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Therapeutics, Targets, and Chemical Biology

Targeting RNA Polymerase I with an Oral Small MoleculeCX-5461 Inhibits Ribosomal RNA Synthesis and SolidTumor Growth

Denis Drygin1, Amy Lin1, Josh Bliesath1, Caroline B. Ho1, Sean E. O’Brien1, Chris Proffitt1, Mayuko Omori1,Mustapha Haddach1, Michael K. Schwaebe1, Adam Siddiqui-Jain1, Nicole Streiner1, Jaclyn E. Quin2,3, Elaine Sanij2,Megan J. Bywater2,3, Ross D. Hannan2,3,4, David Ryckman1, Kenna Anderes1, and William G. Rice1

AbstractDeregulated ribosomal RNA synthesis is associated with uncontrolled cancer cell proliferation. RNA

polymerase (Pol) I, the multiprotein complex that synthesizes rRNA, is activated widely in cancer. Thus,selective inhibitors of Pol I may offer a general therapeutic strategy to block cancer cell proliferation. Couplingmedicinal chemistry efforts to tandem cell- andmolecular-based screening led to the design of CX-5461, a potentsmall-molecule inhibitor of rRNA synthesis in cancer cells. CX-5461 selectively inhibits Pol I–driven transcriptionrelative to Pol II–driven transcription, DNA replication, and protein translation. Molecular studies demonstratethat CX-5461 inhibits the initiation stage of rRNA synthesis and induces both senescence and autophagy, but notapoptosis, through a p53-independent process in solid tumor cell lines. CX-5461 is orally bioavailable anddemonstrates in vivo antitumor activity against human solid tumors in murine xenograft models. Our findingsposition CX-5461 for investigational clinical trials as a potent, selective, and orally administered agent for cancertreatment. Cancer Res; 71(4); 1418–30. �2010 AACR.

Introduction

The rate of ribosome biogenesis controls cellular growthand proliferation (reviewed in ref. 1). It, therefore, is tightlyregulated in mammalian cells and is tuned to respond toextracellular stimuli such as nutrient availability and stress.During tumorigenesis, the tightly regulated relationshipbetween extracellular signaling and ribosome biosynthesisis disrupted, and cancer cells begin the excessive produc-tion of ribosomes necessary for the protein synthesis asso-ciated with unbridled cancer growth. rRNA is a majorcomponent of the ribosome and, as such, carcinogenesisrequires an increase in its synthesis (reviewed in refs. 2–5).Indeed, an increase in the synthesis of rRNA, which istranscribed in the nucleolus by RNA polymerase (Pol) I,correlates with an adverse prognosis in cancer (6). More-over, enlarged nucleoli, reflective of accelerated rRNA

synthesis, have long been recognized as a marker foraggressive tumor cells (7, 8). A number of approved cancertherapeutics reportedly act through inhibition of rRNAsynthesis, but none directly target the Pol I multiproteinenzyme complex (4, 9).

The potential therapeutic benefit of selectively inhibitingthe Pol I target so fundamental to cancer cell survivalprompted the need for identification of small moleculedrugs that selectively inhibit rRNA synthesis. For thispurpose, we fashioned a cell-based quantitative reverse-transcription PCR (qRT-PCR) assay that differentiatesbetween the effects of compounds on Pol I- and Pol II–driven transcription. In this article, we describe the discov-ery and characterization of CX-5461, a potent and selectiveinhibitor of Pol I–mediated rRNA synthesis in cancer cellsthat does not inhibit DNA, mRNA or protein synthesis. Wereveal that CX-5461 induces autophagic cell death in cancercells but not normal cells and exhibits potent in vivo anti-tumor activity in murine xenograft models of human solidtumors with a favorable safety profile. CX-5461 represents afundamentally new class of small molecule–targeted anti-cancer therapeutics.

Materials and Methods

MaterialsCX-5461 and CX-5447 were synthesized by Cylene Pharma-

ceuticals as off-white solid materials (99.2%–99.5% pure) andstored at room temperature as 10 mmol/L stock solutions in

Authors' Affiliations: 1Cylene Pharmaceuticals, Inc., San Diego, Califor-nia; 2Peter MacCallum Cancer Centre; 3Department of Biochemistry andMolecular Biology, University of Melbourne; and 4Department of Biochem-istry and Molecular Biology, Monash University, Melbourne, Victoria,Australia

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: William G. Rice, Cylene Pharmaceuticals Inc.,5820 Nancy Ridge Drive, Suite 200, San Diego CA 92121. Phone: 858-875-5100; Fax: 858-875-5101. E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-10-1728

�2010 American Association for Cancer Research.

