerts an antiproliferative effect on MYC-overexpressing cancer cells [7,10]. First, we found by MTT assay that IOSE-MYC2, relative to IOSE-Ctrl cells ( Figure 9A), display increased sensitivity to the antiproliferative action of the POLRMT inhibitor 2'-C-MeA. Similar findings were also obtained in HOSE-MYC relative to HOSE-Ctrl (Supplementary Figure 2C). The 2'-C-MeA concentration of 40 μM, which determined the most significant difference between IOSE-MYC2 and IOSE-Ctrl proliferation in the MTT assay, also decreased 12-16S mt-rRNA transcription ( Figure  9B) and the number of cells in S phase (assessed by EdU incorporation) ( Figure 9C) significantly more in IOSE-MYC2 versus IOSE-Ctrl proliferation. Moreover, when we tested the effect of 40 μM 2'-C-MeA in combination with CX5461 (ranging from 5 nM to 100 nM) in IOSE-MYC2 cells, we found that 2'-C-MeA significantly enhanced the antiproliferative effect of CX-5461 low concentrations (5-10 nM) ( Figure 9D).
Second, by MTT assay we found that IOSE-MYC2 versus IOSE-Ctrl ( Figure 10A, left), as well as HOSE-MYC versus HOSE-Ctrl (Supplementary Figure 2D), displayed increased sensitivity to the anti-proliferative action of doxycycline across a range of concentrations (0-25 μM). The doxycycline concentration of 15 μM showed the most significant difference between IOSE-MYC2 and IOSE-Ctrl proliferation both in the MTT assay ( Figure  10A, left) and in the EdU incorporation assay ( Figure 10A,  right). Remarkably, when we tested the effect of 15 μM doxycycline in combination with CX5461 (ranging from 5 nM to 100 nM) on IOSE-MYC2 cells, we found that doxycycline significantly enhanced the antiproliferative effect of CX-5461 at concentrations between 5 and 25 nM ( Figure 10B).

Both 2'-C-MeA and doxycycline can enhance the antiproliferative effects of CX-5461 in MYCoverexpressing ovarian cancer cells
In this study we found that CAOV4 ovarian cancer cells overexpressing endogenous MYC show upregulation of nucleolar rRNA and "Pol I regulon" genes ( Figure 2) as well as increased mitochondrial rRNA transcription and upregulation of MYC-target genes involved in mitochondrial rRNA transcription and ribogenesis ( Figure  7). Dose-dependent inhibition of Pol I-mediated rRNA transcription with CX-5461 ( Figure 11A, left) significantly reduced CAOV4 cell proliferation ( Figure 11A, right). Treatment with either 2'C-MeA ( Figure 11B, left) or doxycycline ( Figure 11B, right) also led to dose-dependent inhibition of CAOV4 cell proliferation. Moreover, the anti-proliferative effects of CX-5461 on CAOV4 was significantly enhanced by co-treatment with either 2'C-MeA (80 or 160 μM, Figure 11C) or doxycycline (15 or 25 μM, Figure 11D), which were among the lowest concentrations able to induce significant inhibition of CAOV4 proliferation. In conclusion, combining CX-5461 with drugs inhibiting either mt-rRNA transcription (e.g. 2'C-MeA) or mitoribosome function (e.g. doxycycline) seems to be an effective antiproliferative strategy to treat MYC-overexpressing ovarian cancer.   34,35]. In particular, one of these molecules, CX-5461, which targets Pol I-mediated rRNA transcription, proved to be effective in in vitro and preclinical MYC-driven cancer cell models [16,21,22]. CX-5461 is currently tested in clinical trials for advanced hematological malignancies (Australian New Zealand Clinical Trials Registry 12613001061729) and solid tumors (ClinicalTrials.gov Identifier NCT02719977), and its use could be extended to clinical trials of MYC-driven solid tumors, including breast, ovarian and prostate cancer [19,24,32].

