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. Author manuscript; available in PMC: 2018 Apr 1.
Published in final edited form as: Br J Haematol. 2017 Apr;177(1):80–94. doi: 10.1111/bjh.14525

RNA Polymerase I Inhibition with CX-5461 as a Novel Therapeutic Strategy to Target MYC in Multiple Myeloma

Hans C Lee 1, Hua Wang 1, Veerabhadran Baladandayuthapani 2, Heather Lin 2, Jin He 1, Richard J Jones 1, Isere Kuiatse 1, Dongmin Gu 1, Zhiqiang Wang 1, Wencai Ma 1, John Lim 3, Sean O’Brien 3, Jonathan Keats 4, Jing Yang 1, Richard E Davis 1, Robert Z Orlowski 1,5
PMCID: PMC5695568  NIHMSID: NIHMS836819  PMID: 28369725

Abstract

Dysregulation of MYC is frequently implicated in both early and late myeloma progression events, yet its therapeutic targeting has remained a challenge. Among key MYC downstream targets is ribosomal biogenesis, enabling increases in protein translational capacity necessary to support the growth and self-renewal programmes of malignant cells. We therefore explored the selective targeting of ribosomal biogenesis with the small molecule RNA polymerase (pol) I inhibitor CX-5461 in myeloma. CX-5461 induced significant growth inhibition in wild-type (WT) and mutant TP53 myeloma cell lines and primary samples, in association with increases in downstream markers of apoptosis. Moreover, Pol I inhibition overcame adhesion-mediated drug resistance and resistance to conventional and novel agents. To probe the TP53-independent mechanisms of CX-5461, gene expression profiling was performed on isogenic TP53 WT and knockout cell lines and revealed reduction of MYC downstream targets. Mechanistic studies confirmed that CX-5461 rapidly suppressed both MYC protein and MYC mRNA levels. The latter was associated with an increased binding of the RNA-induced silencing complex (RISC) subunits TARBP2 and AGO2, the ribosomal protein RPL5, and MYC mRNA, resulting in increased MYC transcript degradation. Collectively, these studies provide rationale for the clinical translation of CX-5461 as a novel therapeutic approach to target MYC in myeloma.

Keywords: multiple myeloma, myeloma therapy, myeloma cell lines, oncogenes

INTRODUCTION

The introduction of novel classes of drugs, such as proteasome inhibitors and immunomodulatory agents, has significantly improved outcomes in multiple myeloma over the last decade (Kumar, et al 2014). Nevertheless, myeloma remains largely incurable (Alexanian, et al 2012, Barlogie, et al 2014), and resistance to standard therapies is an inevitable challenge that most patients will encounter during their disease course. Thus, a continued need exists to identify new classes of drugs that target novel pathways for these patients.

The proto-oncogene MYC is a master transcriptional regulator whose target genes affect a diverse array of cellular processes. Dysregulation of MYC has been frequently implicated in malignant transformation through its ability to drive cell growth, metabolism and cell cycle progression (Dang 2012, Meyer and Penn 2008). In multiple myeloma, increases in MYC gene expression signatures and nuclear immunohistochemical staining have been shown in primary samples relative to those with the benign precursor monoclonal gammopathy of undetermined significance (MGUS) (Chiecchio, et al 2009, Chng, et al 2011). This is mirrored in part by the Vk*MYC murine model, in which activation-induced deaminase (AID)-dependent MYC up-regulation in germinal centre B cells recapitulates many features of the human disease, while control mice develop MGUS (Chesi, et al 2008). Finally, amplified MYC expression has been increasingly recognized as a marker of high-risk disease associated with a significantly worse outcome that can be present in up to 50% of myeloma patients (Affer, et al 2014, Glitza, et al 2015, Walker, et al 2014).

Despite knowledge of the oncogenic role of MYC in myeloma and other human cancers, direct therapeutic targeting of MYC at the protein level has remained a challenge due to lack of a clear ligand-binding domain (Verdine and Walensky 2007). Therefore, both upstream and downstream approaches that target MYC indirectly are being explored. These have included disruption of MYC transcription through Bromodomain and extraterminal domain (BET) inhibitors, a strategy now being tested in early phase clinical trials (Delmore, et al 2011). An alternative approach investigated pre-clinically has focused on blocking the heterodimerization of MYC with its binding partner MAX, thereby preventing transactivation of downstream gene targets (Soucek, et al 2008, Yin, et al 2003). Finally, further downstream approaches have investigated the possibility of inhibiting MYC target genes and their effector pathways that may be selectively activated in tumour cells compared to normal cells (Dang 2012). These have included targets of cell cycle progression, such as cyclin dependent kinase 1 (CDK1) (Goga, et al 2007) and 2 (CDK2) (Molenaar, et al 2009) as well as metabolism such as NUAK family kinase 1 (NUAK1, previously termed ARK5) (Liu, et al 2012).