CancerResearch

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Published OnlineFirst December 15, 2010; DOI: 10.1158/0008-5472.CAN-10-1728

http://cancerres.aacrjournals.org/

50 mmol/L NaH2PO4 (pH 4.5). The compounds were diluteddirectly in growth media prior to treatment.

Cell lineshTERT-immortalized BJ-hTERT human fibroblasts were a

gift from Dr. William Hahn, Harvard Medical School (10).Human inflammatory breast cancer cell lines SUM 149PTand SUM 190PT were obtained from Asterand. Humaneosinophilic leukemia cell line EOL-1, human B cell pre-cursor leukemia cell line SEM, and human acute monocyticleukemia cell line THP-1 were obtained from DSMZ. Othercell lines were purchased from American Type CultureCollection. All cell lines were used within 5 to 10 passagesfrom their acquisition. The internal authentication has beenperformed by monitoring growth rate and tracking thechanges in morphology.

qRT-PCRqRT-PCR assays were performed as previously described

(11) with HCT-116 colorectal carcinoma cells and then con-firmed with A375 and MIA PaCa-2 cells.

Cell-free Pol I transcription assayA reaction mixture consisting of 30 ng/mL DNA template

corresponding to (�160/þ379) region on rDNA and 3 mg/mL nuclear extract isolated from HeLa S3 cells in a buffercontaining 10 mmol/L Tris-HCl, pH 8.0, 80 mmol/L KCl,0.8% polyvinyl alcohol, 10 mg/mL a-amanitin was com-bined with different amounts of test compounds and incu-bated at ambient for 20 minutes. Transcription wasinitiated by addition of rNTP mix (New England Biolabs)to a final concentration of 1 mmol/L and was incubated for1 hour at 30�C. Afterward, DNase I was added and thereaction was further incubated for 2 hours at 37�C. DNasedigestion was terminated by the addition of EDTA to finalconcentration of 10 mmol/L, followed immediately by 10-minute incubation at 75�C, and then samples were trans-ferred to 4�C. The levels of resultant transcript were ana-lyzed by qRT-PCR on 7900HT Real Time PCR System(Applied Biosystems).

Chromatin immunoprecipitationCells were treated with 2 mmol/L of CX-5461 for 1 hour and

chromatin immunoprecipitation (ChIP) assay was performedas previously described (11, 12).

Electrophoretic mobility shift assay32P-labeled DNA probe corresponding to the rDNA

promoter was produced by PCR using prHu3 plasmid asa template. The SL1 complex was isolated from HeLaS3 cells nuclear extract as described previously (13). Forthe competition studies, a mixture of 5 nmol/L of DNAprobe and 0.6 mg SL1 complex was incubated in bindingbuffer for 15 minutes at ambient temperature with 0.4 to12.5 mmol/L CX-5461 or CX-5447. The resulting complexeswere resolved using Novex TBE DNA retardation PAGE(Invitrogen). The gels were dried and exposed to X-ray film(Kodak).

Gene expression analysisFor MIA PaCa-2 cells, gene array analyses were performed

at Expression Analysis using Illumina Whole Genome Human-6 v2 beadchips. For A375 cells, the expression analysis wasperformed using Human Cancer Pathway Finder qRT-PCRarray from SABiosciences.

Cell viability assayCells were plated on 96-well plates and treated the next day

with dose response of drugs for 96 hours. Cell viabilitywas determined using Alamar Blue and CyQUANT assays(Invitrogen).

Detection and quantification of acidic vesicularorganelles with acridine orange staining

Cells were plated on 10-cm dishes and treated the nextday with indicated doses of CX-5461 for 24 or 48 hours. Atthe end of treatment, cells were stained for 15 minutes with 1mg/mL of acridine orange, trypsinized, washed twice withice-cold PBS, and analyzed on a BD LSR II flow cytometer(BD Biosciences). The analysis was performed as previouslydescribed (14).

Immunocytochemistry-based autophagydetection assay

Immunofluorescent analysis was performed as previouslydescribed (11) using 1:100 dilution of rabbit polyclonal anti-LC3B-II antibody (Cell Signaling Technology).

Immunocytochemistry-based senescencedetection assay

The assay was performed using Senescence Detec-tion Kit (Calbiochem) according to manufacturer's instruc-tions.

