MYC-Driven Neuroblastomas Are Addicted to a Telomerase- Independent Function of Dyskerin

Author Manuscript Published OnlineFirst on April 13, 2016; DOI: 10.1158/0008-5472.CAN-15-0879 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

MYC-driven neuroblastomas are addicted to a telomerase- independent function of dyskerin

Rosemary O’Brien1#, Sieu L. Tran1#, Michelle Maritz1, Bing Liu1, Cheng Fei Kong1, Stefania

Purgato2, Chen Yang1, Jayne Murray1, Amanda J. Russell1, Claudia L. Flemming1, Georg von

Jonquieres1, Hilda A. Pickett3, Wendy B. London4, Michelle Haber1, Preethi H.Gunaratne5, Murray

D. Norris1, Giovanni Perini2, Jamie I. Fletcher1 and Karen L. MacKenzie1*

1Children’s Cancer Institute Australia, Randwick, Sydney, New South Wales, Australia

2Department of Pharmacy and Biotechnology, University of Bologna, Bologna, Italy

3Telomere Length Regulation Laboratory, Children’s Medical Research Institute, Westmead, New

South Wales, Australia

4Dana-Farber/Boston Children’s Cancer and Blood Disorders Center, Boston, MA, USA

5Department of Biology and Biochemistry, University of Houston, Houston, TX, USA

Running Title: MYC-induced dyskerin supports neuroblastoma tumor growth

Key Words: DKC1, neuroblastoma, telomerase, therapeutic target, Myc

Word count: 5,724, Figures and tables: 7 figures, References: 50

#Equal contribution

*Corresponding Author

Karen MacKenzie, Children’s Cancer Institute Australia

PO Box 81 Randwick NSW 2031 Australia

Tel: 61 (2) 9385-1967, Fax: 61 (2) 9662-2584

Email: [email protected]

The authors disclose no potential conflicts of interest

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2016 American Association for Cancer Research. Author Manuscript Published OnlineFirst on April 13, 2016; DOI: 10.1158/0008-5472.CAN-15-0879 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

ABSTRACT

The RNA-binding protein dyskerin, encoded by the DKC1 gene, functions as a core component of the telomerase holoenzyme as well as ribonuclear protein complexes involved in RNA processing and ribosome biogenesis. The diverse roles of dyskerin across many facets of RNA biology implicate its potential contribution to malignancy. In this study, we examined the expression and function of dyskerin in neuroblastoma. We show that DKC1 mRNA levels were elevated relative to normal cells across a panel of 15 neuroblastoma cell lines where both N-Myc and c-Myc directly targeted the DKC1 promoter. Upregulation of MYCN was shown to dramatically increase DKC1 expression. In two independent neuroblastoma patient cohorts, high DKC1 expression correlated strongly with poor event-free and overall survival (P<0.0001), independently of established prognostic factors. RNAi-mediated depletion of dyskerin inhibited neuroblastoma cell proliferation, including cells immortalized via the telomerase-independent ALT mechanism. Further, dyskerin attenuation impaired anchorage-independent proliferation and tumor growth. Overexpression of the telomerase RNA component hTR demonstrated that this proliferative impairment was not a consequence of telomerase suppression. Instead, ribosomal stress, evidenced by depletion of small nucleolar RNAs and nuclear dispersal of ribosomal proteins, was the likely cause of the proliferative impairment in dyskerin-depleted cells. Accordingly, dyskerin suppression caused p53- dependent G1 cell cycle arrest in p53 wild-type cells, and a p53-independent pathway impaired proliferation in cells with p53 dysfunction. Together, our findings highlight dyskerin as a new therapeutic target in neuroblastoma with crucial telomerase-independent functions and broader implications for the spectrum of malignancies driven by MYC family oncogenes.

INTRODUCTION

The human gene encoding dyskerin, DKC1, was first identified through its involvement in dyskeratosis congenita, a heterogeneous disease characterized by defects in highly regenerative tissues (1). Subsequent studies demonstrated high DKC1 expression in various adult cancers, with the DKC1 promoter shown to be a target of c-Myc in breast cancer (2-4). Gene expression array and proteomic studies identified DKC1 among the suite of genes upregulated in neuroblastoma in association with MYCN amplification (5-7). Despite these findings, the significance of dyskerin expression in cancer has been debated, with some studies suggesting that dyskerin may have tumor suppressor functions (8,9).

Amplification of the MYCN oncogene is a powerful prognostic indicator used in risk stratification of neuroblastoma, in which elevated MYCN expression is known to contribute to the development of aggressive disease (10). Neuroblastoma presents an ideal malignancy for studying

Myc-driven cancer, as cell lines that do not have high N-Myc generally express elevated c-Myc

(11). Neuroblastoma is a malignancy of the developing sympathetic nervous system and the most common extra-cranial solid tumor of childhood (12). It presents as advanced disease in ~50% of all patients and accounts for a disproportionate 15% of pediatric cancer deaths from only 8% of pediatric cancer diagnoses, with children in the high-risk category having less than 50% chance of long-term survival (12). The major challenges in the treatment of high-risk cases are dose-limiting toxicities of chemotherapeutics and drug resistance. Identification and validation of new therapeutic targets will provide opportunity for the development of new drugs to improve outcomes for children with aggressive and drug resistant disease.