Another key MYC-downstream effect is up-regulation of ribosomal biogenesis, resulting in increased protein translational capacity and biomass accumulation that is characteristic of neoplastic cells (Ruggero 2013). Formation of mature 60S and 40S ribosomal subunits occurs in a coordinated series of steps beginning in the nucleolus, where 47S precursor ribosomal RNA (rRNA) is transcribed, processed, and assembled together with ribosomal proteins imported from the cytoplasm (Montanaro, et al 2012). Because the exclusive function of RNA polymerase (pol) I is the transcription of rRNA, which is the rate limiting step of ribosomal biogenesis (Chedin, et al 2007), this provided the rationale of exploring its selective inhibition as a novel approach in cancer therapy (Haddach, et al 2012, Peltonen, et al 2014). Among the first small molecule RNA pol I inhibitors in development has been CX-5461, which binds to Selectivity factor 1 (SL1) of the RNA pol I pre-initiation complex, and prevents recruitment of RNA pol I and other cofactors to the rDNA transcriptional start site (Drygin, et al 2011). A recent study with CX-5461 demonstrated potent activity in an Eμ-myc driven lymphoma model, which was primarily attributed to a TP53-dependent mechanism of action through activation of the ribosomal protein/Mouse double minute 2 homolog (MDM2)/TP53 nucleolar surveillance pathway (Bywater, et al 2012). A separate study also demonstrated its activity in a broad range of solid tumour models, although notably it suggested that CX-5461 acts in a TP53-independent manner mediated through autophagy (Drygin, et al 2011).

In the present study, we sought to characterize the therapeutic potential of RNA pol I inhibition with CX-5461 in multiple myeloma. Our results demonstrated that CX-5461 has potent activity in a variety of myeloma preclinical models both in vitro and in vivo. Mechanistic studies using isogenic TP53 wild-type (WT) and knock-out (KO) myeloma cell line models suggest that the majority of the effects of RNA pol I inhibition are TP53-independent. Notably, we report the unexpected finding that CX-5461 induces rapid down-regulation of MYC transcript levels and global suppression of MYC target genes. Finally, we show that the therapeutic efficacy of CX-5461 is enhanced with increased MYC expression, probably related to basal increases in ribosomal biogenesis that prime MYC-overexpressing cells to the downstream effects of the nucleolar stress response. Taken together, these findings provide pre-clinical evidence that targeting RNA pol I with CX-5461 may be an effective, novel therapeutic strategy in multiple myeloma and other MYC-driven cancers.

MATERIALS AND METHODS

Cell lines and primary patient samples

Five human myeloma cell lines (MM1.S, MOLP-8, RPMI-8226, U266 and H1112) were used for these experiments. Cells were maintained in RPMI 1640 medium (Corning Cellgro®, Manassas, VA, USA) with L-glutamine supplemented with 5% fetal bovine serum and 1% penicillin/streptomycin. Cell lines were authenticated using the University of Texas MD Anderson Characterized Cell Line Core using the short-tandem-repeat method. Drug-resistant (bortezomib, carfilzomib, doxorubicin and lenalidomide) cell lines were generated as described previously (Bjorklund, et al 2011, Dalton, et al 1986, Kuhn, et al 2009). Isogenic MM1.S and MOLP-8 TP53 WT and KO cell lines were generated using sequence-specific zinc-finger nucleases (ZFNs; CompoZr® Knockout ZFNs; Sigma Aldrich, St. Louis, MO, USA) targeting either a non-specific sequence or exon 7 of the TP53 DNA binding domain. MYC overexpression was accomplished using a pCDH-puro-cMyc plasmid, which was a gift from Jialiang Wang (Addgene plasmid # 46970, Addgene, Cambridge, MA, USA) (Cheng, et al 2013), and transduced using a pCDH Lentiviral expression system (Systems Biosciences, Mountain View, CA, USA). Primary myeloma patient samples were purified using magnetic CD138 micro beads (Miltenyi Biotec Inc, San Diego, CA, USA). All primary patient samples were acquired with approval from the Institutional Review Board at the University of Texas MD Anderson Cancer Center after informed consent was obtained in compliance with the Declaration of Helsinki.

Viability assays

Cell viability was assessed using water-soluble tetrazolium (WST1) colourimetric assays (Roche, Indianapolis, IN, USA). Briefly, 10,000 cells in 100 μl of culture media were plated on 96-well plates (Sigma Aldrich) and treated with CX-5461 (Senhwa Biosciences, San Diego, CA, USA) or other agents, including bortezomib, carfilzomib, lenalidomide, doxorubicin and Nutlin-3a (Selleck Chemicals, Houston, TX, USA). Following drug treatment (range, 48–72 h), 10 μl WST1 reagent was added to each well, and absorbance measured at a wavelength of 450 nM using the Victor3V plate reader (Perkin Elmer, Waltham, MA, USA). Experiments were performed in quadruplicate, and absorbance was normalized to vehicle controls, which were arbitrarily set at 100% viability.

Fluorescence-activated cell sorting (FACS)

For apoptosis assays, cells were incubated with Annexin-V Pacific Blue conjugate and TO-PRO-3 (Invitrogen, Waltham, MA, USA) in Annexin-V buffer (Invitrogen). Flow cytometry was performed using an LSR Fortessa cytometer (BD Biosciences, San Jose, CA, USA). FlowJo v10 (Tree Star, Inc., Ashland, OR, USA) was used for data analysis.

Immunoblotting

Cell pellets were lysed with Triton X-100 lysis buffer (Cell Signaling, Beverly, MA, USA) supplemented with protease inhibitors and phosphatase inhibitors (Roche, Indianapolis, IN, USA). Protein concentrations were determined using a colourimetric Bradford assay per the manufacturer instructions (Bio Rad, Hercules, CA, USA). Protein lysates were denatured with sodium dodecyl sulfate (SDS) at 95°C and subjected to electrophoresis on gradient NuPAGE® Bis-Tris gels (Life Technologies, Grand Island, NY, USA). Proteins were then transferred to nitrocellulose membranes (Life Technologies) and probed with primary antibodies to cleaved-PARP, cleaved caspase-3, -8, or - 9, CDKN1A (also termed p21) (Cell Signaling), MYC (Abcam, Cambridge, UK), and ACTB (Sigma Aldrich). Blots were imaged using chemiluminescence solution (GE Healthcare Life Sciences, Pittsburgh, PA, USA) with an SRX-101A processor (Konica Minolta, Tokyo, Japan).

