Suppression of ABCE1-Mediated Mrna Translation Limits N-MYC-Driven Cancer Progression

Author Manuscript Published OnlineFirst on July 10, 2020; DOI: 10.1158/0008-5472.CAN-19-3914 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Submitted Manuscript: Confidential

Suppression of ABCE1-mediated mRNA translation limits N-MYC-driven cancer progression

Running title: ABCE1, a novel therapeutic target in N-MYC-driven cancers

Authors: Jixuan Gao1, 2, MoonSun Jung1, 2, Chelsea Mayoh1, Pooja Venkat1, Katherine M. Hannan3, 4, Jamie I. Fletcher1, 2, Alvin Kamili1, 2, Andrew J. Gifford1, 2, 5, Eric P. Kusnadi6, Richard B. Pearson6, Ross D. Hannan3, 4, 6, 7, 8, 9, Michelle Haber1, 2, Murray D. Norris1,10†, Klaartje Somers1, 2†, and Michelle J. Henderson1, 2* †.

Funding:

Drs M.J. Henderson, K. Somers and all work in this manuscript were supported by grants from the National Health and Medical Research Council, Cancer Institute NSW and Tour de Cure. Dr J. Gao was supported by Australian Postgraduate Award, Children’s Cancer Institute and Cancer Therapeutics CRC (CTx) scholarships.

Affiliations: 1Children’s Cancer Institute Australia for Medical Research, Lowy Cancer Research Centre, University of New South Wales, Sydney, NSW, 2031, Australia; 2School of Women’s and Children’s Health, UNSW Sydney, NSW, Australia, 3The John Curtin School of Medical Research, The Australian National University, Canberra City, ACT, 2601, Australia; 4Department of Biochemistry and Molecular Biology, University of Melbourne, Melbourne, Australia; 5Department of Anatomical Pathology, Prince of Wales Hospital, Randwick, NSW, Australia, 6Division of Cancer Research, Peter MacCallum Cancer Centre, Melbourne, VIC, 3000, Australia; 7Sir Peter MacCallum Department of Oncology, University of Melbourne, Melbourne, Australia; 8School of Biomedical Sciences, University of Queensland, Brisbane, Queensland, Australia; 9Department of Biochemistry and Molecular Biology, Monash University, Clayton, Australia; 10UNSW Centre for Childhood Cancer Research, Sydney, NSW, 2052, Australia. * Corresponding author: Phone: +61 2 9385 1570, mail: Children's Cancer Institute, Lowy Cancer Research Centre UNSW, PO Box 81, Randwick 2031 Australia, email: [email protected] † These authors contributed equally Affiliation statement: The Children’s Cancer Institute is affiliated with UNSW Australia and the Sydney Children’s Hospital Network.

Conflict of interest disclosure statement: The authors have no conflict of interest to declare.

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 10, 2020; DOI: 10.1158/0008-5472.CAN-19-3914 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Abstract:

The ability of the N-MYC transcription factor to drive cancer progression is well- demonstrated in neuroblastoma, the most common extracranial pediatric solid tumor, where MYCN- amplification heralds a poor prognosis with only 11% of patients surviving past 5 years. However, decades of attempts of direct inhibition of N-MYC or its paralogues has led to the conclusion that this protein is 'undruggable'. Therefore, targeting pathways upregulated by N-MYC signaling presents an alternative therapeutic approach. Here we show that MYCN-amplified neuroblastomas are characterized by elevated rates of protein synthesis and that high expression of ABCE1, a translation factor directly upregulated by N-MYC, is itself a strong predictor of poor clinical outcome. Despite the potent ability of N-MYC in heightening protein synthesis and malignant characteristics in cancer cells, suppression of ABCE1 alone selectively negated this effect, returning the rate of translation to baseline levels and significantly reducing the growth, motility, and invasiveness of MYCN-amplified neuroblastoma cells and patient-derived xenograft tumors in vivo. The growth of non-malignant cells or MYCN-non-amplified neuroblastoma cells remained unaffected by reduced ABCE1, supporting a therapeutic window associated with targeting ABCE1. Neuroblastoma cells with c-MYC overexpression also required ABCE1 to maintain cell proliferation and translation. Taken together,

ABCE1-mediated translation constitutes a critical process in the progression of N-MYC-driven and c-MYC-driven cancers that warrants investigations into methods of its therapeutic inhibition.

Significance:

Findings demonstrate that N-MYC-driven cancers are reliant on elevated rates of protein synthesis driven by heightened expression of ABCE1, a vulnerability that can be exploited through suppression of ABCE1.

Introduction

Deregulated expression of the MYC family of transcription factors (N-MYC, C-MYC and L-

MYC) is associated with the development of a majority of human cancers [1]. MYC proteins activate or repress the transcription of numerous genes that encode proteins involved in fundamental cellular processes, including cell proliferation and growth, ribosome biogenesis, metabolism, apoptosis and differentiation [1]. Although the MYC gene, encoding c-MYC, is the best characterized of this family, N-MYC has been established as a driver of multiple tumor types and its increased expression is often associated with a poor cancer prognosis (reviewed in Rickman et al. 2018)[2]. N-MYC is found to be overexpressed in neuroblastoma, medulloblastoma, retinoblastoma, astrocytoma, glioblastoma multiforme, castration-resistant prostate cancer, neuroendocrine prostate cancer, rhabdomyosarcoma, Wilms tumor, small cell lung cancer, pancreatic tumors and hematological malignancies (lymphoma, acute myeloid leukemia) [2-5]. The potential of N-MYC hyperactivity to initiate and drive human cancers is most clearly exemplified in neuroblastoma, the most common extracranial solid tumor in children which accounts for 15% of all childhood cancer deaths [5, 6].

Enhanced MYCN expression has been well established as an initiating event and driver of high-risk neuroblastomas with MYCN-amplification present in about 50% of high-risk neuroblastoma patients and strongly associated with worse prognosis [7-9].

However, despite the well-described contribution of N-MYC to cancer initiation and progression, development of clinically viable inhibitors of N-MYC has not been achieved. This is likely due to the fact that the transcription factor is inherently unstructured, making its direct inhibition difficult [10]. As an alternative approach to disable N-MYC signaling, several indirect N-

MYC-targeting strategies are currently being explored, such as (1) the targeting of proteins that enhance N-MYC stability (e.g. Aurora A inhibitors), (2) the targeting of the transcription of MYCN

(e.g. inhibitors of bromodomain and extra-terminal (BET) proteins), (3) the inhibition of proteins required for N-MYC–driven transcription (e.g. CDK7 inhibitors) as well as (4) the targeting of N-

MYC-driven genes encoding targetable proteins critical for N-MYC-induced oncogenesis and tumor progression [10-15].