Dyskerin is a highly conserved RNA-binding and modifying protein that is expressed in a broad range of normal tissues (1,13). It functions in ribonuclear protein (RNP) complexes by directly binding and thereby stabilizing small non-coding RNAs, including the RNA component of telomerase (hTR), H/ACA-box small nucleolar RNAs (snoRNAs) and Cajal body RNAs

(scaRNAs) (14,15). In complexes with hTR, dyskerin is essential to the biogenesis and function of the active telomerase enzyme complex, which plays a well-characterized role in immortalization and the development of 85–90% of all cancers (16). Telomerase activity has been reported to be highest in advanced stages of neuroblastoma (stages 3 & 4) and is an independent prognostic indicator of poor outcome (17,18).

RNP complexes that include dyskerin and H/ACA snoRNAs play essential roles in ribosome biogenesis, functioning to modify rRNA and in the processing of rRNA precursors (15).

Accordingly, mice featuring Dkc1 mutations harbor defects in rRNA processing and protein translation, as well as telomerase activity (19,20). In complexes with H/ACA-box scaRNAs, dyskerin is also involved in the processing of precursor mRNAs. Studies showing that some snoRNAs encode microRNA precursors allude to additional means by which dyskerin may influence mRNA processing and the abundance of specific proteins in cancer cells (21,22). The diverse functions of dyskerin highlight multiple mechanisms through which it may contribute to malignancy, and further add to the rationale for investigating the regulation, function and potential for therapeutic exploitation of dyskerin.

This study shows that the DKC1 gene is targeted by both c-Myc and N-Myc, and that high

DKC1 expression is an independent prognostic indicator for adverse clinical outcome in neuroblastoma. Furthermore, we show for the first time that down-regulation of dyskerin arrests the replication of neuroblastoma cells, impairs anchorage independent growth and suppresses tumor formation. The inhibitory effect of dyskerin depletion on neuroblastoma cell proliferation was accompanied by a reduction in H/ACA snoRNAs and hallmarks of ribosomal stress, and found to be independent of telomerase suppression.

METHODS Cell culture

IMR32, HEK293T cells, Phoenix-A packaging cells and MRC-5 fetal lung fibroblasts were purchased from American Type Culture Collection (Manassas, VA), and SK-N-AS, SK-N-BE(2),

SK-N-DZ, SK-N-FI, SK-N-SH, CHP-134, Kelly, NB69 from European Collection of Cell Cultures

(Porton Down, UK). BE(2)-C, LAN-1, SH-EP, and SH-SY5Y were provided by Dr June Biedler,

Memorial Sloan-Kettering Cancer Centre, New York, NY and NBL-S and NBL-W by Dr Susan

Cohn, University of Chicago, IL. These cell lines were authenticated by short tandem repeat profiling (CellBank Australia, Westmead Australia) in May 2012 and were cultured for no more than six weeks. Neuroblastoma cell lines were cultured in Dulbecco’s Modified Eagles Medium

(DMEM) or RPMI-1640. HEK293T, Phoenix-A and HeLa cells were cultured in DMEM and

MRC-5 in α-Minimal Essential Medium (all media from Life Technologies, Grand Island, NY).

Culture medium was supplemented with 10% fetal bovine serum (FBS; ThermoTrace, Nobel Park,

Australia), 2mM L-glutamine, 100U/ml penicillin and 100µg/ml streptomycin (Invitrogen, CA).

Cells were cultured at 37°C with 5% CO2.

Targeted gene silencing

Cells were transfected with 50–100nM siRNA (Supplementary Table 1) using Lipofectamine

RNAiMAX reagent (Life Technologies, Mulgrave Australia). Vectors and gene transfer methods are detailed in the Supplementary Methods. Transduced cells were selected by fluorescence- activated cell sorting (FACS) for GFP expression (LMS-based retroviral vectors) or by growth in either 0.5 mg/mL genetecin (MND retroviral vectors) or 0.5 μg/mL puromycin (BABEpuro-shRNA retroviral vectors). Expression of shRNA from Fh1t-UTG-based lentiviral vectors was induced by addition of 1 µg/ml doxycycline-hyclate (dox; Sigma-Aldrich) to the growth medium every 3 days.

Gene and protein expression, telomerase activity and telomere length analyses

Gene and protein expression were assayed by immunoblot blot and quantitative real-time PCR according using standard procedures. Antibodies and primers are detailed in the Supplementary

Methods and Supplementary Table 2 respectively. Telomerase enzyme activity and telomere length were measured using the quantitative telomeric amplification protocol (qTRAP) and Southern blot- based telomeric restriction fragment analysis, respectively, as described previously (23). For the qTRAP assay, negative controls were MRC-5 cells, heat-treated SK-N-SH lysate (Heat-Tx), and lysis buffer without sample (LB).

Chromatin Immunoprecipitation Assays

Chromatin Immunoprecipitation (ChIP) assays were performed as previously described (24) using antibodies and primers listed in Supplementary Table 3. Relative enrichment of specific promoter regions was determined by normalization to input DNA.

Cell cycle analysis

Cells were washed in cold phosphate-buffered saline (PBS), then fixed in cold 70% ethanol before permeabilization with 0.5% Triton-X/0.1% bovine serum albumin (BSA) and staining with 25µg/ml propidium iodide (Sigma-Aldrich)/100µg/ml DNAse-free RNAse (Roche, Germany) in 0.1%

BSA/PBS. Stained cells were analyzed by flow cytometry (FACSCalibur, BD Biosciences) using

ModFit LT v3.0 software (Verity Software House).