Co-culture assays

Green fluorescent protein (GFP)+ HS5 stromal cells (50,000 cells/well) and MM1.S plasma cells (200,000 cells/well) were plated on 24-well plates (Sigma Aldrich) one day prior to treatment with CX-5461. After 48-h of drug treatment, cells were harvested, and apoptosis was assessed using flow cytometry as described earlier. HS5 stromal and MM1.S plasma cells were sorted by GFP+ positivity, allowing for separate analyses of the cell populations.

Gene expression profiling

Isogenic TP53 WT and TP53 KO MM1.S (5 pairs of single-cell clones) and MOLP-8 (3 pairs) cell lines were treated with 200 nM CX-5461 or vehicle for 48 h. RNA was extracted from cell pellets using the RNAqueous kit (Ambion, Grand Island, NY, USA), amplified with biotin-labelled primers using the Illumina TotalPrep RNA Amplification kit (Ambion, Grand Island, NY, USA), and hybridized to the Illumina HT12 version 4 human whole-genome arrays (Illumina, San Diego, CA, USA). Data processing and normalization were performed as previously described (Ma, et al 2010). After selecting for genes expressed above background in at least 25% of samples, log2-transformed expression values of the vehicle samples were subtracted from those of the corresponding CX-5461-treated samples. Subtracted values for different probes for the same gene were averaged, and then data from the TP53 KO clones were averaged and ranked for Gene Set Enrichment Analysis (GSEA) using the C2 and C5 categories in the Molecular Signatures Database (MSigDB; http://software.broadinstitute.org/gsea/msigdb/).

Real time polymerase chain reaction (qPCR)

Cells were lysed and total RNA extracted and purified using the Direct-zol™ RNA MiniPrep kit (Zymo Research, Irvine, CA, USA) per the manufacturer’s instructions and quantified using a spectrophotometer (NanoDrop 2000; Thermo Scientific, Wilmington, DE). cDNA synthesis was performed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Grand Island, NY, USA) per the manufacturer’s protocol. qPCR was carried out with the TaqMan Gene Expression (Life Technologies) or SYBR Green (Life Technologies) assays using probes for MYC (Applied Biosystems) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Applied Biosystems). For pre-rRNA qPCR, the following pre-rRNA primers were used: 5′ETS 851 5′GAACGGTGGTGTGTCGTT, 5′ 1297 CAGGTGTTTCCTCGTACCG, and 45S rRNA ITS1 5′ CCGGCTTGCCCGATTT. qPCR reactions were performed in triplicate on the StepOnePlus PCR System (Applied Biosystems). The comparative CT method was used for relative quantification of CX-5461- or vehicle-treated samples, which were normalized to GAPDH.

Murine xenograft models

Six- to 8-week-old female non-obese diabetic, severe combined immunodeficiency, interleukin (IL)-2 receptor γ null (NOD-scidIL2Rgnull) mice (Jackson Laboratory, Bar Harbor, ME, USA) were injected via tail vein with 1 ×106 isogenic MM1.S TP53 WT and KO cells containing a dual GFP/luciferase Lentiviral vector derived from pLV-TH (Trono vector) (Wiznerowicz and Trono 2003). Twice weekly bioluminescent imaging was performed after administration of 10 μl/g D-luciferin substrate (Life Technologies) using the IVIS Spectrum (Perkin Elmer) in vivo imaging system. On day +8, treatment with either weekly vehicle (50 mM sodium phosphate buffer, pH 4.5) or CX-5461 (50 mg/kg) by intraperitoneal injection was initiated in a 1:1 randomization (5 mice/treatment group). Twice weekly bioluminescent imaging was continued to monitor tumour growth, and mice were euthanized by CO2 inhalation per institutional guidelines when showing signs of distress. All experiments were performed in accordance with procedures and protocols approved by the MD Anderson Animal Care and Use Facility.

Co-immunoprecipitation (co-IP) assay

Pellets from cells treated with CX-5461 or vehicle were lysed using Triton X-100 lysis buffer with protease and phosphatase inhibitors. Each lysate (500 μg) was then immunoprecipitated overnight at 4°C with 5 μg of anti-trans-activation-responsive RNA-binding protein (TARBP2; Lifespan Biosciences, Seattle, WA, USA) or anti-mouse IgG control (Sigma Aldrich). Immune complexes were captured using magnetic Protein A/G beads (Thermo Scientific) per manufacturer’s instructions. Protein was denatured using SDS buffer and immunoblotted using antibodies to TARBP2 (Abcam) or Ribosomal protein L5 (RPL5; Abcam).

RNA immunoprecipitation (RIP) assay

RIP on vehicle- or CX-5461-treated cells was performed using the Imprint® RNA immunoprecipitation kit (Sigma-Aldrich) per the manufacturer’s instructions. Antibodies to Argonaute RNA-induced silencing complex (RISC) catalytic component 2 (AGO2; Sigma-Aldrich) or IgG control (Sigma Aldrich) were used for immunoprecipitation. cDNA synthesis and qPCR of immunoprecipitated RNA were performed for MYC and GAPDH as described earlier. Calculation of mRNA fold-enrichment of each RIP reaction was performed using a Microsoft® Excel worksheet template provided on the manufacturer’s product website (Sigma Aldrich).