Our previous work in neuroblastoma demonstrated that N-MYC directly up-regulates the expression of several members of the ATP-binding cassette (ABC) transporter family [16]. This family of transporters, including P-gp (ABCB1) and multidrug resistance associated protein (MRP1), is best known for the ability to perform ATP-dependent efflux of structurally dissimilar drugs from cells to create multidrug resistance in cancers such as neuroblastoma [16-18]. As enzymatic domains are readily inhibitable by small molecule inhibitors, the presence of ATPase domains on these transporters makes them highly druggable [19, 20]. Interestingly, two ABC transporters directly upregulated by N-MYC, ABCE1 and ABCF1, are reported to exert a completely different function namely in supporting mRNA translation. ABCE1 is essential to multiple stages of translation including translation termination, ribosome recycling, translation re-initiation and ribosome biogenesis while ABCF1 is thought to promote translation initiation, but their roles in N-MYC driven cancers are currently unknown [21-23].

Therefore, in this study, we examined the role of ABC proteins that regulate mRNA translation in relation to N-MYC-driven cancer progression in neuroblastoma. We demonstrate that high expression of ABCE1 is strongly correlated with poor clinical outcome and that N-MYC-driven neuroblastoma is dependent upon elevated levels of ABCE1. This in turn is essential to N-MYC hyperactivity. ABCE1 suppression also impaired the growth and translation in c-MYC overexpressing cells. We thereby identified, for the first time, that the progression of N-MYC-driven and possibly c-MYC-driven cancers can be halted by targeting ABCE1-mediated protein synthesis.

Material and methods:

Patient data:

Neuroblastoma patient survival and RNA sequencing data were obtained from the R2

Genomics SEQC 498 RPM seqcnb1 cohort containing 498 patients (Genomics Analysis and

Visualization Platform, GEO accession number GSE62564, https://hgserver1.amc.nl/cgi- bin/r2/main.cgi). Patient data for the analysis of ChIP-seq experiments were obtained from Zeid et al., 2018 (GEO accession number: GSE80154) [24].

Immunohistochemistry (IHC):

Tumor tissue microarray sections were obtained from the tumor bank of Children’s Hospital at Westmead, Sydney, Australia. Tumors were fixed in formalin before sections of the tumors were taken and embedded in paraffin blocks. Cut sections were stained with hematoxylin and eosin (H&E) and with anti-ABCE1 (#Ab185548, Abcam, 1 in 500 dilution), anti-cleaved PARP clone E51 (#: ab32064, Abcam, RRID:AB_777102, 1:2000 dilution); anti-N-MYC clone NCM II 100 (#: ab16898,

Abcam, RRID:AB_443533, 1:200 dilution); and anti-c-MYC (#ab32072, clone Y69, Abcam, 1 in

200 dilution) antibodies. Photos of the tumor sections were taken with Olympus BX53 microscope with Olympus DP73 camera at 600x and 50x magnification. ABCE1 protein expression level was graded as described in Gamble et al., 2019 [25]. Tumors were considered c-MYC positive if score

>0. Tissues were considered to express high levels of ABCE1 if the score of the tissue was higher than the median score. Blocks from SK-N-BE(2) Luc cells with or without shRNA-induced ABCE1 suppression were used as reference samples.

Tissue culture for cell line maintenance:

SK-N-BE(2) (# 95011815, RRID:CVCL_0528), Kelly (# 92110411, RRID:CVCL_2092),

CHP-134 (# 06122002,), SK-N-AS (# 94092302, RRID:CVCL_1700) and SK-N-FI (# 94092304,

RRID:CVCL_1702) neuroblastoma cell lines were obtained from Sigma-Aldrich. MRC5 fibroblast cells were from American Type Culture Collection (# CCL-171, ATCC, RRID:CVCL_0440). hTERT RPE.1 (RRID:CVCL_4388) cells were a kind gift from Dr Marcia Munoz. The SK-N-BE(2)

Luc, SH-EP (RRID:CVCL_0524), SHEP Tet21N (RRID:CVCL_9812) and COG-N-519

(RRID:CVCL_LF62) cell lines were kind gifts from Dr Jamie Fletcher [26], Dr June Biedler [27], Dr

Susan Cohn [28] and Dr C. Patrick Reynolds, respectively.

SK-N-BE(2) and SK-N-BE(2) Luc were cultured as described by Byrne et al., 2014 [26].

CHP-134, Kelly, SK-N-AS, SK-N-FI, SH-EP and SH-EP Tet21N were cultured as described in Lau et al., 2015 [29]. COG-N-519 cells were cultured as described in Harenza et al., 2017 [30]. MRC5 fibroblast cells were grown in MEM-α with 10% FBS and split twice weekly using trypsin. hTERT

RPE.1 cells were grown in DMEM F12 with 10% FBS and split 2-3 times weekly using trypsin. All cell lines were mycoplasma negative (tested every 6 months, using MycoAlertTM Mycoplasma

Detection Kit from Lonza, # LT07-218) and verified by short tandem repeat (STR) profiling and when compared to reference profiles found to have 100% match. All cell lines were passaged for no more than 6 months after thawing. siRNA transfections:

10-20nM of ABCE1-specific siRNA duplex 1 (siRNA1; 5’-

UGUCUCAGCUUGAAAUUAC-3’, #: DHA-J-008702-06, Dharmacon) and duplex 2 (siRNA2; 5’-

CAAAGACACAGGCAAUUGU-3’, # DHA-J-008702-07, Dharmacon), alongside 10-20 nM of control, non-targeting siRNA (sequence proprietary, # D-001810-10-20, Dharmacon) were transfected into cells at 50-60% confluency using LipofectamineRNAimaxTM (Thermo Fisher

Scientific) or Lipofectamine2000TM (Thermo Fisher Scientific) according to manufacturer’s instructions.

Protein extraction and Western blots:

Protein extraction and Western blots were performed as described in Gamble et al. 2019 [25].