DISCUSSION
Interestingly, CX-5461 was shown to be even more effective when used in combination with other drugs, such as drugs targeting PI3K-AKT-mTORC1dependent nucleolar ribosome biogenesis and translation [21]. By the same tenet, we reasoned that CX-5461 could also be more effective if combined with drugs targeting mitochondrial ribogenesis processes and translation in MYC-overexpressing cells. Indeed, recent studies highlighted the anticancer potential of several antiviral and antibacterial molecules capable of targeting either POLRMT-mediated mitochondrial rRNA transcription or mitoribosome function [7,10,26]. Specifically, there are several ribonucleosides analogues, originally developed for antiviral therapy, which are substrates and inhibitors of human mitochondrial RNA polymerase (POLRMT) [33], and can be repurposed for cancer therapy. In preclinical studies the 2-C-methyladenosine ribonucleoside (2'-C-MeA) was shown to reduce mitochondrial gene expression and increase cell death in acute myelogenous leukemia (AML), while treatment of normal human hematopoietic cells did not alter clonogenic growth [27]. Due to the ancestral relationship between bacteria and mitochondria, several classes of antibiotics, including tetracyclines (e.g. Doxycycline and Tigecycline) capable of targeting mitoribosome function, are also re-evaluated as potential anti-cancer drugs ( [26] and references within).
This study provides the proof of concept that available drugs targeting mitochondrial ribogenesis or mitoribosome function can improve the anticancer effects of emerging new drugs capable of selectively targeting nucleolar Pol I rRNA synthesis in cancer and, in particular, MYC-driven cancer of different histotypes due to concomitant MYC deregulation of both nucleolar and mitochondrial rRNA synthesis and ribogenesis processes.
Based on preliminary observations that ovarian cancer cells overexpressing MYC (CAOV4) displayed upregulation of POLRMT and mt-rRNA synthesis relative to ovarian cancer cells that do not overexpress MYC (CAOV3), we assessed the antiproliferative effects of targeting POLRMT-mediated mt-rRNA transcription in two human ovarian epithelial cell contexts (IOSE and HOSE), and in human fibroblasts (HFF) overexpressing MYC. Consistent with genetic studies (either MYC or POLRMT knock down), we found that the POLRMT inhibitor 2'-C-MeA significantly reduced both mt-rRNA transcription and the number of cells in S phase. Remarkably, even if MYC-overexpressing ovarian epithelial cells were more sensitive to the anti-proliferative action of the Pol I inhibitor CX-5461 alone, 2'-C-MeA significantly enhanced the effects of CX-5461. Indeed, in the presence of 2'-C-MeA, CX-5461 exerted an antiproliferative effect at lower concentrations (5-10 nM).
Further, based on the fact that MYC upregulation induces PTCD3, MRPS5, and MRPS27 proteins necessary for mitoribogenesis in MYC-overexpressing CAOV4 cancer cells, as well as in HFF fibroblast and different ovarian epithelial cell contexts with MYC overexpression, we tested if doxycycline, a tetracycline that targets the 28S subunit of mitochondrial ribosomes, was capable of reducing MYC-induced proliferation. Indeed, we found that MYC-overexpressing fibroblasts and ovarian epithelial cells were more sensitive than control cells to the anti-proliferative action of doxycycline alone. Moreover, doxycycline, when used in combination with low doses of CX-5461 Pol I inhibitor, enhanced CX-5461 antiproliferative effect in MYC overexpressing ovarian epithelial cells. In the same cell contexts, we also tested Tigecycline, another tetracycline that was recently shown to curb proliferation of MYC-overexpressing lymphoma and osteosarcoma cells [7,10], but we did not achieve the anti-proliferative effects of doxycycline (data not shown).
Finally, we found that CX-5461 significantly inhibits CAOV4 ovarian cancer cell proliferation, and that the anti-proliferative effect of CX-5461 is significantly enhanced by co-treatment with either 2'C-MeA or doxycycline. Since CX-5461 was recently shown to have a positive effect on both chemoresistant ovarian epithelial cells and patient derived xenografts [36], it is possible that drugs targeting mt-rRNA and mitoribogenesis, when combined with drugs inhibiting Pol I like CX-5461, can be also effective in chemoresistant MYC-overexpressing cancer cell populations.
Overall these findings indicate that leveraging a) MYC-regulated rRNA transcription by RNA polymerases in distinct cell sites (nucleolar Pol I and mitochondrial POLRMT) and b) other MYC-regulated processes and functions of ribogenesis and mitoribogenesis, can be an offbeat approach to control MYC-driven cancers.