Statistics

For in vitro experiments, statistical analyses and figure generations were performed using Excel (Microsoft Corporation, Redmond, WA, USA) and GraphPad Prism 6 (GraphPad Software Inc, La Jolla, CA, USA). Error bars were represented as the standard error of the mean (SEM), and significance of drug effects was further evaluated using a one-tailed Student’s paired t-test. The Kaplan-Meier method was used to estimate the distribution of overall survival (OS), and log-rank testing was performed to evaluate the difference in survival between groups, with OS defined as the time from tail-vein injection of myeloma cells to the time of death or of euthanasia. SAS version 9.2 (SAS Institute, Inc., Cary, NC, USA) and S-Plus version 8.04 (TIBCO Software, Inc., Palo Alto, CA, USA) were used to carry out the computations for in vivo experiments.

RESULTS

CX-5461 demonstrates anti-myeloma activity in TP53 wild-type and mutant models

To evaluate the efficacy of CX-5461 in multiple myeloma, we initially exposed a panel of TP53 WT (MM1.S and MOLP-8) and TP53 mutant (U266 and RPMI-8226) myeloma cell lines to the drug. Treatment with CX-5461 induced a time- and dose-dependent decrease in cell viability, with half-maximal inhibitory concentrations (IC50) after 72 h in the nM range for all cell lines (Figure 1A, MM1.S, 55 nM; MOLP-8, 157 nM; U266, 691 nM; RPMI 8226, 430 nM; time-dependence not shown). Flow cytometry revealed that this was due to induction of apoptosis based on the presence of greater Annexin-V staining after 48-h of treatment with CX-5461 in both TP53 WT and mutant cells (Figure 1B). This corresponded with an increased abundance of cleaved forms of poly (ADP-ribose) polymerase (PARP), caspase-9 and caspase-3 with CX-5461 treatment, though this was more pronounced in the MM1.S and MOLP-8 TP53 WT cells (Figure 1C). Finally, CX-5461 treatment also showed activity in primary patient samples, including a heavily pre-treated patient with relapsed/refractory myeloma, and a de novo plasma cell leukaemia patient with del 17p corresponding to loss of the TP53 gene (Figure 1D).

Figure 1. CX-5461 and TP53 wild-type and mutant myeloma models.

Figure 1

Figure 1

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(A) Myeloma cell lines shown were treated with varying concentrations of CX-5461 for 72 h. Cell proliferation was assessed using WST1 tetrazolium salt assays in quadruplicate experiments, and values were normalized to the vehicle control. (B) Assessment of apoptosis by staining for Annexin-V and TO-PRO-3 using flow cytometry after 48 h of exposure to 500 nM CX-5461. (C) Immunoblotting of markers of apoptosis including cleaved PARP, caspase-9, and caspase-3 after treatment with 1000 nM CX-5461. (D) Cell viability of primary patient samples after 72-h CX-5461 treatment assessed using WST-1 tetrazolium salt assay. Patient 1 was a 71-year-old female with relapsed/refractory IgA kappa myeloma, with prior treatments including lenalidomide, bortezomib, carfilzomib, pomalidomide, doxorubicin and high-dose melphalan and del 13q on fluorescence in situ hybridization (FISH). Patient 2 was a 76-year-old female with de novo plasmacytic leukaemia with 80.5% interphase nuclei containing del 17p on FISH.

CX-5461 retains activity in drug-resistant myeloma models

The role of the bone marrow microenvironment has been well established in potentiating cell adhesion-mediated drug resistance through the interaction of myeloma cells with bone marrow stromal cells and the extracellular matrix (Hideshima, et al 2007). Therefore, to mimic the bone marrow microenvironment, MM1.S cells were co-cultured with HS5 stromal cells, and subsequently treated with CX-5461 (Figure 2A). The presence of HS5 stromal cells did abrogate some of the pro-apoptotic effect of CX-5461, leading to a 25% absolute increase in cell viability compared to treatment without HS5 stromal cells. However, this was markedly lower than the 58% absolute increase in cell viability that the presence of HS5 cells conferred when dexamethasone was used as a positive control.

Figure 2. CX-5461 in drug-resistant myeloma models.

Figure 2

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(A) MM1.S cells were treated with vehicle or 500 nM CX-5461 for 48 h in the absence or presence of GFP+ HS5 stromal cells. Cell viability was assessed using flow cytometry with Annexin-V (A5)/TO-PRO-3 (PI) staining in triplicate experiments. Treatment with 10 μM dexamethasone (dex) was used as a positive control. (B) RPMI 8226 and RPMI 8226 bortezomib-resistant (BR), (C) KAS-6/1 and KAS-6/1 carfilzomib-resistant (CR), (D) RPMI 8226 and RPMI 8226 doxorubicin-resistant (DoxR), and (E) MM1.S and MM1.S lenalidomide-resistant (R10R) cell lines were treated with CX-5461 for 72 h. Cell proliferation was assessed using WST1 tetrazolium salt assays in quadruplicate experiments, and values were normalized to the vehicle control.

Next, we tested CX-5461 on a panel of cell lines resistant to standard myeloma therapeutic agents, including bortezomib, carfilzomib, doxorubicin and lenalidomide, which were generated by gradually exposing the parental cell lines to increasing drug concentrations (Figure S1). Treatment with CX-5461 induced a dose-dependent decrease in cell viability in all drug-resistant cell line models, with IC50 values in the nM range (Figures 2B–2E, RPMI-8226 BR, 639 nM; RPMI-8226 CR, 55 nM; RPMI-8226 Dox40, 464 nM; MM1.S R10R, 44 nM). These results suggest that RNA pol I inhibition with CX-5461 does not demonstrate significant cross-resistance to other myeloma drugs.