Antibodies include anti-N-Myc (B8.4B) (#: sc-53993, Santa-Cruz, RRID:AB_831602, 1:500 dilution); anti-ABCE1 (#: ab32270, Abcam, RRID:AB_722514, 1:3,000 dilution); anti-puromycin (#:

MABE343, Merck Millipore, RRID:AB_2566826, 1:8,000 dilution); anti-c-Myc clone 9E10 (#: sc-

40, Santa-Cruz, RRID:AB_627268, 1:500); anti-actin (#: A2066, Sigma-Aldrich, RRID:AB_476693,

1:2,000) primary antibodies and anti-rabbit-HRP (#: A0545, Sigma-Aldrich, RRID:AB_257896,

1:10,000) or anti-mouse-HRP (#: NXA931, VWR, RRID:AB_772209, 1:5000) secondary antibodies.

Puromycin incorporation assay:

Puromycin incorporation assays were performed as described in Schmidt et al., 2009 [31]. To measure the effects of ABCE1 suppression on protein synthesis rate, 1-2x105 cells/mL, cells were pulsed with 0.5-2 μg/mL of puromycin for 10 minutes before incubated without puromycin in plates for 1 hour.

Polysome profiling:

Polysome profiling was performed as described by Somers et al., 2019 [32] with minor modification of adding 50 μg/mL cycloheximide (# C4859, Sigma-Aldrich), 1:100 dilution of protease inhibitor (Sigma-Aldrich; # P8340), 3mM DTT (# D0632, Sigma-Aldrich) and 120 U/mL of

RNasin (# N2515, Promega) to the lysis buffer after it has been heated to 60oC for 10mins to activate the RNAse Secure. Cellular extracts were prepared from 3x107 SK-N-BE(2) cells. N=3.

BrdU incorporation assays:

Cell proliferation was measured using a BrdU incorporation assay kit from Sigma-Aldrich (#:

1647229001) according to manufacturer’s instructions, except for inclusion of a blocking step in PBS

containing 10% FBS following fixation. N=3 for all experiments except for the Kelly cells used to assess the effects of ABCE1 knockdown for which 4 independent runs were performed.

Clonogenic assays:

Clonogenic assays were performed on neuroblastoma cells at 24-48 hours after transfection according to Henderson et al., 2011 [33]. N=3 for all cell lines.

Migration and invasion assays:

Cell migration and invasion assays were as described by Byrne et al. 2014 [26]. The amount of migration or invasion was expressed as the migration or invasion index which refers to the number of migrated or invaded cells expressed as a percentage of total number of cells and thus takes into account any changes in cell proliferation. N=3 for all cell lines.

Production of lentiviral plasmids:

Lentiviruses carrying non-targeting control (sequence propriety, #: RHS4750, Dharmacon) or

ABCE1-specific shRNA (5’-ATTAAATAGACATCAGCAG-3’, #: RHS4696-200683418,

Dharmacon) were produced by transfecting 2 µg of plasmid into HEK293T cells using 2 µg of

Translentiviral Packaging MixTM (#: TLP5913, Dharmacon) according to manufacturer’s instructions. Both lines were mycoplasma negative when tested 3 weeks after removal of puromycin.

Animal models and experiments:

Female Balb/c nu/nu (nude; ID: BALB/c-Fox1nu/Ausb), NOD-SCID (ID: NOD/ShiLtJAusb) and NOD-SCID IL2Rγ null (NSG; ID: NOD.CgPrkdcIL2rgSzJAusb) mice at 5-6 weeks of age were purchased from Australian Bio Resources (ABR; Moss Vale, NSW, Australia) while NOD-SCID mice were purchased. All mice were housed in pathogen-free animal facility at Children’s Cancer

Institute and acclimatized for a week prior to experimentation. Regular mouse food was purchased from Gordon's Specialty Stock Feeds. Irradiated rat or mouse food (with or without 600mg/kg of 8

doxycycline) was purchased from Specialty Feeds. All animal experiments were conducted under the ethics approvals 15/139A and 17/42B, as approved by the UNSW Animal Care and Ethics

Committee in Sydney, Australia.

Effects of ABCE1 silencing on subcutaneous tumor growth were assessed by engrafting

5x106 SK-N-BE(2) Luc or 1x106 COG-N-519 cells into one of the two dorsal flanks of female Balb/c nude mice or NSG mice, respectively. Prior to injection, both cell lines were transduced with control

(Ctrl) or ABCE1-specific shRNAs (ABCE1-shRNA) described above. Once each tumor reached

50mm3, mice were placed into one of 4 groups (10 mice per group), receiving the following: (i)

ABCE-specific shRNA: food without doxycycline, (ii) ABCE1-specific shRNA; food with doxycycline, (iii) Control shRNA; food without doxycycline and (iv) Control shRNA; food with doxycycline.

Tumor dimensions were measured every second day using Vernier calipers and sizes were calculated using (height x width x length)/2. When tumors reached 1000mm3, mice were euthanized.

Parts of tumors were snap-frozen for Western blot analysis of ABCE1 suppression or fixed in 10% neutral buffered formalin and stained with anti-Ki67 antibody (clone SP6, Thermo Fisher Scientific,

Cat#: RM-9106, 1 in 400 dilution). Ki67 expression was measured by the number of Ki67 positive cells per field of view under 400x magnification.

To determine the impact of ABCE1 suppression on metastasis, 1x106 SK-N-BE(2) Luc cells carrying Ctrl or ABCE1-specific shRNAs were injected intravenously into NOD/SCID mice [34].

Cells or mice destined to receive doxycycline were given doxycycline (# 0219504405MP

Biomedicals,) 1 week prior to inoculation of cells. A week after inoculation of cells, mice were intraperitoneally administered with 150mg/kg D-luciferin (# LUCK, Gold Biotechnology) and imaged using the Xenogen IVIS Spectrum imaging system. Mice were euthanized 7 weeks after

inoculation or earlier if breathing difficulties were observed. Western blots, Ki67 staining and mouse groups were as described above.

Gene Set Enrichment analysis:

To assess the enrichment of cellular processes in tumors collected from neuroblastoma patients with or without MYCN-amplification, normalized expression values from the Kocak et al.

2013 dataset (GEO accession number: GSE45480) were used as input into Gene Set Enrichment

Analysis (GSEA) on 649 patients across 32,900 probes annotated with genes [35]. Analysis was performed using Gene Set Enrichment Analysis software from the Broad (GSEA v3.0;

RRID:SCR_003199) using C5: GO and C6: Oncogenic (v6.2) in the Molecular Signatures Database.