In vivo efficacy in murine xenograft modelAnimal experiments were performed with 5- to 6-week-

old female athymic (NCr nu/nu fisol) mice of Balb/c origin(Taconic Farms) in accordance with approved standardoperating protocols of Cylene Pharmaceuticals that wereapproved by the Institutional Animal Care and Use Com-mittee. Mice were inoculated with 5 � 106 in 100 mL of cellsuspension subcutaneously in the right flank. Tumor mea-surements were performed by caliper analysis, and tumorvolume was calculated using the formula (l � w2)/2, wherew ¼ width and l ¼ length in mm of the tumor. Establishedtumors (�110–120 mm3) were randomized into vehicle(50 mmol/L NaH2PO4, pH 4.5), gemcitabine, or CX-5461treatment groups. Tumor growth inhibition (TGI) wasdetermined on the last day of study according to theformula: TGI (%) ¼ [100 � (VfD � ViD)/ (VfV � ViV) �100], where ViV is the initial mean tumor volume in vehicle-treated group, VfV is the final mean tumor volume invehicle-treated group, ViD is the initial mean tumor volumein drug-treated group, and VfD is the final mean tumorvolume in drug-treated group.

Selective Inhibitor of RNA Pol I Complex Exhibits Antitumor Activity

www.aacrjournals.org Cancer Res; 71(4) February 15, 2011 1419




Results

Discovery of CX-5461To screen for inhibitors of rRNA synthesis, we developed a

cell-based screening assay capable of identifying agents thatselectively inhibit Pol I transcription relative to Pol II tran-scription. Two short-lived RNA transcripts (half-lives �20–30minutes), one produced by Pol I and another by Pol II, werequantitated by qRT-PCR as a measure of drug-related effectson transcription. The 45S pre-rRNA served as the Pol Itranscript and the mRNA for the protooncogene c-myc servedas the comparator Pol II transcript. Both Pol I and Pol IItranscription are known to be affected by general cellularstress. To minimize the potential effects of such stress, cellswere exposed to test agents for only a short period of time (2hours). This is sufficient time for these transcripts to bereduced by greater than 90% if a drug affects their synthesis.

Among numerous molecules screened, CX-5461 (Fig. 1A)was found to selectively inhibit rRNA synthesis (Pol I IC50¼ 142nmol/L; Pol II IC50 > 25 mmol/L; selectivity � 200-fold) in theHCT-116 cells (Fig. 1B). Selective inhibition of rRNA synthesisby CX-5461was confirmed in two other human solid tumor celllines; melanoma A375 (Pol I IC50¼ 113 nmol/L; Pol II IC50 > 25mmol/L) and pancreatic carcinomaMIAPaCa-2 (Pol I IC50¼ 54nmol/L; Pol II IC50 � 25 mmol/L; Fig. 1C).

Characterization of CX-5461To determine if CX-5461 directly targets the Pol I machinery

and to identify which specific step in Pol I transcription it mayaffect, we performed cell-free transcription "order of addition"studies (Fig. 1D). CX-5461 was preincubated with eithernuclear extract (NE) or with DNA template (Template) priorto the preinitiation complex (PIC) formation, after the PICformation but prior to initiation of transcription (NE/Tem-plate), or after the initiation of the transcription (NE/Tem-plate/NTP). Analysis of the resulting rRNA transcripts by qRT-PCR (Fig. 1D) revealed that addition of 80 nmol/L CX-5461prior to PIC formation resulted in considerable inhibition ofPol I transcription, whereas addition after PIC formation onlyminimally affected Pol I transcription. These data indicate CX-5461 directly targets the Pol I machinery and inhibits Pol Itranscription at the initiation stage.

CX-5461 inhibits Pol I via disruption of the SL1-rDNAcomplex

ChIP analysis was employed to investigate the effects of CX-5461 on the association of various components of the Pol Imultiprotein complex with the rDNA promoter. Treatment ofHCT-116, A375, or MIA PaCa-2 with 2 mmol/L CX-5461resulted in 40% to 60% reduction of the Pol I enzyme associa-tion with the rDNA promoter (Fig. 2A). Further, CX-5461significantly depleted the binding of Pol I transcription factors(TF) to the rDNA promoter in HCT-116 cells, with the TBP andTAFI110 subunits of SL1 being most overtly affected (Fig. 2B).As SL1 is required for stabilization of UBF on and recruitmentof Pol I to the rDNA promoter, the effect of CX-5461 on thebinding of UBF and Pol I to rDNA is most likely secondary tothe compound's effect on SL1 (15).