Anchorage-independent growth and tumorigenicity

Anchorage-independent growth was assayed by colony formation in soft agarose as described (25).

Tumorigenicity was assessed with approval of the Animal Care and Ethics Committee of the

UNSW Australia (ACEC #11/5B). Female 6-8 week old BALB/c nu/nu (nude) mice (Animal

Resource Centre; WA, Australia) were injected with 2×106 cells suspended in 200µl DMEM into the hind flank. Mice with palpable tumors were switched to food laced with 600mg/kg doxycycline

(Specialty Feeds, WA, Australia) to induce transgene expression. Mice were sacrificed before tumor volume reached 1.5cm3, calculated as: Volume (mm3)=[Length (mm)×Width (mm)×Height

(mm)] /2.

Detection of ribosomal proteins by immunofluorescence

Cells cultured on glass coverslips were fixed with 4% paraformaldehyde, permeabilized with 0.1%

Triton X-and blocked for 50 minutes with 10% FCS before staining with anti-rpl11 (Abcam; ab86863) or anti-rpl5 (Abcam; ab79352) for 50 minutes and then secondary antibody (Life

Technologies; A-11012) for 50 minutes. Coverslips were mounted in Prolong-Gold (Life

Technologies; P36930). Images were captured using a Zeiss Axiovert 200M inverted microscope with a LCI Plan-Neolfluar 63x/1.3 objective and an AxioCam HRm camera (Carl Zeiss Inc.,

Oberkochen, Germany).

RESULTS The DKC1 gene is up-regulated in neuroblastoma cells and is a direct target of N-Myc and c-

Myc

Quantification of DKC1 mRNA in 15 neuroblastoma cell lines (8 with MYCN amplification and 7 lacking MYCN amplification) revealed that DKC1 mRNA was elevated in the neuroblastoma cells relative to normal human myofibroblasts (MRC-5 and WI-38) and bone marrow endothelial cells

(BMEC) (Fig. 1A, upper panels). On average, DKC1 expression was four-fold higher in neuroblastoma cells than in normal cells (P<0.01), and was higher in cells with MYCN amplification compared to those without amplification (P<0.05). Immunoblot analysis showed that dyskerin protein levels were highest in cells with MYCN amplification, and was also elevated in cells with high c-Myc (Fig. 1B). There was a significant correlation between dyskerin protein and

DKC1 mRNA levels (Supplementary Fig. 1; r=0.639, P<0.05).

Neuroblastoma cell lines were also characterized for expression of the catalytic component of telomerase, TERT, as well as telomerase enzyme activity and telomere length. TERT mRNA expression was detected in all but one of the neuroblastoma cell lines (SK-N-FI) (Fig. 1A, lower graphs). Telomerase activity varied across the panel of cell lines (Fig. 1C), correlating more closely with expression of DKC1 (r=0.575, P<0.05) than TERT (r=0.375, P=0.169) (Supplementary Fig.

2A-B, respectively). There was also heterogeneity in telomere length length among the neuroblastoma cell lines (Fig. 1D). The SK-N-FI cell line, which had no detectable TERT expression and negligible telomerase activity, exhibited very long and heterogeneous telomeres, which is typical of cells immortalized by the telomerase-independent Alternate Telomere

Lengthening mechanism (26).

Having shown that elevated DKC1 expression was associated with high MYC and MYCN expression, ChIP assays were performed to determine whether N-Myc and c-Myc proteins directly interact with the DKC1 gene. Using BE(2)-C, a MYCN-amplified neuroblastoma cell line, and SH-

SY5Y, which has high c-Myc expression and lacks MYCN amplification (Fig. 1B), we found that both Myc proteins (together with their obligate binding partner, Max), bound DKC1 at sites proximal to the DKC1 promoter (Fig. 2A). We identified a non-canonical E-box within a CpG island downstream of the DKC1 transcription start site (Fig. 2A, Amplicon B) and two canonical E- boxes within the first intron (Fig. 2A, Amplicon C) as sites of N-Myc and c-Myc binding in BE(2)-

C and SH-SY5Y cells, respectively. N-Myc and c-Myc also bound at canonical E-boxes proximal to the transcriptional start site of the TERT gene (Fig. 2B). No c-Myc or N-Myc binding was observed at control amplicons upstream of the DKC1 and TERT promoters (Amplicon A in Fig. 2A

& 2B).

The relationship between MYCN expression and that of DKC1 and TERT was further tested using a well-characterized tetracycline-modulated vector system that induces MYCN expression to very high levels in SH-EP neuroblastoma cells (27). These experiments showed that MYCN levels were dramatically up-regulated following removal of tetracycline (Fig. 2C). Up-regulation of

MYCN was accompanied by a 50- to 100-fold increase in DKC1 expression, and a 10- to 20-fold increase in TERT expression. Together these data provide the first functional evidence of N-Myc and c-Myc targeting the DKC1 gene in neuroblastoma cells.