TP53 status and myeloma sensitivity to CX-5461

A prior study suggested that the activity of CX-5461 was largely TP53-dependent due to sequestration of MDM2, the E3 ubiquitin ligase of TP53, by ribosomal proteins released through the nucleolar stress response, ultimately leading to TP53 accumulation (Bywater, et al 2012). While decreased drug sensitivity was seen in our TP53 mutant compared to TP53 WT cell lines, their IC50 values remained in the nanomolar range, suggesting that TP53-independent mechanisms must also play a key role. To explore TP53-independent effects of CX-5461, isogenic TP53 WT and KO cell line models were generated by introducing sequence-specific ZFNs targeting exon 7 of the TP53 DNA binding domain, or a non-targeted sequence, into TP53 WT MM1.S and MOLP-8 cells. After single cell cloning, colonies of interest were selected by TP53 gene resequencing, and Western blot confirmed the presence of an absent or truncated TP53 protein in several KO clones (Figure 3A). Phenotypically, the TP53 KO cells demonstrated resistance to drugs known to act through TP53-dependent mechanisms, such as the MDM2 inhibitor Nutlin-3a (Vassilev, et al 2004) (Figure 3B). Also, in the case of doxorubicin, whose activity is strongly influenced by TP53 status (el-Deiry, et al 1993), TP53 KO cells were relatively resistant (Figure 3C, left panel), and showed decreased CDKN1A transactivation with drug treatment (Figure 3C, right panel).

Figure 3. CX-5461 and isogenic TP53 WT and KO myeloma models.

Figure 3

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Isogenic TP53 wild type (WT) and knockout (KO) MM1.S and MOLP-8 cell lines were generated using sequence specific ZFNs targeting TP53 exon 7 or a non-targeting sequence. (A) Immunoblotting of basal TP53 expression in MM1.S and MOLP-8 TP53 WT and KO clones. (B) Assessment of cell viability with WST1 tetrazolium salt assays in TP53 WT and KO isogenic cell lines after exposure to Nutlin-3a. (C) Cell viability in MOLP-8 TP53 WT and KO cells after 48-h treatment with doxorubicin (left panel), and immunoblotting for CDKN1A expression after 48-h treatment with 50 nM doxorubicin (right panel). (D) Assessment of cell viability in isogenic MM1.S and MOLP-8 TP53 WT and KO cells treated with CX-5461 for 72 h. (E) Assessment of apoptosis in isogenic cell lines treated with vehicle or 200 nM CX-5461 for 48 h by Annexin-V and TO-PRO-3 staining. (F) Immunoblotting of downstream markers of TP53 activation and apoptosis after 500 nM CX-5461 treatment in isogenic MM1.S TP53 WT and KO cells.

We next evaluated the effect of TP53 status in relation to CX-5461 sensitivity using our isogenic myeloma cell line models. Overall, CX-5461 demonstrated potent anti-proliferative effects regardless of TP53 WT or KO status, with a mild decrease in sensitivity in the TP53 KO cells (Figure 3D). Notably, IC50 values for the MM1.S TP53 WT and KO cell lines were 71 and 82 nM, respectively, and 63 and 141 nM for the MOLP-8 TP53 WT and KO cells, respectively. Consistent results were seen when measuring apoptosis with Annexin-V staining by flow cytometry (Figure 3E). As expected, CX-5461 treatment led to strong induction of TP53 and CDKN1A in the TP53 WT cells, probably due to activation of the ribosomal protein-MDM2-TP53 nucleolar surveillance pathway, which was absent in the TP53 KO cells (Figure 3F). Nevertheless, expression of downstream markers of the intrinsic apoptotic pathway were similar regardless of TP53 status although, notably, the extrinsic pathway, represented by cleaved caspase-8 expression, was abrogated in TP53 KO cells. This may be explained by the role of TP53 in regulating TNFRSF10B (previously termed DR5) expression, which directly mediates downstream caspase-8 activation (Surget, et al 2012, Wu, et al 1997).

CX-5461 demonstrates therapeutic activity in vivo

To test the anti-myeloma activity of CX-5461 in vivo, we established systemic isogenic GFP+ MM1.S TP53 WT and KO myeloma murine xenografts injected via tail-vein. Mice were treated intraperitoneally with vehicle or CX-5461 50 mg/kg once weekly starting one week after cell injection, and tumour volume was assessed by serial bioluminescent imaging (Figure 4A). Median overall survival in the vehicle-treated MM1.S TP53 WT cohort was 41 days, while median survival in the drug-treated mice was not reached (P=0.05, Figure 4B). In the MM1.S TP53 KO mice, the effect of CX-5461 was more modest as there was only a trend towards improved survival in the drug-treated mice (P=0.1, Figure 4C). Overall, the vehicle-treated TP53 KO mice had a decreased median overall survival compared to the vehicle-treated TP53 WT mice (37.5 days versus 41 days, P=0.02, Figure S2), recapitulating the inferior clinical outcomes of TP53 mutant or deleted myeloma (Avet-Loiseau, et al 2012, Chng, et al 2007).

Figure 4. CX-5461 activity in an in vivo murine model.