Because TBP is a member of both Pol I and Pol II tran-scription machineries (as a unit of the TFIID transcriptionfactor), we compared the effects of CX-5461 on binding of TBPwith the Pol I promoter on rDNA relative to its binding to thepromoter of 2 genes transcribed by Pol II (p21 and histoneH2B). Treatment with CX-5461 had no effect on the binding ofTBP to the Pol II promoter regions in HCT-116 cells, but didinhibit binding of TBP to the Pol I promoters (Fig. 2C). To testif CX-5461 could directly compete with SL1 for the rDNApromoter, we performed an electrophoretic mobility shiftassay (EMSA) measuring the interaction of purified humanSL1 with a radiolabeled, double-stranded DNA fragment cor-responding to the promoter region of rDNA. As a negativecontrol, we used a close analogue of CX-5461, CX-5447, that isinactive against Pol I transcription (IC50 > 25 mmol/L).Although CX-5447 had no detectable effect on the stabilityof the SL1/rDNA complex, CX-5461 clearly disrupted the SL1/rDNA complex (Fig. 2D). Such dissociation was likely due todisruption of the protein–rDNA interaction rather than dis-sociation of SL1 itself, as we detected no decrease in protein–protein interactions within SL1 after treatment with CX-5461(data not shown).

Biological characterization of CX-5461To ensure that CX-5461 selectively inhibits rRNA transcrip-

tion but not DNA or protein synthesis, HCT-116, A375, andMIA PaCa-2 cells were preincubated with 10 mmol/L CX-5461for 60 minutes and pulsed for an additional 120 minutes withmodified precursors BrdU or 35S-methionine. Mitoxantroneand cycloheximide served as positive controls for inhibitionof DNA and protein synthesis, respectively. At this concen-tration, which is approximately 100-times higher than its IC50

for the inhibition of Pol I transcription, CX-5461 had onlymodest effects on global DNA or protein synthesis (Fig. 3A).In dose–response studies, the IC50 for inhibition of DNAsynthesis in A375 and MIA PaCa-2 cell lines ranged from16.8 to 27.9 mmol/L, whereas a high-test dose of 30 mmol/LCX-5461 had no significant effect on proteins synthesis (Sup-plementary Fig. S1). Thus, CX-5461 possesses 250- to 300-foldselectivity for inhibition of rRNA transcription versus DNAreplication and protein translation.

Using Gene Expression Arrays (Illumina or qRT-PCR based)as a tool to probe for any potential effect of CX-5461 on Pol IItranscription, we observed that 1-hour exposure of MIA PaCa-2 (Fig. 3B) or A375 (Fig. 3C) cells to 300 nmol/L CX-5461 (thatresulted in 63% and 55% reductions in pre-rRNA, respectively),an equal number of Pol II–transcribed genes were significantlyupregulated as were downregulated. These changes in Pol IItranscription were most likely secondary to the inhibition ofPol I by CX-5461, as an inhibition of Pol II transcription resultsin a distribution significantly skewed to the negative (16). Tofurther illustrate selectivity of CX-5461 for Pol I versus Pol IItranscription, we compared it with another drug (actinomycinD) known to inhibit Pol I transcription. Treatment of MIAPaCa-2 cells with either 1 mmol/L CX-5461 or 1 mmol/Lactinomycin D causes rapid inhibition of Pol I transcription,with an observed half-life of 21 minutes for 45S pre-rRNA inboth cases (Fig. 3D). However, actinomycin D inhibited Pol II

Drygin et al.

Cancer Res; 71(4) February 15, 2011 Cancer Research1420




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Figure 1. CX-5461 selectively inhibits rRNA synthesis. A, structure of CX-5461. B, inhibition of Pol I (pre-rRNA) versus Pol II (c-myc mRNA) transcriptionin HCT-116 cells by CX-5461. C, qRT-PCR analysis of rRNA, c-myc mRNA, and b-actin mRNA transcription after 2-hour treatment with CX-5461in A375 and MIA PaCa-2 cells. D, order of addition study setup and results.






transcription as well, as judged by the depletion of c-mycmRNA (half-life¼ 35 minutes), whereas CX-5461 had nomajoreffect on Pol II transcription even after 24-hour treatment(Fig. 3D).

In vitro antiproliferative activity of CX-5461To evaluate the range of antiproliferative activity of CX-

5461, we measured the antiproliferative EC50 across a panel of50 human cancer cell lines and 5 nontransformed cell lines.