High DKC1 gene expression is an independent prognostic indicator of poor event-free and overall survival in neuroblastoma

To investigate the clinical significance of DKC1 expression levels, we combined publically available microarray data from a large cohort of neuroblastoma patients and published clinical data for a subset of these patients (n=477, the Kocak cohort)(28). Our analyses revealed that DKC1 expression was significantly higher in tumors with amplification of the MYCN gene compared to those without (Fig 3A; P<0.001). Moreover, DKC1 expression strongly correlated with MYCN expression in the subset of patients with MYCN amplification (Fig 3B; r=0.531 P<0.001). The

strength of this association was notable in light of a poor correlation between TERT and MYCN expression in this sample subset (Supplementary Fig. 3; r=0.215, P=0.071). When DKC1 gene expression was dichotomized around the upper quartile, high expression of DKC1 was significantly associated with poorer event-free survival (EFS, Fig. 3C) and poorer overall survival (OS, Fig. 3D), with five-year EFS rates of 76%±2% and 28%±5% and 5-year OS of 91%±2% and 36%±5% for tumors with low and high DKC1 expression respectively. These correlations were also highly statistically significant when DKC1 expression was dichotomized at the median or upper decile

(Supplementary Fig. 4A-B, p<0.0001). The clinical significance of high DKC1 expression was further validated by analysis of an independent cohort of 197 primary tumors from neuroblastoma patients enrolled in COG protocol 9047. In the COG data set, high expression of DKC1 was again associated with poorer EFS (P<0.0001, Fig. 3E) and poorer OS (P<0.0001, Fig. 3F).

Multivariate analysis was performed using the larger cohort (Kocak) to determine whether the prognostic significance of high DKC1 was independent of established prognostic indicators for neuroblastoma. Age at diagnosis (<18 mo vs ≥18 mo), tumor stage (1, 2, or 4s vs 3 or 4), MYCN status (amplified vs single copy) and DKC1 gene expression (upper quartile) were tested in a Cox’s proportional hazards regression model (Supplementary Table 4). DKC1 expression retained strong independent prognostic significance for EFS (hazard ratio [HR]=2.21; 95% CI 1.41 to 3.45;

P<0.0005) and OS (HR=2.60; 95% CI 1.43 to 4.71; P=0.0017). Consistent with previous reports

(18), high TERT expression also correlated with poor patient outcomes in univariate analysis of the

Kocak cohort (Supplementary Fig. 5A-B), however TERT expression was not significant in mulitvariate analyses with independent prognostic factors (Supplementary Table 4; P=0.8066 for

EFS and P=0.5561 for OS).

Down-regulation of DKC1 inhibits the replication and tumorigenic growth of neuroblastoma cells

The functional significance of high DKC1 expression was investigated using two siRNAs (D2 and

D3) designed to effectively down-regulate the DKC1 gene (Fig. 4A-B). In short-term cell proliferation assays, down-regulation of DKC1 to levels similar to those detected in normal cells significantly inhibited the replication of neuroblastoma cells (Fig. 4C). Across 10 neuroblastoma cell lines, there was an overall correlation between the extent of dyskerin suppression and the magnitude of the proliferative impairment (Spearman r=0.431 P<0.05).

The impact of dyskerin expression levels on the malignant properties of neuroblastoma cells was further investigated by transducing SK-N-BE(2) cells with either an LMS-based retroviral vector encoding a miR30-based shRNA targeting DKC1, a control vector encoding non-silencing shRNA (NS) or the empty vector (EV). DKC1 mRNA was reduced in cells transduced with LMS-

DKC1 shRNA to 35–40% of that in control-vector transduced cells (Fig. 4D). When plated in semi- solid media, the LMS-DKC1 shRNA-transduced cells generated significantly fewer colonies than control vector-transduced cells (Fig. 4E; P<0.01), demonstrating that high levels of dyskerin are required for anchorage independent growth.

To test the impact of dyskerin suppression on tumor growth, DKC1 shRNA (shD3) and non- silencing shRNA (NS) were cloned into a doxycycline-inducible expression cassette within the

Fh1t-UTG lentiviral vector. Upon addition of doxycycline to culture media, DKC1 mRNA was initially reduced by ~90% in shD3-transduced cells, however there was a gradual recovery of DKC1 mRNA until expression eventually plateaued at 40-50% of basal levels (Fig. 4F). Tumorigenicity was then tested by subcutaneous engraftment of BALB/c nu/nu mice with transduced SK-N-BE(2).

Upon establishment of a palpable tumor, mice received dietary doxycycline to suppress DKC1 expression. Over a four week period, tumor growth was markedly suppressed in mice implanted with shD3 cells compared to those implanted with control vector transduced cells (Fig. 4G;

P<0.05). Indeed tumor growth did not progress after initiation of doxycycline in 2 out of 8 mice transplanted with shD3 cells. In contrast all tumors grew at a steady and rapid rate in mice transplanted with control cells.

Down-regulation of dyskerin halts cell replication independently of telomerase suppression

Consistent with previous studies showing that dyskerin insufficiency destabilizes hTR and dampens telomerase activity (14), NB69 and SK-N-BE(2) cells transfected with DKC1 siRNAs had lower hTR levels and telomerase activity than control cells (Fig. 5A-B). In contrast, TERT mRNA levels were not impacted by depletion of DKC1 mRNA in either cell line (Fig. 5C). To test whether suppression of hTR and telomerase activity was necessary for the growth arrest induced by dyskerin depletion, NB69 cells were transduced with a retroviral vector expressing hTR (MND-hTR). siRNA-mediated repression of DKC1 reduced both hTR and telomerase activity in control vector

(MND) and hTR overexpressing cells (Fig. 5D), however telomerase activity in hTR- overexpressing cells transfected with dyskerin siRNA remained substantially above that detected in control vector-transduced cells (Fig 5Diii). Strikingly, despite the maintenance of high telomerase activity, MND-hTR cells still underwent proliferative arrest upon siRNA-mediated repression of dyskerin (Fig. 5E). These results demonstrate that neuroblastoma cells are addicted to a telomerase- independent function of dyskerin.