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(A) Bioluminescent in vivo imaging of GFP/luciferase+ MM1.S TP53 wild type (WT) xenograft models in NOD-SCID IL2Rgnull mice at day +39 after tail-vein injection. Mice (N=10) were randomized to receive either vehicle (V) or CX-5461 (CX), 50 mg/kg, by weekly intraperitoneal injection beginning on day +8. (B) Overall survival of MM1.S TP53 WT murine xenografts (N=10) randomized to vehicle or CX-5461 treatment. (C) Overall survival of MM1.S TP53 knockout murine xenografts (N=10) randomized to vehicle or CX-5461 treatment.

CX-5461 induces MYC down-regulation through enhanced MYC mRNA degradation

In order to further characterize the TP53-independent effects of CX-5461, we treated the MM1.S and MOLP-8 isogenic TP53 WT and KO cells with vehicle or 200 nM CX-5461 for 48 h prior to gene expression profiling (GEP). Comparisons between treated and untreated cells demonstrated that the transcriptional changes induced by CX-5461 were largely TP53-independent (data not shown). GSEA of data from TP53 KO clones, using the C2 and C5 categories in the Molecular Signatures Database (MSigDB), enriched gene sets consistent with significant down-regulation of MYC transcriptional targets in both the TP53 WT and KO cell lines (Figure 5A, Figure S3) (Schuhmacher, et al 2001). To assess if MYC itself was down-regulated by RNA poI I inhibition, isogenic TP53 WT and KO cell lines were treated with CX-5461, and MYC transcript and protein levels were assessed by qPCR and Western blotting, respectively. Indeed, down-regulation of MYC mRNA (Figure 5B) and protein (Figure 5C) levels was observed, with decreases in MYC protein levels lagging behind reductions in MYC mRNA levels, which was observed as early as one-hour post-CX-5461 (Figure S4). Moreover, MYC down-regulation was also demonstrated after CX-5461 treatment in the previously described plasma cell leukaemia patient sample with deletion 17p (Figure 5D).

Figure 5. CX-5461 and MYC mRNA and protein levels.

Figure 5

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(A) Gene expression profiling was performed on paired isogenic TP53 wild type (WT) and knockout (KO) MM1.S and MOLP-8 isogenic cell lines after 48 h treatment with 200 nM CX-5461 or vehicle. Gene expression values from untreated samples were subtracted from treated samples in TP53 KO cell lines. Probes of subtracted data were then merged and ranked for Gene Set Enrichment Analysis (GSEA) to identify TP53-independent effects of CX-5461. Shown is the GSEA enrichment plot for subtracted probes of MM1.S TP53 KO cells after treatment with CX-5461 for the SCHUHMACHER_MYC_TARGETS_UP gene set. (B) Isogenic TP53 WT and KO MM1.S and MOLP-8 cell lines were treated with 200 nM CX-5461, and MYC mRNA levels were evaluated at different time points with quantitative polymerase chain reaction using the comparative CT method. Values were normalized to GAPDH as an internal control. (C) MYC protein expression after treatment with 500 nM CX-5461 in MM1.S TP53 WT and KO cell lines. (D) Cell viability and MYC mRNA levels in a primary patient sample after treatment with 1 μM CX-5461. (E) Immunoblotting of RPL5 after co-immunoprecipitation with TARBP2 of cell lysates treated with vehicle or 500 nM CX-5461 with murine IgG as a negative control. (F) Relative enrichment of MYC mRNA after RNA immunoprecipitation (RIP) with AGO2 at T=0 and T=30 minutes after 500 nM CX-5461. CT values of each RIP reaction were normalized to input fractions to obtain ΔCT, and MYC fold-enrichment represent ΔΔCT values derived from normalized ΔCT values of AGO2 and IgG RIPs at each time point.

Recent studies have suggested ribosomal biogenesis is tightly controlled by an auto-regulatory feedback mechanism in which ribosomal proteins RPL5 and RPL11 bind to the 3′ untranslated region (UTR) of MYC mRNA and facilitate its degradation through RISC (Dai, et al 2007, Liao, et al 2014). Because RNA pol I inhibition is known to induce a nucleolar stress response and increase the availability of free ribosomal proteins, RISC-mediated degradation of MYC mRNA was explored as one possible mechanism of CX-5461-mediated MYC down-regulation. Indeed, treatment with CX-5461 led to increased pull-down of RPL5 when immunoprecipitated with the RISC subunit TARBP2 compared to vehicle-treated controls (Figure 5E). Moreover, RNA immunoprecipitation assays with the catalytic RISC subunit AGO2 demonstrated enrichment of MYC mRNA with CX-5461 treatment (Figure 5F). These results suggest that CX-5461 may induce degradation of MYC transcripts by enhancing the cooperative binding of ribosomal proteins, RISC subunits and MYC mRNA.

MYC overexpression enhances sensitivity to RNA polymerase I inhibition

Previous studies demonstrated profound sensitivity to RNA pol I inhibition in pre-malignant and malignant B cells derived from an Eμ-MYC murine lymphoma model compared to normal B cells from wild-type littermates (Bywater, et al 2012). Because premalignant B cells in this model are characterized by their constitutive activation of the MYC transgene, these data suggested that MYC dysregulation may play a central role in determining sensitivity to RNA pol I inhibition (Bywater, et al 2012). To test this hypothesis in myeloma, we overexpressed MYC in the H1112 myeloma cells (Figure 6A), which lack a MYC translocation at baseline and have relatively low basal levels of MYC expression compared to other myeloma cell lines (Dib, et al 2008). As expected, this resulted in a corresponding increase in basal pre-rRNA transcript levels in the H1112pCDH-MYC compared to H1112pCDH cells (Figure 6B). Moreover, the H1112pCDH-MYC cells also demonstrated markedly increased sensitivity to CX-5461 (IC50 76 nM) compared to their non-MYC overexpressed (IC50 708 nM) counterparts (Figure 6C), suggesting a critical role of MYC dysregulation in sensitizing cells to RNA pol I inhibition.