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Figure 2. CX-5461 selectively competes SL1 from the Pol I promoter on rDNA. A, ChIP analysis of Pol I associations with Pol I promoter in HCT-116,A375, and MIA PaCa-2 cells. B, ChIP analysis of Pol I TF SL1 (TBP, TAFI110, TAFI63) and UBF associations with Pol I promoter in HCT-116 cells. C, ChIPanalysis of TBP associations with Pol I, histone H2B, and p21 promoters in HCT-116 cells. D, EMSA of SL1-rDNA complex in presence of increasingconcentrations of CX-5461 and CX-5447. UTC, untreated control.

Drygin et al.





The EC50 values ranged from 3 nmol/L against the EOL-1eosinophilic leukemia cell line to 5,500 nmol/L against theLNCaP prostate carcinoma cell line (Fig. 4A). The median EC50

across all tested cell lines was 147 nmol/L, yet all normal celllines had EC50 values of approximately 5,000 nmol/L. Evalua-tion of the antiproliferative dose response (Fig. 4B, left) forHCT-116, A375, and MIA PaCa-2 cell lines yielded EC50 valuesof 167, 58, and 74 nmol/L, respectively. In contrast, an EC50 of

approximately 5,000 nmol/L was observed for the BJ-hTertnormal cell line (nontransformed hTERT-immortalizedhuman foreskin fibroblasts). The lesser sensitivity of thenormal cells was not due to a reduced uptake of CX-5461,as the IC50 for rRNA synthesis for the BJ-hTert normal cell lineof 74 nmol/L correlated well with the IC50 values of the solidtumor cell lines (IC50 ¼ 142, 113, and 54 nmol/L, respectively;see Fig. 4B, right). These findings illustrate that CX-5461 can

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Figure 3. CX-5461 inhibits Pol I transcription without having an effect on DNA replication, protein translation, and general cellular transcription. A, effectof CX-5461 on total DNA and protein synthesis in HCT-116, A375 and MIA PaCa-2 cells. Histogram showing the proportion of genes that were up- ordownregulated upon treatment of (B) MIA PaCa-2 or (C) A375 cells with 300 nmol/L of CX-5461 for 1 hour. Each column represents a bin of 0.25 units(log2 change in gene expression) and its height is proportional to the number of genes showing such a change in detected mRNA levels. D, comparativeanalysis of the effects of CX-5461 and actinomycin D (act D) on Pol I– and Pol II–driven transcription.






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Figure 4. CX-5461 exhibits broad antiproliferative potency in a panel of cancer cell lines in p53-independent manner, but has minimal effect on viabilityof nontransformed human cells. A, panel of cancer and normal cell lines were treated with various doses of CX-5461 for 96 hours and the resultingeffects on cell viability were measured with CyQUANT assay. B, effect of CX-5461 on cell viability and Pol I transcription of HCT-116, A375, MIA PaCa-2, andBJ-hTERT cells. C, effect of CX-5461 on p53 stabilization in A375 cells. D, CX-5461 exhibit similar activity against p53wt and p53mutant solid cancer cell lines.

Drygin et al.





equivalently inhibit Pol I transcription in cancer and normalcells, but the normal cells can tolerate reductions in rRNAsynthesis without induction of cell death whereas the cancercells cannot.Inhibition of Pol I transcription has been previously

demonstrated to cause nucleolar stress that leads to stabi-lization of p53 and induction of p53-dependent apoptosis(17). We therefore wanted to determine if CX-5461 promotesstabilization of p53. For this purpose, we performed a dose–response analysis of CX-5461 and measured levels of p53 at24 and 48 hours in wild-type (wt) p53 A375 cells. As shown inFigure 4C, whereas CX-5461 slightly increased p53 levels, itdid so at concentrations 3- to 10-fold higher than its IC50 forPol I transcription. Actinomycin D, used as a positive con-trol, caused a more robust increase in p53 levels. In addition,we analyzed the correlation between the sensitivity of celllines to CX-5461 and their p53 genetic status. Among the 44cell lines with known p53 status, 18 had wt p53 and 26carried some form of mutation in the p53 gene (18). Theanalysis revealed that across all tested cell lines CX-5461exhibits similar sensitivity in wt p53 cell lines (median IC50¼144 nmol/L) as it does in cell lines with mutant p53 (medianIC50 ¼ 138 nmol/L), P ¼ 0.56 (Fig. 4D). Interestingly, the 3cell lines most sensitive to CX-5461 were derived fromhematologic malignancies that had wt p53. When we thenfurther analyzed data for correlations of p53 status withsensitivity to CX-5461 among hematologic cancers, we didobserve a significant difference (P ¼ 0.003) between theactivity of CX-5461 in cell lines with wt p53 (3 cell lines,median IC50 ¼ 5 nmol/L) and those with mutant p53 (8 celllines, median IC50 ¼ 94 nmol/L). Although the sample size istoo limited to make strong conclusions, it does hint at anintriguing possibility that nucleolar stress signaling in hema-tologic cancers is more dependent on p53 function and isworthy of future investigation.