Drugs and treatments
CX-5461 (ApexBio) was dissolved in 50 mM NaH 2 PO 4 (pH 4.5) to obtain a 1 mM stock solution.
2'-C-Methyladenosine (Santa Cruz Biotechnologies) was dissolved in DMSO to obtain a 100 mM stock solution. Doxycycline (Fisher Scientific) was dissolved in H 2 O to obtain a 100 mM stock solution. Drugs were stored in aliquots at -20°C. Treatments were performed with drugs or vehicle diluted in medium at the indicated concentrations as described in the Results. For qRT-PCR analysis, EU incorporation, and EdU incorporation, 30-50% confluent cells were treated for 24 h. For MTT analysis, cells were treated for 3-5 days refreshing the medium every 48 hours for the duration of the treatment. siRNA 40-60% confluent cells seeded on 24-well plates were transiently transfected with 10 nM Silencer Select pre-designed siRNAs (Thermo Fisher) targeting MYC (s9129+s9131), UBTF (s14613+s14615), RRN3 (s29324) or POLRMT (s10825+s10826) by using RNAi max Lipofectamine (Thermo Fisher) according to the manufacturer's instructions. As a negative control, cells were transfected with 10 nM siRNA Negative Control (Thermo Fisher). 24-48h after transfection, cells were analyzed by qRT-PCR or EdU incorporation.

Western blot
Cells were lysed in RIPA Buffer (Cell Signaling) supplemented with 1 mM PMSF and EDTA-free Protease Inhibitor Cocktail (Roche). Total protein concentration was measured by using Coomassie Plus Protein Assay Reagent (Thermo Fisher). Equal amounts of proteins were separated by SDS PAGE electrophoresis and transferred onto a PVDF membrane according to standard procedures. Membranes were blocked with 5% non-fat dry milk diluted in TBS + 0.05% Tween 20 for 1 h at room temperature, incubated over night at 4 °C with anti-MYC (C33) antibody (Santa Cruz Biotechnology) diluted in 5% milk, washed, incubated for 1 h at room temperature with HRP-conjugated anti-mouse antibody (GE Healthcare) diluted in 5% milk, washed, and incubated with Clarity Western ECL Substrate (Bio-Rad). Chemiluminescence was detected and quantified by using ChemiDoc Touch Imaging System (Bio-Rad).

Assessment of nucleolar EU incorporation
5-ethynyl uridine (EU) incorporation assay was performed by using Click-iT RNA Alexa Fluor Imaging Kit (Thermo Fisher). Subconfluent cells were incubated with 1 mM EU under standard growth conditions for 2 h, fixed with 4% paraformaldehyde for 10 min., permeabilized with PBS + 0.5 % Triton X100 for 15 min. and incubated with Click-iT reaction cocktail for 30 min., blocked with PBS+ (PBS plus 1% BSA, 1% FBS, 0.05% Tween 20) for 20 min, incubated with anti-B23 (Santa Cruz) diluted in PBS+ for 1 h at room temperature, washed four times with PBS+, incubated with antirabbit AlexaFluor 546 (Thermo Fisher) diluted in PBS+ for 1 h, washed four times with PBS, counterstained with 330 nM DAPI for 10 min., and mounted with Vectashield (Vector Laboratories). Cells were imaged with a fluorescence microscope using the same exposure time for each fluorescent channel. EU incorporation in the nucleolar compartment (identified by B23 staining) was assessed with Adobe Photoshop by multiplying the EU-positive nucleolar area (number of pixels) by the EU signal intensity (mean) in the B23-positive nucleolar compartment normalized to the EU signal intensity (mean) in the surrounding nucleoplasm. Statistical significance was calculated by Student's t-test.

MTT assay
2-5 x 10 3 cells/well were seeded in 24-well plates in four replicates, let attach overnight, and treated with the indicated drugs diluted in growth medium. After 3-5 days of treatment, cells were incubated for 2 hours under standard growth conditions with 0.5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma) and lysed with DMSO. Cell lysates were transferred into a 96-well plate, and absorbance at 570 nm was measured on a microplate spectrophotometer. Statistical significance was calculated by Student's t-test.

EdU incorporation assay
EdU-incorporation was assessed with Click-iT EdU imaging kit (Thermo Fisher), according to the manufacturer's instructions. Briefly, cells were incubated under standard growth conditions with 10 μM EdU for 1h. After EdU incorporation, cells were fixed with 4% paraformaldehyde for 10 min., incubated with PBS plus 0.5 % Triton X100 for 10 min., washed with PBS plus 3% BSA, reacted with Click-iT reaction cocktail for 30 min., counterstained with 330 nM DAPI for 10 min, and mounted with Vectashield (Vector Laboratories). Cells were imaged with a fluorescence microscope. DAPIpositive cells and EdU-positive cells were counted in at least 5 random fields. Statistical significance was calculated by using the Student's t-test.