Figure 6. MYC overexpression and sensitivity to RNA polymerase I inhibition.

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(A) Immunoblotting of MYC in H1112 cell lines with vector only (pCDH) or the MYC overexpression vector (pCDH-MYC). (B) Basal pre-rRNA transcript levels in H1112pCDH and H1112pCDH-MYC cells assessed by real time polymerase chain reaction (qPCR). (C) Cell viability of H1112pCDH and H1112pCDH-MYC after a 72-h treatment with CX-5461; *P < 0.05, **P < 0.01, ***P < 0.001.

DISCUSSION

The role of dysregulation of the proto-oncogene MYC in both early and late multiple myeloma progression events has been well established, and the tumourigenicity of increased MYC expression is often attributed to its function as a master transcriptional regulator of cellular growth processes (Dang 2013). Emerging evidence has suggested that MYC is a key modulator of ribosomal biogenesis, enabling global increases in protein translational capacity to support the growth and self-renewal programs of malignant cells (van Riggelen, et al 2010). Thus, given the relationship between myeloma pathobiology, MYC dysregulation, and ribosomal biogenesis, we hypothesized that selective inhibition of RNA pol I with the small molecule inhibitor CX-5461 may represent a novel therapeutic strategy in multiple myeloma.

In this study, we show that treatment with CX-5461 led to growth inhibition and apoptosis in both drug-naïve and drug-resistant myeloma preclinical models. The effect of RNA pol I inhibition had been previously reported as largely TP53-dependent through induction of the nucleolar stress response, where free ribosomal proteins released from the nucleolus into the nucleoplasm bind to MDM2, leading to increased stabilization of TP53 (Bywater, et al 2012). Consistent with these prior studies, there was enhanced sensitivity to CX-5461 in TP53 WT compared to TP53 mutant myeloma cell lines, although the IC50 values in the TP53 mutant cell lines remained in the nanomolar range. This is in contrast to our earlier studies with the second-generation MDM2 inhibitor MI-63, in which TP53 mutant myeloma models showed clear resistance to the drug (Gu, et al 2014). Therefore, this suggested that other mechanisms besides activation of the MDM2-TP53 axis could strongly contribute to the therapeutic efficacy of RNA pol I inhibition with CX-5461. Indeed, GEP and GSEA performed on isogenic TP53 WT and KO myeloma cell lines revealed that the majority of changes in gene expression were TP53-independent, including a strong signature of down-regulation of MYC-downstream targets in both TP53 WT and KO isogenic cell line models.

Recent studies have demonstrated that ribosomal proteins may play a central role in an auto-regulatory feedback loop to limit MYC-dependent growth. Specifically, RPL11 has been shown to inhibit MYC transactivation of its target genes by competing with the critical coactivator Transformation/transcription domain-associated protein (TRRAP) for MYC box II binding sites (Dai, et al 2007). As a result, TRRAP-mediated histone H4 acetylation at MYC target gene promoters is reduced, and it is plausible that RNA pol I inhibition may amplify this negative feedback mechanism of MYC transactivation due to the increased availability of unused ribosomal proteins. Moreover, our data demonstrate that RNA pol I inhibition with CX-5461 also led to direct down-regulation of MYC itself, initially at the transcript level, followed by subsequent decreases at the protein level. As another auto-regulatory mechanism of MYC and ribosomal biogenesis, ribosomal proteins such as RPL5 and RPL11 have been shown to interact with the 3′UTR of MYC mRNA and facilitate its degradation through the RISC (Dai, et al 2007, Liao, et al 2014). In our studies, CX-5461 treatment led to increased binding of RPL5 with the RISC protein TARBP2, and furthermore, led to enrichment of MYC mRNA with the catalytic RISC subunit AGO2. The observation that MYC transcript levels were unaffected by CX-5461 in H1112pCDH-MYC cells which lack the MYC 3′UTR lends further credence to this mechanism of MYC down-regulation (Figure S5). Together, these data support the idea that RNA pol I inhibition and induction of the nucleolar stress response may be one strategy to directly target MYC by hijacking its natural auto-regulatory feedback mechanisms mediated by ribosomal proteins.

Interestingly, overexpression of MYC in the non-MYC translocated parental H1112 myeloma cell lines led to enhanced sensitivity to CX-5461 treatment, which was independent of direct down-regulation of MYC itself. This suggests that alternative mechanisms exist in explaining the sensitivity of RNA pol I inhibition in MYC overexpressed cancers. Because MYC overexpression leads to marked upregulation of ribosomal biogenesis as measured by increases in basal pre-rRNA transcription, this may prime MYC-driven cancer cells to the effects of the nucleolar stress response after inhibition of RNA pol I. Indeed, a recent study demonstrated a direct relationship between the rate of rRNA synthesis and the level of TP53 stabilization after treatment with drugs that target ribosomal biogenesis (Scala, et al 2015). In this model, cells with high rRNA synthesis rates generated more ribosomal proteins, which when no longer needed for ribosome assembly after ribosomal biogenesis was disrupted, could then bind to a greater number of MDM2 molecules. Given the relationship between rRNA synthesis and MYC, it is tempting to extrapolate that MYC expression levels may directly relate to the level of TP53 stabilization after RNA pol I inhibition. While this would have been interesting to evaluate in the H1112pCDH and H1112pCDH-MYC cell lines, sequencing of the TP53 gene in the H1112 cell line revealed a 119C>G mutation in exon 4 and a 70C>Y mutation in exon 7, probably disrupting its native interaction with MDM2. Consequently, no changes in TP53 expression were seen with CX-5461 treatment in the H1112pCDH and H1112pCDH-MYC cells (data not shown). Thus, while the presence of WT TP53 undoubtedly augments the cytotoxicity of CX-5461, these data would suggest that the excess of unused elements of the ribosome assembly machinery when RNA pol I is inhibited may also activate other pathways that promote growth arrest and apoptosis aside from those mediated through the MDM2-TP53 axis.