CX-5461 induces autophagy and senescence in solidtumor cancer cellsNext, we investigated the mode of cell death promoted by

CX-5461 in the A375 and MIA PaCa-2 cells. Previously, inhibi-tion of rRNA synthesis using "semi-selective" drugs, such asactinomycin D, was shown to induce apoptosis (19). Notsurprisingly, we found that actinomycin D induced apoptosis,as judged by Western blot analysis of PARP cleavage andcaspases-3/7/9 cleavage (Fig. 5A), as well as caspase activationanalysis and Terminal deoxynucleotidyl transferase dUTP nickend labeling (TUNEL) assay (Supplementary Fig. S2). However,no changes in these apoptotic markers were observed for CX-5461, with the exception of very modest cleavage of PARP after48-hour treatment with 1 mmol/L of CX-5461 in MIA PaCa-2(Fig. 5A). These data indicated that apoptosis is not theprimary pathway through which CX-5461 affected cellularviability. In addition, no evidence of necrotic cell death wasdetected, as measured by lactate dehydrogenase (LDH) releaseas late as 48 hours after the initiation of treatment (data notshown). These data led us to consider the possibility that CX-5461 might be inducing autophagy to kill these solid tumorcell lines.

Using immunofluorescence cytometry to monitor the pro-duction of LC3B-II, a known marker of autophagy (20), weobserved that a 24-hour treatment with 300 nmol/L CX-5461caused a significant increase from 15% � 3% to 67% � 2% inLC3B-II staining (P ¼ 0.0041) in A375 and from 19% � 2% to62% � 3% in LC3B-II staining (P ¼ 0.002) in MIA PaCa-2 cells(Fig. 5B). In addition, CX-5461 promoted a concentration-dependent increase in acidic vesicular organelles (AVO)including autophagolysosomes, another marker of autophagicresponse (refs. 14, 21; Fig. 5C, see Supplementary Materialsand Methods). To exclude the possibility that induction ofautophagy by CX-5461 resulted from a decrease in the proteintranslation rate, we performed a time-course analyses tomonitor the effects of CX-5461 on protein synthesis in A375and MIA PaCa-2 cells (Supplementary Fig. S3). Although 24-hour treatment of A375 or MIA PaCa-2 cells with CX-5461dramatically reduced Pol I transcription (Fig. 3D; data notshown), it had only a minor effect on overall protein synthesis(13%–19% reduction at 300 nmol/L and 24%–27% reduction at1 mmol/L). Several reports that have investigated the effects ofoverall protein synthesis inhibition on the viability of cancercells demonstrated that such a reduction (i.e., <30%) shouldnot have a significant effect on cell viability (22). As autophagiccells can themselves have reduced protein synthesis rate (23),the observed effect of CX-5461 on protein synthesis is likelysecondary to the induction of autophagy and not vice versa.

Several recent reports link autophagy to the induction ofcellular senescence (24). The analysis of senescence-asso-ciated marker b-galactosidase demonstrated that, in additionto autophagy, CX-5461 is able to induce senescence in bothcell lines. Treatment with 300 nmol/L CX-5461 for 24 hourscaused an increase in b-galactosidase staining from 6% � 1%to 79% � 1% (P < 0.0001) in A375 cells and from 14% � 2% to55% � 3% (P < 0.0001) in MIA PaCa-2 cells (Fig. 6). Together,the mechanistic data demonstrate that treatment of solidtumor cell lines with CX-5461 induces cellular senescence andautophagy through selective inhibition of rRNA synthesis.