Down-regulation of dyskerin depletes H/ACA snoRNAs and causes ribosomal stress

Aside from its interaction with hTR, dyskerin binds and stabilizes H/ACA snoRNAs that guide rRNA modification and function in the processing of rRNA precursors (15). We therefore postulated that the proliferative arrest induced by dyskerin depletion resulted from reduced availability of snoRNAs, and consequently ribosomal stress. We therefore quantified the abundance of three H/ACA snoRNAs, SNORA16, SNORA42 and SNORA75 (U23), which are involved in

rRNA processing in human cancer cells, in siRNA transfected NB69 cells (29,30). qRT-PCR analyses confirmed that the level of each of these snoRNAs was substantially less in dyskerin- depleted cells than in control siRNA-transfected cells (Fig. 6A).

A hallmark of disrupted ribosome biogenesis is dispersal of ribosomal proteins from nucleoli into the nucleoplasm (31). Accordingly, immunofluorescence staining showed diffuse nucleoplasmic staining of ribosomal protein rpl11 (Fig. 6B) in D2 and D3 transfected cells. This contrasted with Sc-transfected, in which there was very little rpl11 in the nucleoplasm and more obvious staining of rpl11 in nucleoli. Similar results were obtained from immunofluorescence staining of rpl5 (Supplementary Fig. 6). The effect of dyskerin depletion on ribosomal proteins mirrored the impact of low dose treatment with actinomycin D (5nM), which disrupts ribosome biogenesis by inhibiting rRNA synthesis (Fig. 6B and Supplementary Fig. 6). Thus together these results provide compelling evidence that downregulation of dyskerin-induced disrupted ribosome biogenesis in NB69 neuroblastoma cells, thus implicating ribosomal stress as a cause of proliferative arrest in dyskerin-depleted cells.

Dyskerin depletion activates p53 and G1 cell cycle arrest in NB69 cells

During ribosomal stress, ribosomal proteins within the nucleoplasm recruit hdm2, resulting in p53 stabilization, up-regulation of p21cip1 and G1 cell cycle arrest (32). Consistent with this mechanism, we detected an accumulation of p53 (Fig. 7A left panel) in NB69 cells transfected with DKC1 siRNA, coinciding with up-regulation of p21cip1 mRNA (Supplementary Fig. 7A) and protein (Fig.

7A left panel). In contrast to NB69 cells, there was no induction of p21cip1 protein or mRNA when dyskerin was repressed in SK-N-BE(2) cells, which express a mutant p53 protein defective in transactivation properties (Fig. 7A right pane, Supplementary Fig. 7B)(33). Propidium iodide staining showed there was a significant increase in the proportion of NB69 cells with sub-G1 DNA content following dyskerin repression (Fig. 7B, left graph & Supplementary Fig. 8A), consistent

with a loss of viability evidenced by trypan blue staining (Supplementary Fig. 8B, P<0.05).

Dyskerin-depleted NB69 cells accumulated in G1 phase, with a corresponding decrease in S and

G2/M phase (Fig. 7C, left graph, Supplementary Fig. 8C). In contrast to NB69 cells, the viability and cell cycle kinetics of SK-N-BE(2) cells were unaffected by dyskerin repression (Fig. 7B & 7C right graphs, Supplementary Fig. 8D-F), despite a marked effect on cell proliferation (Fig. 4C).

In light of the above results with SK-N-BE(2) cells, suggesting that p53 function was not necessary for dyskerin repression to impact on cell proliferation, we further investigated the role of p53 in the response of dyskerin depleted NB69 cells. For these investigations, NB69 cells were transduced with BABEpuro-based retroviral vectors encoding p53 shRNA or control shRNA

(GFPshRNA). Suppression of p53 and p21cip1 in the p53 shRNA-transduced cells was confirmed by immunoblotting (Fig. 7D) and cell counts showed that the p53 shRNA and control vector- transduced cells proliferated with identical kinetics. Proliferation was impaired by D2 and D3 irrespective of p53 and p21cip1 expression status (Fig. 7E). Therefore, while dyskerin depletion induced ribosomal stress and a robust upregulation of p53 and p21cip1 in p53 competent cells (Figs.

6B and 7A), p53 function was not essential for proliferative impairment proliferation.

4. DISCUSSION

Past studies suggest that elevated dyskerin expression levels correlate with more aggressive disease

(2,3,6), however prior to the current investigation there was a paucity of functional evidence supporting the significance of dyskerin expression levels in any cancer type, and no previous report of dyskerin suppression inhibiting tumor growth in vivo. The present study not only identifies

DKC1 expression as a powerful independent prognostic indicator of poor clinical outcome, but also shows that high DKC1 expression has functional significance and potential as a therapeutic target in the Myc-driven malignancy neuroblastoma. We have shown for the first time that suppression of dyskerin dramatically and immediately inhibits neuroblastoma cell proliferation, impairs anchorage independent growth and reduces tumor progression in a xenograft model. Notably, the effect of dyskerin depletion on tumor cell proliferation was mediated by a mechanism that is independent of telomerase suppression.