A False discovery rate (FDR) cutoff below 0.25 was applied to indicate significant enrichment.

Statistical analysis:

P-values of <0.05 were considered statistically significant. Data analysis for all in vitro and in vivo experiments was performed with GraphPad Prism v6.01 for Windows (GraphPad software,

Sydney, Australia; RRID:SCR_002798).

For survival analysis, patient cohorts were dichotomized at the median level of ABCE1 mRNA and protein expression. High MYC expression or activity were defined as above the median of the cohort, as described in Jung et al., 2017 [36]. Cox proportional hazards model was used for univariate and multivariate comparisons to generate, P-values, HR and 95% confidence intervals

(95% CI) using SPSS software v22 (IBM; RRID:SCR_002865). Comparisons of gene expression values between tumor groups were made using student’s t-test.

For ChIP-seq, Bowtie2 (version 2.1.0; RRID:SCR_005476) aligner was used to align raw data to the human genome (GRch38). Binding peaks were called by MACS2 (version 2.1.1;

RRID:SCR_013291). The resulting peaks were annotated using Homer. MACS2 (version 2.1.1) was

used to generate fold enrichment and P-values. FASTQC (RRID:SCR_014583) was used to assess the quality of reads. Reads with Phred quality score below 30 were excluded from the analysis. A false discovery rate (FDR) and P-value of <0.05 were considered significant.

For in vitro experiments, one sample t-tests were used for all experiments where normalization to the control was performed. For in vitro experiments where data were not expressed as a percent change of control, One-Way ANOVA with Dunnett’s multiple corrections tests (> 2 groups) or unpaired, two-tailed t-tests (for 2 groups) were performed.

For in vivo experiments, differences in survival rates between groups were measured by

Mantel-Cox regression. Bioluminescence from tumors were measured in radiance and log2 transformed before unpaired, two-tailed t-tests (for 2 groups) or One-way ANOVA with Dunnett’s multiple corrections tests (> 2 groups) were performed.

Densitometric analysis of all Western blots was performed using ImageJ. Band intensity was normalized to the loading control, total actin. Statistical tests for Western blots varied with experiments and were performed as indicated in figure legends using GraphPad Prism v6.01 for

Windows.

Differences in protein expression determined by immunohistochemical analyses were assessed using unpaired, two-tailed t-tests. The numbers of c-MYC positive tumors with and without

MYCN-amplification were compared through Chi-squared test.

Results

Heightened protein synthesis is a feature of MYCN-amplified neuroblastoma

Previously, up-regulation of genes involved in translation has been reported following N-

MYC overexpression in neuroblastoma cell culture but this observation has never been examined in patient samples [16]. To collect evidence of a role for N-MYC in driving protein synthesis in 11

neuroblastoma tumors, GSEA was used to compare transcriptomes of MYCN-amplified versus non- amplified tumors in a cohort of 649 patients. Differential gene expression analysis identified protein synthesis amongst the top three most highly up-regulated processes in neuroblastoma tumors with

MYCN-amplification (Figure 1A-B). The most highly enriched gene set in MYCN-amplified neuroblastoma was ‘structural constituent of the ribosome’ that encodes the translational machinery

(Fig. 1C; FWER P-value = 0.04; false discovery rate (FDR) Q-value = 0.035). To gain further support we subsequently investigated the effect of forced N-MYC expression on protein synthesis rates in N-MYC-inducible SH-EP Tet21N neuroblastoma cells. Overexpression of N-MYC resulted in increased protein synthesis rates, as measured by puromycin incorporation assays (Figure 1D;

P=0.001). Consistent with this, MYCN-amplified neuroblastoma cell lines had significantly higher basal rates of protein synthesis compared to neuroblastoma cells without MYCN-amplification

(Figure 1E; P=0.005). Taken together, these data demonstrate that N-MYC overexpression is associated with increased global levels of protein synthesis.

ABCE1 as a target to inhibit N-MYC-driven protein synthesis

Given the previous association between N-MYC and ABC protein expression [16], we subsequently examined the specific role of ABCE1 and ABCF1 that are both known regulators of mRNA translation. Analysis of a publicly available ChIP-Seq dataset with Phred quality scores threshold of q30 [24], showed strong binding of N-MYC at the promoter of ABCE1 in BE(2)C (26- fold, FDR<0.0001, P<0.0001), Kelly (6-fold, FDR<0.0001, P<0.0001) and NGP (8-fold,

FDR<0.0001, P<0.0001) MYCN-amplified neuroblastoma cell lines (Figure 2A). Although N-MYC bound to the promoter of ABCF1 in BE(2)C (13-fold, FDR<0.0001, P<0.0001) and NGP (4-fold,

FDR<0.0001, P<0.0001) cells, this observation was lost upon application of a quality score of q30, indicating the observed binding at ABCF1 may be a false positive finding or at least considerably weaker (Supplementary Figure S1A-B). Consistent with this, analysis of an independent publicly

available RNAseq dataset of 498 neuroblastoma patients showed that the mRNA expression of

ABCE1 was significantly higher in patients with tumor MYCN-amplification but not for ABCF1

(ABCE1 P<0.0001, Figure 2B). Moreover, high expression of ABCE1 was correlated to reduced event-free survival (EFS), with a stronger correlation observed for ABCE1 compared to ABCF1

(ABCE1 P<0.0001, ABCF1 P=0.014, Figure 2C and Supplementary Table S1). This was further supported by a multivariate analysis indicating that high ABCE1 mRNA expression remained an independent predictor of EFS after adjustment for MYCN-amplification, age and INSS stage while this was not observed for high expression of ABCF1 (ABCE1: P<0.001, HR = 2.46 with 95% CI:

1.71-3.54 for EFS; Supplementary Table S2). The prognostic significance of ABCE1 expression was further examined at the protein level by tumor tissue microarray (TMA) analysis on 81 neuroblastoma samples. The analysis showed that elevated protein expression of ABCE1 was correlated with reduced patient survival (P=0.02, HR=2.09 with 95% CI: 1.11-3.92; Figure 2D) and

MYCN-amplified tumors expressed significantly higher levels of ABCE1 protein compared to tumors lacking MYCN-amplification (P<0.001; Figure 2E).