In vivo antitumor activity of CX-5461The antitumor activity of CX-5461 was evaluated in two

murine xenograft models of human cancers, pancreatic car-cinoma (MIA PaCa-2) and melanoma (A375). In these xeno-graft models, CX-5461 was administered orally (50 mg/kg)either once daily or every 3 days. Untreated animals receivedvehicle orally on the equivalent schedule, whereas the positivecontrol gemcitabine was administered intraperitoneally onceevery 3 days at 120 mg/kg. CX-5461 demonstrated significantMIA PaCa-2 TGI with TGI equal to 69% on day 31 (Fig. 7A),comparable to that of gemcitabine (63% TGI). Likewise, CX-5461 demonstrated significant A375 TGI (Fig. 7B) with TGIequal to 79% on day 32. Unpaired t test revealed statisticallysignificant differences between the vehicle-treated and CX-5461–treated groups throughout both studies. CX-5461 waswell tolerated at all tested schedules as judged by the absenceof significant changes in animal body weights. These datademonstrate that a selective inhibitor of Pol I transcriptioncan produce in vivo antitumor responses against solid tumors,with a favorable therapeutic window.






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Figure 5. CX-5461 induces autophagy but not apoptosis in A375 and MIA Paca-2 cancer cells. A, A375 and MIA PaCa-2 cells were treated withvarious doses of CX-5461 for 24 hours and the induction of apoptosis was measured by monitoring activation of caspases-3/7/8/9 and PARP cleavage.Actinomycin D (act D) was used as a positive control. B, A375 andMIA PaCa-2 cells were treated with 300 nmol/L of CX-5461 for 24 hours and the induction ofautophagy was measured by detection of LC3B-II using fluorescent microscopy. C, A375 and MIA PaCa-2 cells were treated with a dose response ofCX-5461 for 24 or 48 hours and the induction of autophagy was measured by acridine orange staining followed by flow cytometry. Statistically significantdifferences from untreated control (UTC) are marked by asterisks (unpaired two-tailed t-test; *, P < 0.05; **, P < 0.01, ***, P < 0.001).

Drygin et al.





Discussion

Deregulated rRNA synthesis plays a fundamental role intumorigenesis (reviewed in refs. 1–5), and Pol I is the keyregulatory enzyme required for production of rRNA (reviewedin refs. 25–27). Herein, we present the results from a discoveryprogram that identified CX-5461, a small molecule that selec-tively inhibits Pol I–driven transcription of rRNA but does notinhibit Pol II–driven mRNA synthesis or DNA or proteinsynthesis. CX-5461 must be viewed globally as targeting aspecific transcriptional event. A host of currently marketeddrugs target transcription, including tamoxifen, cyclosporins,salicylates, and others (28). In the case of CX-5461, it disruptsthe binding of the SL1 transcription factor to rDNA promoter,which inhibits initiation of rRNA synthesis by the Pol I multi-protein complex. The inhibition of rRNA synthesis leads toinduction of senescence and autophagy in a p53-independentmanner in solid tumor cell lines. This response is not driven byreductions in ribosomes or protein synthesis, as the cancercell induces the death pathway signaling long before anyreduction in ribosomes or protein content can occur (29).SL1, a protein complex containing TATA-binding protein–

associated factors, is responsible for Pol I promoter specificity(27, 30, 31). SL1 performs important tasks in transcriptioncomplex assembly, mediating specific interactions betweenthe rDNA promoter region and the Pol I enzyme complex,thereby recruiting Pol I, together with a collection of Pol I–associated factors, to rDNA. Our data indicate that disruption

of the SL1/rDNA complex by CX-5461 results from the inter-ference between SL1 and rDNA and not through dissociationof protein–protein interactions, as was shown for severalknown tumor suppressors (p53, Rb, PTEN; refs. 32–34).

Numerous reports have linked the inhibition of rRNAsynthesis to induction of apoptosis. Actinomycin D, a rela-tively selective inhibitor of rRNA synthesis, inhibits Pol Itranscription during the elongation step (35) and inducesapoptosis in cancer cell lines (36). Inhibition of Pol I tran-scription at the elongation step leads to the formation oftruncated pre-rRNA, which can be interpreted by the cell asbreaks in rDNA. The cell attempts to repair such breaks andultimately triggers the apoptotic response (37). In contrast,CX-5461 inhibits Pol I transcription prior to/during the PICformation step and induces autophagy in solid tumor celllines. When Pol I transcription is inhibited at the initiationstep, cells may interpret this as decreased nutrient availability.The process of autophagy is known to be used by cancer cellsto survive times of limited nutrient availability, but whendriven to the extreme it causes cell death (38). Cancer cells arecontinuously subjected to autophagy inducing stress (e.g.,unfolded protein response; reviewed in ref. 39), and thismay prime them to become more susceptible to certain typesof metabolic stress such as inhibition of rRNA synthesis. Thismay be one of the reasons why rapamycin that targets mTOR,a protein kinase that serves as a sensor for nutrient availabilityand in turn controls both rRNA as well as protein synthesis(40), can also induce autophagy (reviewed in ref. 41). Likewise,

Figure 6. CX-5461 inducessenescence in A375 and MIAPaca-2 cancer cells. A375 andMIA PaCa-2 cells were treatedwith 300 nmol/L of CX-5461 for 24hours and the induction ofsenescence was measured byb-galactosidase staining.