Although dyskerin repression effectively lowered hTR levels and suppressed telomerase enzyme activity in neuroblastoma cell lines, several lines of evidence indicate that suppression of telomerase activity and telomere shortening was not the cause of the proliferative impairment.

Firstly, impaired proliferation was evident immediately upon dyskerin repression, and there was no correlation between the proliferative response and telomere length among the different neuroblastoma cell lines. Secondly, down-regulation of dyskerin inhibited proliferation of the SK-

N-FI cell line, which does not express the catalytic component of telomerase and exhibits ALT-like telomeres (34). Finally, the proliferative defect induced by dyskerin depletion was not rescued by overexpression of hTR and elevation of telomerase activity to levels significantly exceeding those of control cells.

The alternate mechanistic explanation for the acute proliferative arrest induced by downregulation of dyskerin is destabilization of H/ACA snoRNAs and consequential disruption of ribosome biogenesis inducing a ribosomal stress response. This conclusion is supported by our

results showing reduced levels of snoRNAs (SNORA16A, SNORA42A and SNORA75A) that guide modification of rRNAs (18s and 28s) and dispersal of the ribosomal proteins rpl11 and rpl5. A body of literature has shown that free rpl11 and rpl5, liberated by disruption of ribosome biogenesis, bind the p53-targeting E3 ubiquitin ligase Mdm2, resulting in robust activation of p53 (31,35,36). Thus the p53-mediated G1 arrest and cell death demonstrated in NB69 neuroblastoma cells is consistent with the known effects of rpl11 and rpl5 dispersal. The consequences of dyskerin ablation on ribosomal proteins, p53 and G1 cell cycle progression also concur with the reported effects of disrupting ribosome biogenesis by pharmacologic inhibition of rRNA transcription (37).

TP53 mutation is infrequently observed at diagnosis in neuroblastoma (38), however TP53 mutations and defects in the p53 pathway, including inactivation of tumor suppressor p14ARF, have been reported in recurrent and drug-resistant disease (38). Our results show that dyskerin repression inhibited proliferation of cell lines with known defects in TP53 (SK-N-BE(2), SK-N-FI, SK-N-AS) and genomic loss of p14ARF (SK-N-AS), as well as NB69 cells transduced with p53 shRNA (39).

Thus, in addition to showing that p53 is activated in response to dyskerin depletion, our investigations provide evidence of a secondary p53-independent mechanism that inhibits proliferation of neuroblastoma cells with defective p53 pathways. It is shown that downregulation of dyskerin in NB69 cells, which express wild-type p53 and p14ARF (39,40), caused a G1 cell cycle arrest and cell death. In contrast, SK-N-BE cells, which express mutant p53 (33), remained viable but exhibited dramatically impaired proliferation following downregulation of dyskerin. These results concur with a study that showed p53-independent proliferative arrest, without cell death, induced by ribosomal stress following treatment of cancer cell lines with an inhibitor of rRNA synthesis (41).

Contrary to the conclusions of the present study, it was previously proposed that dyskerin functions as a tumor suppressor, a hypothesis drawn from studies that showed a hypomorphic

DKC1 mutation impaired IRES-mediated translation of tumor suppressors p27kip1 and p53 (8,9,42).

However, consistent with the present findings, an independent study demonstrated that an alternate

Dkc1 mutation in mice induced a proliferative defect associated with p53 activation (43). One possible explanation for these apparently conflicting findings is that the distinct DKC1 mutations modeled in mice were functionally different. The present study, which used dyskerin knockdown rather than gene mutation, is uncomplicated by potential gain-of-function effects of DKC1 mutations, and unequivocally shows that down-regulation of dyskerin halts cell replication and induces a robust p53 response. These effects would not be expected if dyskerin functioned as a tumor suppressor, but rather are consistent with addiction to a gene up-regulated during the transformation process. Studies that found p53 was not induced in U2OS and MCF-7 cell lines subject to siRNA-mediated downregulation of dyskerin may be explained by the defects in p14ARF known to be harbored by those cells (9,44-46). This possibility is supported by a recent investigation that showed p14ARF bolsters p53 activation during ribosomal stress (47). It will be of interest for future studies to further characterize the mechanism of proliferative arrest induced by ribosomal stress in cancer cells with defects in the p53/p14ARF pathway.

In the context of previous investigations of dyskerin suppression, our data suggest that neuroblastoma cells may be particularly sensitive to the depletion of dyskerin. Contrasting with the robust effect on neuroblastoma cell proliferation demonstrated herein, previous investigations have reported no apparent effect of dyskerin depletion on proliferation of UM-SCC1 squamous cell carcinoma or HeLa cervical carcinoma cells (22), a moderate effect on the proliferation of U2OS osteosarcoma and MCF-7 breast cancer cells (9,44) and a varied response in two different prostate cancer cell lines (3). Here we have shown that repression of dyskerin impaired the proliferation of several neuroblastoma cell lines that represent the molecular heterogeneity of the disease, including cells with or without MYCN amplification, TP53 mutation, p14ARF deletion and telomerase enzyme activity. Among the neuroblastoma cell lines tested, the one cell line that consistently did not respond to robust dyskerin mRNA suppression was the Kelly cell line (data not shown). Kelly cells

feature extremely high MycN expression due to extraordinary amplification of the MYCN locus

(>100 copies), as well as an activating mutation in the oncogenic tyrosine kinase Anaplastic

Lymphoma Kinase (ALK) (48). The molecular basis for the apparent resistance of the Kelly cell line to dyskerin depletion warrants future investigation.