We next sought to determine the functional role of ABCE1 in N-MYC enhanced protein synthesis. We therefore silenced ABCE1 expression by siRNA in a panel of neuroblastoma cell lines and observed a significant reduction in protein synthesis only in MYCN-amplified cell lines (Figure

3A, 3B; Supplementary Figure 2). This was further substantiated using the N-MYC overexpressing

SH-EP Tet21N cells where loss of ABCE1 returned the elevated protein synthesis in N-MYC overexpressing cells to baseline levels (Figure 3C). Translation efficiency was then determined using polysome profile analysis and demonstrated that ABCE1 suppression in MYCN-amplified neuroblastoma cells reduced translation efficiency, indicated by a reduction in the polysome to monosome ratio, while translation in non-malignant MRC5 fibroblasts and retinal epithelial cells

(hTERT RPE.1) was unaffected (Figure 3D, 3E; Supplementary Figure 2). Taken together, these data

provide strong evidence that ABCE1 expression is necessary to sustain N-MYC-driven protein synthesis.

ABCE1 suppression impairs N-MYC-driven malignant phenotypes

To determine whether ABCE1 plays a role in mediating the aggressive characteristics of N-

MYC-driven cancer, we assessed the effects of siRNA-mediated ABCE1 suppression in a panel of neuroblastoma cell lines. Interestingly, ABCE1 suppression in neuroblastoma cell lines with MYCN- amplification significantly reduced cell proliferation (Figure 4A), clonogenic capacity (Figure 4B) and migration (Figure 4C) whereas no effect was observed in neuroblastoma cell lines without

MYCN-amplification or the non-malignant MRC5 and hTERT RPE.1 cell lines (Figure 4A-C).

ABCE1 suppression did not appear to affect apoptosis in the MYCN-amplified SK-N-BE(2) neuroblastoma cells as evaluated by flow cytometry measuring levels of cell surface Annexin V and

7-AAD expression (Supplementary Figure S3), indicating the changes observed in BrdU incorporation assays and clonogenic assays following ABCE1 suppression are likely due to impaired cell proliferation. ABCE1 suppression also significantly reduced the invasion of extracellular matrix by MYCN-amplified SK-N-BE(2) cells (Figure 4D; P≤0.001).

ABCE1 is required for N-MYC-driven tumor progression

To investigate the impact of ABCE1 suppression on N-MYC-driven tumor growth in vivo,

ABCE1-specific or non-targeting (control) lentiviral shRNAs were transduced into bioluminescent neuroblastoma SK-N-BE(2) Luc or patient-derived neuroblastoma COG-N-519 cells. The impact of

ABCE1 suppression by ABCE1-specific shRNA on malignant phenotype of these cells was first confirmed in vitro (Supplementary Figure S4). Following subcutaneous engraftment of SK-N-BE(2)

Luc or COG-N-519 cells into Balb/c nude or NSG mice respectively, doxycycline-induced ABCE1 suppression significantly extended the survival of tumor-bearing mice compared to control groups

(Figure 5A). In mice with SK-N-BE(2) Luc tumors, ABCE1 suppression lengthened median survival by 2.0-fold (median survival of 16.4 days (control) and 32.75 days (treatment group); Figure 5A;

P=0.001). A significant (30%) extension in median survival was also observed in mice with COG-N-

519 tumors following ABCE1 suppression (median survival is 22.75 days (control) and 29.55 days

(treatment group); Figure 5A; P=0.005). The presence of doxycycline itself had no effect on tumor growth (Supplementary Figure S5A). Induction of ABCE1-specific shRNA led to a marked inhibition in tumor cell proliferation, as indicated by a significant reduction in Ki67 staining (Figure

5B; P=0.002), while no change in apoptosis levels was observed (Supplementary Figure S5B), consistent with findings from in vitro experiments. Western blot analysis confirmed reduced expression of ABCE1 in shRNA-treated tumors, indicating that the delay in tumor progression can be attributed to ABCE1 suppression (Figure 5C; P<0.001).

To test whether ABCE1 suppression also affected cancer spread and colonization at distant sites, SK-N-BE(2) Luc cells were xenografted intravenously in NOD/SCID mice and the formation of metastases evaluated by bioluminescent imaging. Over the course of the experiment, mice bearing neuroblastoma cells with induced ABCE1 down-regulation consistently showed lower metastatic tumor burden compared to mice in the control groups (Figure 5D-E; P<0.001, Supplementary Figure

S5C). Tumors from these mice also showed reduced cell proliferation (as indicated by Ki67 staining) and exhibited fewer liver metastases compared to the control group (Figure 5F-G). As anticipated, the shRNA-treated tumors also displayed lower ABCE1 expression (Figure 5H; P<0.001).

High ABCE1 expression is correlated to poor outcome and malignant characteristics in c-

MYC overexpressing neuroblastomas.

N-MYC and c-MYC share a number of transcriptional targets including several ribosomal proteins and translation initiation factors. Due to negative feedback loops between expression of these two transcription factors, when either is expressed at high levels expression of these proteins is 15

typically inversely correlated and this has been shown by us and others in tumors from neuroblastoma patients [36, 37]. We found that in neuroblastoma patient tumors c-MYC positivity is more often observed in neuroblastomas lacking MYCN-amplification (11% in MYCN-amplified vs

22% in non-MYCN-amplified tumors; P=0.036). The overlapping functions and reciprocal expression of these proteins implies that c-MYC overexpression can functionally replace N-MYC to drive the malignant transformation and progression of neuroblastoma in a similar manner to N-MYC, as demonstrated in preclinical models [9]. It also indicates that these proteins may rely on similar downstream processes, such as ABCE1-mediated translation, to fuel neuroblastoma progression. To investigate the ABCE1 to c-MYC overexpressing neuroblastomas, we firstly examined the association between ABCE1 expression and clinical outcome in neuroblastoma patients in relation to

N-MYC and c-MYC expression levels. High ABCE1 expression was predictive of poor outcome in

MYCN non-amplified neuroblastoma patients with either high MYC expression (defined by expression above the median) or high MYC activity based on a MYC signature score above the median of the cohort as described in Jung et al. 2017 (Figure 6A) [36]. High ABCE1 expression was not associated with poor outcome in neuroblastomas with low MYC activity (Figure 6A, right panel), consistent with the absence of a role for ABCE1 in the malignant phenotype of neuroblastoma cell lines with negligible levels of N-MYC or c-MYC (SH-EP, SK-N-AS and SK-N-FI). The relationship between expression of ABCE1 and MYC was further examined in tumor tissue microarrays stained with anti-ABCE1, c-MYC or N-MYC antibodies. High ABCE1 expression was observed in tumors that lacked MYCN-amplification but expressed high levels of c-MYC (Figure 6B, 6C). There was a similar trend in cell lines where those expressing c-MYC or N-MYC displayed higher levels of