Indeed, several TP53-independent effects of the nucleolar stress response have been reported involving ribosomal proteins, in addition to downregulation of MYC as described earlier. For example, E2F1, which regulates the expression of genes important for cell cycle progression through S-phase, is protected by MDM2 against proteasomal-mediated degradation. Silencing of Polymerase (RNA) I polypeptide A (POLR1A), the catalytic subunit of RNA pol I, has been shown to induce G1/S cell cycle arrest in a TP53-independent manner by increasing E2F1 degradation due to its displacement from MDM2 by RPL11 (Donati, et al 2011). Another recent study demonstrated that the ribosomal protein RPL3 may play a role in cell-cycle regulation by up-regulating CDKN1A expression through a multi-protein complex involving RPL3, SP1, and NPM that localizes to the CDKN1A promoter site (Russo, et al 2013). Overexpression of RPL3 led to increased G1/S cell cycle arrest in a CDKN1A-mediated, TP53-independent manner. Collectively, these studies and our results suggest that basal increases in ribosomal biogenesis sensitize MYC-driven cancer cells to both the TP53-dependent and TP53-independent effects of the nucleolar stress response that ultimately lead to growth inhibition and apoptosis.

The role of MYC overexpression in the sensitivity of CX-5461 may inform rational drug combinations with other novel myeloma agents, particularly proteasome inhibitors such as bortezomib, carfilzomib or ixazomib. Inhibition of the ubiquitin proteasome pathway may lead to MYC protein stabilization, and thus enhance myeloma sensitivity to CX-5461 (Gregory and Hann 2000, Holien, et al 2012), making this a potentially attractive regimen. In contrast, it is plausible that down regulation of the IKZF1/3-IRF4-MYC axis by immunomodulatory drugs, such as lenalidomide and pomalidomide, may reduce the efficacy of CX-5461 (Gopalakrishnan, et al 2016, Kronke, et al 2014). Indeed, when the BET bromodomain inhibitor JQ1, which suppresses MYC transcription, was combined with CX-5461 in myeloma cell lines as part of our preliminary studies, an antagonistic effect was observed (data not shown).

In summary, our results demonstrate that CX-5461 has potent antitumour activity in vitro in both TP53 WT and mutant myeloma models. Activity in vivo was more modest in a TP53 KO xenograft model compared to the TP53 WT model, which probably reflects the consequence of TP53 loss in accelerating tumour migration and invasion, particularly in the setting of a severely immunodeficient mouse model (Pichiorri, et al 2010, Powell, et al 2014). While this also reflects the role of an intact MDM2-TP53 axis in enhancing the therapeutic efficacy of CX-5461, our findings also suggest a prominent role of TP53-independent effects of RNA pol I inhibition in its therapeutic activity, and a central role of MYC overexpression in conferring sensitivity to CX-5461. Taken together, these findings provide a preclinical rationale for RNA pol I inhibition as a novel therapeutic strategy to treat myeloma and other MYC-driven cancers.

Supplementary Material

Supp FigS1-S5

Acknowledgments

We thank the Flow Cytometry and Cellular Imaging Facility, Sequencing and Microarray Core Facility, Small Animal Imaging Facility, Research Animal Support Facility, and the Cell Line Characterization Core, all funded under The University of Texas MD Anderson Cancer Center (MDACC) Support Grant (P30 CA16672). RZO, the Florence Maude Thomas Cancer Research Professor, would also like to acknowledge support from the National Cancer Institute (MDACC SPORE in Multiple Myeloma (P50 CA142509) and R01 CA184464 and U10 CA032102, and thank the Brock Family Myeloma Research Fund, the Yates Ortiz Myeloma Fund, the Jay Solomon Myeloma Research Fund, and the Diane and John Grace Family Foundation. HCL acknowledges support from an American Society of Clinical Oncology Young Investigator Award, American Association for Cancer Research Fellowship in Myeloma Research, and the MDACC Advanced Scholars Program supported by The Jeannine T. Rainbolt Fund and CG Johnson Foundation.

Footnotes

CONFLICT OF INTEREST DISCLOSURE

John Lim and Sean O’Brien are employees of Senhwa Biosciences, Inc.

AUTHOR CONTRIBUTIONS

HCL conceived and performed the majority of experiments, analysed data, prepared figures, and wrote the manuscript. HW generated zinc-finger nuclease cell lines. VB and HYL provided statistical analysis of murine xenograft experiments. JH and JY performed murine xenograft experiments. RJJ, DG, and IK provided valuable comments and technical support. SO, JL, and JK provided reagents and cell lines. ZW, WM, and RED performed gene expression profiling, and RED edited the manuscript. RZO provided research guidance, supervised the work herein, and edited the manuscript.

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