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Figure 7. CX-5461 demonstratesin vivo anticancer activity. MIAPaCa-2 human pancreatic cancer(A) and A375 humanmelanoma (B)xenograft models in which micewere treated with vehicle, CX-5461, or gemcitabine. Theresulting changes in tumorvolumes and body weights aredemonstrated. Statisticalsignificance in tumor volumebetween vehicle-treated and CX-5461–treated groups is marked byasterisks (*, P < 0.05; **, P < 0.01).

Drygin et al.





this might illustrate how CX-5461 takes advantage of theunique qualities of cancer cells and elicits potent and selectiveantitumor activity, whereas having minimal effect on non-transformed cells.CX-5461 exhibited a broad-spectrum antiproliferative activ-

ity of cancer cells in vitro, whereas having a lesser effect on theviability of nontransformed cells, indicating that certain can-cer cells are significantly more susceptible to inhibition ofrRNA synthesis than nontransformed cells. Further, we ana-lyzed if sensitivity of CX-5461 correlates with the p53 muta-tional status of cells, as inhibition of Pol I was previouslyreported to cause nucleolar stress that leads to stabilization ofp53 and induction of p53-dependent apoptosis (17). Across allcell lines, CX-5461 exhibited similar sensitivity in wt p53 andmutant p53 cell lines. However, wt p53 cell lines derived fromhematologic malignancies appeared highly sensitive to CX-5461 and this apparent trend is the focus of additionalinvestigations.Herein, we present the results of a discovery and develop-

ment program that identified CX-5461, a small molecule thatselectively inhibits Pol I transcription of rRNA. CX-5461 doesnot inhibit Pol II transcription of mRNA or inhibit the synth-esis of DNA or proteins. CX-5461 targets the SL1 transcriptionfactor of the Pol I complex and induces autophagy andsenescence among solid tumor cell lines and selectively killscancer cells relative to normal cells. Studies with human solidtumors grown in murine xenograft models revealed that CX-5461 can be orally administered with favorable pharmacoki-netics and an antitumor efficacy without changes in bodyweight or overt toxicity. In addition, our data demonstratethat the anticancer activity of CX-5461 against solid tumors

does not depend on the p53 pathway that is frequentlymutated in many types of cancer. Thus, CX-5461 representsa first-in-class oral small molecule therapeutic agent thatselectively targets Pol I transcription and induces autophagyin solid tumors.

Disclosure of Potential Conflicts of Interest

D. Drygin, J. Bliesath, C.B. Ho, S. O’Brien, C. Proffitt, M. Omori, M. Haddach,M. Schwaebe, A. Siddiqui-Jain, N. Streiner, D. Ryckman, K. Anderes and W.G.Rice own stock options in Cylene Pharmaceuticals. Cylene Pharmaceuticals hasa pending patent application covering composition of matter for CX-5461. M.Haddach and M. Schwaebe are named among the inventors. The other authorsdeclared no potential conflicts of interest.

Acknowledgments

We thank Mr. Jerome Michaux for his help in synthesizing CX-5461,Dr. William Hahn from Harvard Medical School for his kind gift of BJ-hTERTcells, and Dr. Lucio Comai from Keck Medical School for his kind gift ofantibodies to SL1 subunits.

Grant Support

This work was supported in part by Cylene Pharmaceuticals Inc. and in partby grants to R.D. Hannan and E. Sanij from the National Health and MedicalResearch Council (NHMRC) of Australia. R.D. Hannan was supported by aresearch fellowship from the NHMRC. M.J. Bywater was supported by anNHMRC postgraduate scholarship. J.E. Quin was supported by an Australianpostgraduate award.

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received May 14, 2010; revised November 11, 2010; accepted November 15,2010; published OnlineFirst December 15, 2010.

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2011;71:1418-1430. Published OnlineFirst December 15, 2010.Cancer Res Denis Drygin, Amy Lin, Josh Bliesath, et al. Inhibits Ribosomal RNA Synthesis and Solid Tumor GrowthTargeting RNA Polymerase I with an Oral Small Molecule CX-5461

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