ABCE1 protein (Supplementary Figure S6A, S6B). The observed elevated ABCE1 expression may stem from the direct up-regulation of ABCE1 by c-MYC as we have previously found in chronic myeloid leukemia [18]. We further confirmed this for neuroblastoma cells by showing the direct

binding of c-MYC to the ABCE1 gene promoter using a publicly available ChIP-seq dataset produced from the c-MYC overexpressing NB69 cells (Supplementary Figure S6C)[38]. To formally test whether ABCE1 suppression also affects the cell proliferation and protein synthesis in c-MYC- expressing neuroblastoma cells, we suppressed ABCE1 expression in NB69 cells. In agreement with the observations in N-MYC overexpressing neuroblastoma cells, suppression of ABCE1 in c-MYC overexpressing NB69 neuroblastoma cells led to significant reductions in protein synthesis and proliferation (Figure 6D-E). Together these findings indicate that, in addition to N-MYC-driven neuroblastoma, ABCE1 is required to sustain efficient elevated protein synthesis to support the growth of neuroblastoma cells with high c-MYC expression.

Discussion

Despite the critical importance N-MYC plays in the progression of multiple types of cancer, clinically efficacious methods of targeting N-MYC are lacking. Our study has uncovered that N-

MYC drives elevated mRNA translation and that direct suppression of translation factor ABCE1 can disable N-MYC-driven translation and tumor progression. Of further interest, although ABCE1 is known to be fundamentally important for mRNA translation and the growth of Archaean, yeast and mammalian cells [21, 23], its suppression selectively dampens the aggressive characteristics of N-

MYC-driven cancers without affecting non-malignant cells.

Translation factors can contribute to cancer progression by enhancing the translation of specific transcripts important in cancer biology (sequence-specific translation) or by enhancing the translation of transcripts indiscriminately (global protein synthesis) [39, 40]. The observed decrease in global protein synthesis, measured by puromycin incorporation assays, suggests that ABCE1 suppression may impair translation in MYCN-amplified neuroblastoma cells in a global manner.

Based on our data, the selectivity of ABCE1 suppression for MYCN-amplified neuroblastoma cells stems from their reliance on elevated rates of protein synthesis which causes these cells to be more 17

vulnerable to translation impairment compared to those without MYCN-amplification. This difference in dose requirement for translation factors concurs with observations made in c-MYC driven B-cell lymphomas, where haploinsufficiency in factors such as ribosome protein L24 or translation initiation factor eIF4E only becomes rate-limiting during c-MYC-driven malignant transformation and progression [41, 42]. Many translation factors that contribute to the progression of c-MYC- driven cancers can also up-regulate sequence-specific translation [39]. For example, eIF4E promotes the translation of mRNAs encoding proteins required for elimination of reactive oxygen species, critical for c-MYC or RAS-driven malignant progression and transformation [41]. Previous studies examining ABCE1 in yeast or chronic myeloid leukemia cells support our hypothesis by demonstrating that impaired ribosome recycling following the loss of ABCE1 occurs in a non- sequence-specific manner [43, 44]. Hence, the difference in dose-requirement for translation mediated by heightened ABCE1 expression is most likely responsible for the observed differential response to ABCE1 suppression in MYCN-amplified and non-amplified neuroblastoma cells.

The elevated rates of translation in MYCN-amplified neuroblastomas and their dependence on heightened ABCE1 expression raise the question of whether existing translation inhibitors can offer the same selective effects as ABCE1 suppression. Translation factors eIF4A and eIF4E may be of particular relevance to neuroblastoma as they have been implicated in N-MYC-driven diseases. A study by Rust et al. (2018) revealed that inhibition of eIF4A activity, either through a bacterial toxin

(BLF1) or through the eIF4A inhibitor, rocaglamide, has a greater growth-impairing effect on N-

MYC overexpressing neuroblastoma cells compared to cells lacking N-MYC overexpression [45].

Similarly, elevated expression of eIF4E has been associated with MYCN-amplified neuroblastoma, late stage disease and poorer clinical outcome [46]. Although there is no published report demonstrating the efficacy of eIF4E inhibitors, unpublished pilot data from our laboratory indicates that treatment with ribavirin can confer stronger anti-proliferative effects in the N-MYC

overexpressing SH-EP Tet21N neuroblastoma cells compared to cells without N-MYC expression

(P=0.01, N=3). These data provide encouraging evidence that agents targeting these translation initiation factors may be re-purposed for neuroblastoma, although extensive testing in preclinical models of neuroblastoma would be required to assess their in vivo efficacy. This will be particularly important for eIF4A inhibitors as they are known to possess low bioavailability (as low as <2%) and are highly effluxed through the P-gp membrane transporter, implying cells with multidrug resistance, through expression of the drug efflux pumps, may be unresponsive to such treatment [47, 48].

Nevertheless, preliminary in vitro investigations with these agents in neuroblastoma complement our findings by showing that directly targeting translation factors may be a potential therapeutic strategy for MYCN-amplified neuroblastoma. By demonstrating the potent in vivo anti-growth effects of

ABCE1 suppression and its selectivity against MYCN-amplified neuroblastoma cells, we have identified a novel translation factor that can be exploited for neuroblastoma therapy. Although specific ABCE1 inhibitors have yet to be described, the ATPase domains of ABCE1, that provide the energy for mRNA translation, are generally highly druggable, which supports the potential of developing highly specific inhibitors of the protein [19, 23].

Beside direct inhibitors of translation, several anti-cancer agents that exert their function partly through inhibiting mRNA translation have recently been proposed to impair disease progression. However, the ability to specifically impair N-MYC-driven translation is a characteristic that distinguishes ABCE1 suppression from these agents. These agents include difluoromethylornithine (DFMO), which inactivates eIF5A by inhibiting polyamine production, and

CX-5461, which blocks rRNA transcription by targeting RNA Pol I [25, 49]. Despite their ability to inhibit translation, there is no evidence that these compounds work through impairing N-MYC-driven translation in the same manner as ABCE1 suppression. A possible reason is that these agents affect other specific cellular pathways, with the inhibition of protein synthesis a consequence of this action.