The capacity for dyskerin ablation to arrest proliferation of neuroblastoma cells with defective p53, loss of p14ARF expression or amplified MYCN highlight the potential for therapeutic effect in neuroblastoma cases that are the most difficult to treat. Toward the therapeutic exploitation of dyskerin, further studies are needed to explore the impact of dyskerin depletion on various types of normal cells. Although inactivation of the DKC1 locus is embryonic lethal in mice (49), the pattern of inheritance of DKC1 mutations observed in individuals with dyskeratosis congenita indicate that a partial reduction in dyskerin is compatible with normal cell replication and development, at least in the short to medium term. Consistent with that notion, fibroblasts from patients with DKC1 mutations were shown to proliferate at a similar rate to normal fibroblasts, even though long-term cell replication was limited by eventual critical telomere shortening (50).

Collectively, the results from these investigations demonstrate that neuroblastoma cells are addicted to a telomerase-independent pathway that is activated by Myc-induced upregulation of dyskerin. This study reveals dyskerin as an exciting new therapeutic target that is not dependent upon p53 function or telomerase, with potential for exploitation in the broad spectrum of paediatric and adult cancers that are driven by MYC oncogenes.

FUNDING

This work was supported by funds from the Cancer Council New South Wales (Project Grants RG

11-01 and RG 15-16), National Health and Medical Research Council (Career Development

Fellowship 510378), Cancer Institute New South Wales (Career Development Fellowship

09CDF217) and the Italian Association for Cancer Research (AIRC).

ACKNOWLEDGMENTS

Children’s Cancer Institute Australia is affiliated with UNSW Australia and the Sydney Children’s

Hospitals Network. We thank Elysse McIlwain and Paul Young for technical assistance.

REFERENCES

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FIGURE LEGENDS

Figure 1: Expression of DKC1, TERT and telomerase enzyme activity in neuroblastoma cell lines

A) DKC1 and TERT gene expression in neuroblastoma cell lines determined by quantitative real- time PCR (qRT-PCR) analysis. Values are mean±standard error (SEM) from 3–5 independent assays, with duplicates in each assay. **P<0.01, *P<0.05 and n.s. is not significantly different in

Man-Whitney test. B) Representative immunoblot showing dyskerin, N-Myc and c-Myc protein, with GAPDH as a loading control. The vertical white line indicates rearrangement of the image presentation purposes. C) Telomerase enzyme activity measured using the telomeric repeat amplification protocol (qTRAP). Heat-Tx; heat treated SK-N-SH lysate, LB; Lysis buffer with no sample. Values are mean±SEM from three independent assays, with duplicates in each assay. The graph at the right shows mean telomerase activity in subgroups of the cells. D) Telomeric restriction fragment length (TRF) analysis of telomere length in neuroblastoma cell lines.

Figure 2: The DKC1 gene is a target of Myc-family oncogenes in neuroblastoma cells

Chromatin immunoprecipitation performed to assess binding of N-Myc and c-Myc proteins to the promoters of (A) DKC1 and (B) TERT in BE(2)-C (left schemes) and SH-SY5Y (right schemes).

Schemes beneath each graph represent human DKC1 and TERT promoters showing the canonical and non-canonical E-box sites, and positions of the PCR amplicons (A, B and C). Values represent mean±SEM from three independent experiments. (C) qRT-PCR analysis of MYCN, DKC1 and

TERT expression in SHEP-TET21/N cells previously transfected with a MYCN vector induced by tetracycline withdrawal.

Figure 3: High DKC1 expression is a marker of poor prognosis in neuroblastoma

(A) DKC1 expression in neuroblastoma tumor samples with (n=71) and without (n=405) MYCN amplification. The graph shows log-transformed, zero-centered expression levels obtained from microarray dataset (Kocak cohort)(28). Boxes indicate 25th–75th percentiles and whiskers illustrate minimum to maximum values. P-value from two-tailed Mann-Whitney tests. (B) Correlation between DKC1 and MYCN expression in tumors from the Kocak cohort with MYCN amplification. r is Spearman coefficient and P-value from two-tailed correlation tests. (C-F) Kaplan-Meier curves showing the probability of EFS and OS for the Kocak (C, D) and Children’s Oncology Group

(COG) (E, F) cohorts. Values were dichotomized into “high” and “low” DKC1 expression around the upper quartile

Figure 4: Down-regulation of dyskerin suppresses neuroblastoma cell proliferation in vitro and tumor growth in mice

A) qRT-PCR analysis of DKC1 expression in neuroblastoma cells transfected with siRNAs targeting DKC1 mRNA (D2 and D3) or a control oligonucleotide (Sc). Values are mean±SEM from

3–5 independent transfections. Upper and lower horizontal dotted line indicate DKC1 expression in

HeLa and normal MRC5 myofibroblasts, respectively. B) Immunoblot dyskerin protein in a representative experiment where SK-N-AS cells were transfected with siRNA. C) Proliferation of siRNA-transfected cells. Values are mean±SEM from 3–7 independent transfections, quantified as expansion. D) SK-N-BE(2) cells were transduced with retroviral vectors that constitutively express shRNA targeting dyskerin mRNA (LMS-DKC1 shRNA), non-silencing control shRNA (LMS-NS) or GFP only (LMS-EV). qRT-PCR was performed on GFP+ cells within 15 days of transduction.