For example, RNA pol I inhibition by CX-5461 impairs the production of ribosomes and thereby induces several cellular responses including nucleolar stress while DFMO starves cells from polyamines that exert numerous roles within a cell, in addition to protein synthesis. These data emphasize the knowledge gap in finding translation inhibitors that specifically abolish N-MYC- driven translation in neuroblastoma and reiterates the need to find therapeutics that exploit this dependence.

The implications of our findings could extend beyond neuroblastoma as a variety of cancers are driven by N-MYC [11]. By identifying elevated ABCE1-mediated translation as a potentially targetable vulnerability of MYCN-amplified neuroblastoma, the epitomic model for N-MYC-driven cancers, our work indicates that targeting ABCE1 may be an effective therapeutic strategy for many other cancers driven by N-MYC. Furthermore, as c-MYC-driven cancers are also dependent on efficient translation for their malignant progression and ABCE1 expression is also up-regulated by c-

MYC [18], it is possible that ABCE1 constitutes a therapeutic target for c-MYC driven cancers as well. Our data suggest that ABCE1 is elevated in c-MYC overexpressing tumors and that it is required to maintain translation and proliferation in these cells. Since c-MYC hyperactivity is implicated in 50% of human cancers [50] examining whether ABCE1 is a therapeutic target for

MYC-driven cancers other than neuroblastoma is a priority and might identify cancers that can benefit from agents targeting ABCE1.

The contribution of ABCE1 to cancers driven by oncogenes other than c-MYC and N-MYC has not been reported. The regulation of ABCE1 expression at the transcriptional and translational levels is an under-investigated area of research such that besides MYC-driven transcriptional up- regulation of ABCE1, no other regulatory mechanisms have been reported. Investigating this concept could potentially uncover other diseases that are dependent on elevated ABCE1 expression.

In conclusion, we have identified elevated translation through ABCE1 as a vulnerability of N-

MYC-driven neuroblastoma. Exploitation of this vulnerability by the development of ABCE1 inhibitors may lead to a novel therapeutic approach for MYCN-amplified neuroblastoma. Due to the overlapping functions of N-MYC and c-MYC transcription factors, our work also warrants further investigations into the possibility of targeting ABCE1 to attenuate the growth of other MYC-driven cancers.

Author Contributions: M.J.H., K.S. and M.D.N. conceived and supervised the project. J.G., M.J.H. and K.S. designed the experiments. J.G. performed the in vitro and in vivo experiments. M.J. performed the patient survival analysis. C.M. and P.V. performed the analysis of the GSEA data.

J.I.F. provided the SK-N-BE(2)-Luc cells, provided advice and assisted in the imaging of the systemic xenograft model. A.K. provided and advised on experimental design for the COG-N-519 cells. E.P.K., K.M.H., R.B.P. and R.D.H. advised on the project and assisted in designing polysome profiling experiments. M.H. advised on the project. A.J.G. advised on the histological staining and analysis. J.G., K.S and M.J.H wrote the manuscript. All authors reviewed the manuscript and provided input. Data and materials availability: The COG-N-519 patient-derived cell line model was obtained under a material transfer agreement (MTA) between the Children’s Oncology Group and the Children’s Cancer Institute. The microarray data for the 649 neuroblastoma patients cohort is available at the Gene Expression Omnibus database (accession no. GSE45480) along with age, stage, and MYCN-amplification status. The RNA sequencing data along with age, stage, and MYCN- amplification status for the 498 neuroblastoma patients were obtained from publicly available datasets in R2 Genomics Analysis and Visualization platform (https://hgserver1.amc.nl/cgi- bin/r2/main.cgi). All the remaining data used for the study are present in the main text or in the

Supplementary Materials.

Acknowledgements: We acknowledge Dr Jayne Murray, Dr Emanuele Valli, Dr Denise Yu, Dr

Tzong-Tyng Hung, Dr Brendan Lee, Mr Janith Seneviratne, Miss Lara Martin and Miss Ashley Weir for their technical advice or help with performing certain experiments. We would like to thank Dr

Charles De Bock and Dr Tao Liu for their review of the manuscript. Funding: This work was supported by the National Health and Medical Research Council (APP1016699 and APP1132608 to

M.H, M.D.N.), Cancer Institute NSW (10/TPG/1-03 and 14/TPG/1-13 to M.H., M.D.N.), Tour de

Cure (RG162423 to M.J.H.), the Australian Postgraduate Award (to J.G.), Children’s Cancer Institute and Cancer Therapeutics CRC PhD Top-up Scholarship (to J.G.).

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Figure legends:

Figure 1. N-MYC overexpression heightens protein synthesis in neuroblastoma cells. (A) Gene set enrichment analysis (GSEA) comparing gene ontology (GO) datasets enriched in neuroblastoma 25

patient tumors with MYCN-amplification compared to those without. Enriched datasets were classified by the processes to which they contribute. (B) GSEA comparing oncogenic signature datasets enriched in neuroblastoma patient tumors with MYCN-amplification compared to those without. Enriched datasets were classified by the processes to which they contribute. (C) GSEA enrichment plot of the most enriched GO dataset in MYCN-amplified neuroblastoma patient tumors when ranked by normalized enrichment score (NES). This dataset consists of genes that contribute to the structural integrity of the ribosome. (D) Representative Western blot with densitometric analysis of puromycin incorporation assays. N=4. (E) Representative Western blot and densitometric analysis of puromycin incorporation assay. N=3. Data represent the mean ± SEM. P-values derived from unpaired t-test. Clinical data for (A-C) were obtained from a cohort of 649 neuroblastoma patients described in Kocak et al. 2013 [35].

Figure 2. High ABCE1 expression is correlated with poor patient outcome and MYCN gene expression. (A) ChIP-seq tracks of ABCE1 after pulldown with anti-N-MYC antibody in three MYCN-amplified neuroblastoma cell lines, BE(2)C, Kelly and NGP. Phred quality scores with a threshold of q30 were used to eliminate false positives. (B) Expression of ABCE1 or ABCF1 mRNA in neuroblastoma patients with or without tumor MYCN-amplification. RNA sequencing data obtained from 493 neuroblastoma patients in the R2 Genomics and Visualization platform, SECQ 498 RPM seqcnb1 cohort. P-value derived from student’s t-test. (C) Kaplan-Meier survival analysis of 498 neuroblastoma patients in the cohort described in (B). Groups were dichotomized at the median level of ABCE1 expression in the cohort. P-values derived from Log-rank test. (D) Prognostic significance of ABCE1 protein expression in tumor tissue microarrays (TMAs) obtained from the Children’s Hospital at Westmead, Sydney, Australia. P-value derived from Log-rank test. N=81. (E) ABCE1 protein expression in the neuroblastoma patients with or without tumor MYCN-amplification. P-value derived from unpaired t-test. N=77. Representative images of samples with or without MYCN-amplification. Scale bar represents 100μm. Photos taken at 600x magnification with 50x magnification for inset. Please see Supplementary Tables S1 and S2.

Figure 3. ABCE1 suppression selectively reduces protein synthesis in MYCN-amplified neuroblastoma cells. (A) Western blots from puromycin incorporation assays measuring protein synthesis in MYCN-amplified neuroblastoma cell lines that were transfected with non-targeting control (Ctrl) or ABCE1-specific siRNAs (siRNA1 and siRNA2). (B) Western blots from puromycin incorporation assays in neuroblastoma cell lines lacking MYCN-amplification with or without siRNA-induced ABCE1 suppression. (C) Western blot and respective densitometric analysis of puromycin incorporation assays performed in the SH-EP Tet21N neuroblastoma cells with or without ABCE1 suppression. * P-value compares difference between control siRNA transfected cells with and without N-MYC expression. ^ P-value compares the difference between N-MYC negative cells transfected with control or ABCE1-specific siRNAs. Dox – doxycycline. (D) Polysome profiling experiments measuring translation efficiency in the SK-N-BE(2) neuroblastoma cells. (E) Western blots from puromycin incorporation assays in MRC5 (non-malignant, fibroblast) and hTERT RPE.1 (non-malignant, retinal epithelial) cells with or without siRNA-mediated ABCE1 suppression. N=3 for all experiments, except for puromycin incorporation assays for hTERT RPE.1 cells for which 4 26

independent experiments were performed. All data represent means ± SEM. P-values for all comparisons were derived from one sample t-test, except for samples without MYCN-amplification in part (C) where One-Way ANOVA with Dunnett’s multiple corrections test was performed.

Figure 4. ABCE1 suppression selectively impairs the malignant phenotypes of MYCN-amplified neuroblastoma cells. (A) BrdU proliferation assays performed on cells transfected with control (Ctrl) or ABCE1-specific siRNAs (siRNA1 and siRNA2). N=4 for Kelly cells; N=3 for all other cell lines. (B) Colony forming assays following siRNA-mediated ABCE1 suppression. N=3. (C) The ability of cells to migrate, expressed as the migration index, was measured using cell culture inserts following siRNA-mediated ABCE1 suppression. Migration index refers to the number of migrated cells expressed as a percentage of total number of cells. N=3. (D) The ability of cells to invade extracellular matrix, expressed as the invasion index, was measured using cell culture inserts coated with type IV human collagen following ABCE1 suppression. Invasion index refers to the number of invaded cells expressed as a percentage of total number of cells. N=3. (A-B) P-values obtained from one sample t-test. (C-D) P-values obtained from One-Way ANOVA with Dunnett’s multiple corrections test. All data represent means ± standard error (SEM).

Figure 5. ABCE1 suppression impairs the progression of MYCN-amplified neuroblastoma tumors. (A) Tumor growth curves and Kaplan-Meier survival curves of mice xenografted with SK- N-BE(2) or COG-N-519 neuroblastoma cells as indicated on figure. N=10 per group. Endpoint was when tumors reached ≥ 1000mm3. Days refers to number of days after the mice developed tumors ≥50mm3 and were placed on food with or without doxycycline. P-values were derived from Log-rank test. (B) Ki67 staining showing the number of proliferating cells in subcutaneous tumors with or without induction of ABCE1-specific shRNA. Photos taken at 400x magnification. N=6. P-value was derived from unpaired t-test. (C) Western blots showing ABCE1 expression in subcutaneous tumors. P-value was derived from unpaired t-test. (D) Growth curves of metastatic disease burden over time after engraftment and in vivo induction of ABCE1-specific shRNA. (E) Quantification of metastatic disease burden at 4 weeks after engraftment and in vivo induction of ABCE1-specific shRNA. N=10 per group. P-value was derived from unpaired t-test. (F) Ki67 staining of proliferating cells in liver metastases. Photos taken at 400x magnification. (G) Photos of livers obtained from mice after the endpoint. (H) Western blots showing ABCE1 expression in liver metastases. N=6 for group without doxycycline induction; N=8 for group treated with doxycycline. P-value was derived from unpaired t-test. (B and F) Scale bar represents 100μm. All data represent means ± SEM. See Supplementary Figures S3 and S4.

Figure 6: ABCE1 predicts poor clinical outcome and supports the malignant characteristics of neuroblastomas with c-MYC overexpression. (A) Prognostic significance of ABCE1 in neuroblastoma patients with different levels of c-MYC or MYC activity. RNA sequencing data were obtained from the R2 Genomics and Visualization platform, SEQC 498 RPM seqcnb1 cohort. Out of this 498-patients cohort, 401 tumors that do not possess MYCN-amplification were used for our

analysis. MYC expression or activity levels were segregated at the median of the cohort (N=200 or 201) before the cohort was further segregated based on ABCE1 expression at the median level. P- values were derived from Log-rank test. (B) Photos from TMAs obtained from Westmead Children’s Hospital, Sydney Australia and stained for c-MYC, N-MYC and ABCE1 expression and with H&E. Photos taken at 600x magnification and 50x magnification for inset. Scale bar represents 100um. (C) Quantification of ABCE1 staining in the TMAs as exemplified in (B). P-value was derived from unpaired t-test. N=77. (D) Puromycin incorporation assay measuring protein synthesis in c-Myc over-expressing NB69 neuroblastoma cells. N=5. P-values were derived from one sample t-test. (E) Brdu incorporation assays following ABCE1 suppression in c-Myc over-expressing NB69 neuroblastoma cells. N=3. P-values were derived from one sample t-test.

Suppression of ABCE1-mediated mRNA translation limits N-MYC-driven cancer progression

Jixuan Gao, MoonSun Jung, Chelsea Mayoh, et al.

Cancer Res Published OnlineFirst July 10, 2020.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-19-3914

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2020/10/31/0008-5472.CAN-19-3914.DC1

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