Values are mean±SEM from at least five independent assays. **P<0.01, *P<0.05 from

Bonferroni’s multiple comparisons test. E) Anchorage-independent growth in soft-agarose measured two weeks-post transduction. Values are mean±SEM from four independents

experiments. **P<0.01 Holm-Sidak's multiple comparisons test. F–G) SK-N-BE(2) cells were transduced with FH1t shRNA lentiviral vectors encoding shRNA under control of a doxycycline- inducible promoter. F) qRT-PCR analysis of DKC1 expression (left axis) and cell viability determined by trypan blue (right axis) in cells cultured with or without doxycycline. Values are the average of duplicates at each time point. G) Tumor growth in BALB/c nu/nu mice engrafted with

FH1t-transduced SK-N-BE(2) cells (10 mice per group). P-value two-way ANOVA.

Figure 5: Proliferative arrest following repression of dyskerin does not require depletion of hTR or telomerase siRNA transfected NB69 and SK-N-BE(2) cells assayed for A) hTR by qRT-PCR, B) telomerase activity by qTRAP and (C) hTERT mRNA by qRT-PCR 48h and 96h post-transfection. D-E)

NB69 transduced with MND-hTR or an empty vector (MND), selected in Geneticin, then transfected with siRNA. D) DKC1 mRNA (i) and hTR levels (ii) quantified by qRT-PCR. (iii) telomerase activity measured by qTRAP. E) Proliferation expressed relative to cell number at the time of transfection. Values are mean±SEM from 3–5 experiments. ****P<0.0001, **P<0.01,

*P<0.05, Two-way ANOVA with Bonferroni’s multiple comparison of D2 and D3 with Sc.

Figure 6: Depletion of dyskerin reduces availability of H/ACA snoRNAs and induces ribosomal stress

A) Relative abundance of DKC1 mRNA and H/ACA snoRNAs (SNORA16A, SNORA42 and

SNORA75) in siRNA transfected cells determined by qRT-PCR. Values are mean±SEM from three independent experiments. ***P<0.001, **P<0.01, *P<0.05, Holm-Sidak’s multiple comparison test of D2 and D3 with Sc. B) Representative images from immunofluorescence staining of rpl11 in

NB69 cells 96h post-transfection with siRNA. Treatment with 5nM actinomycin D (ActD) for 24

hours was used as a positive control (bottom panel). Merged images show rpl11 in red and nuclei in blue (DAPI stained). The size bar represents 10μm.

Figure 7: p53 is activated, but is not required for the proliferative impairment induced by dyskerin depletion

A) Immunoblot of dyskerin, p53 and p21cip1 in siRNA-transfected NB69 (left) and SK-N-BE(2)

(right). M; mock-transfected cells. GAPDH was used as a loading control. The white vertical line indicates rearrangement of the image for presentation purposes. B-C) Flow cytometric analysis of siRNA-transfected NB69 (left graphs) and SK-N-BE(2) (right graphs) stained with propidium iodide (B) subG1/G0 (dead and dying cells) and (C) viable G1, S and G2/M phase cells. Bar graphs show mean±SEM from 4 independent experiments. ***P<0.001 *P<0.05, Holm-Sidak's multiple comparisons. D-E) NB69 transduced with retroviral vectors encoding p53 shRNA or GFP shRNA

(control), then subject to siRNA transfection. D) Immunoblot of transduced NB69 cells following transfection with siRNA. E) Proliferation assessed using trypan blue. Values are mean±SEM from 4 independent experiments, ***P<0.001 **P<0.01, Bonferroni’s multiple comparisons test of D2 and

D3 against Sc.

MYCN amplified Non amplified

4 4 B

A B

Ave DKC1

DKC1 E(2)

DZ SH AS FI - 134 - - - - W (n) 1 S

3 3 -

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0 4.2

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1.5

Figure 1: Expression of DKC1, TERT and telomerase enzyme activity in neuroblastoma cell lines

A DKC1

BE(2)-C SH-SY5Y

SKN-BE2 SY5Y

TERT B BE(2)-C SH-SY5Y

Figure 2: The DKC1 gene is a target of Myc-family oncogenes in neuroblastoma cells

A P < 0.001 B 1.5

n 1.5 r = 0.531

o n

i 1.0 o

s P < 0.001

e s

r 0.5 1.0

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D Low DKC1

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High DKC1 High DKC1

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E F

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High DKC1 High DKC1

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Figure 3: High DKC1 expression is an indicator of poor prognosis in neuroblastoma

A B 2.0 2.0 SK-N-AS NB69 SK-N-BE(2) 1.5

1.5

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Figure 4: Down regulation of dyskerin suppresses neuroblastoma cell proliferation in vitro and tumor growth in mice Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2016 American Association for Cancer Research. Author Manuscript Published OnlineFirst on April 13, 2016; DOI: 10.1158/0008-5472.CAN-15-0879 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

A B C hTR Telomerase activity TERT mRNA 1.5 1.0 NB69 NB69 NB69 20

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Figure 5: Down regulation of dyskerin inhibits proliferation via a telomerase-independent mechanism

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5 5

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Figure 7: p53 is activated, but not required for the proliferative impairment induced by dyskerin depletion

MYC-driven neuroblastomas are addicted to a telomerase-independent function of dyskerin

Rosemary O'Brien, Sieu L Tran, Michelle Maritz, et al.

Cancer Res Published OnlineFirst April 13, 2016.

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