Inhibition of mitochondrial translocase SLC25A5 and histone deacetylation is an effective combination therapy in neuroblastoma

Abstract

The mitochondrion is a gatekeeper of apoptotic processes, and mediates drug resistance to several chemotherapy agents used to treat cancer. Neuroblastoma is a common solid cancer in young children with poor clinical outcomes following conventional chemotherapy. We sought druggable mitochondrial protein targets in neuroblastoma cells. Among mitochondria-associated gene targets, we found that high expression of the mitochondrial adenine nucleotide translocase 2 (SLC25A5/ANT2), was a strong predictor of poor neuroblastoma patient prognosis and contributed to a more malignant phenotype in pre-clinical models. Inhibiting this transporter with PENAO reduced cell viability in a panel of neuroblastoma cell lines in a TP53-status-dependant manner. We identified the histone deacetylase inhibitor, suberanilohydroxamic acid (SAHA), as the most effective drug in clinical use against mutant TP53 neuroblastoma cells. SAHA and PENAO synergistically reduced cell viability, and induced apoptosis, in neuroblastoma cells independent of TP53-status. The SAHA and PENAO drug combination significantly delayed tumour progression in pre-clinical neuroblastoma mouse models, suggesting that these clinically advanced inhibitors may be effective in treating the disease.

Keywords: PENAO; SAHA; SLC25A5; neuroblastoma.

FIGURE 1

A subset of mitochondria‐associated genes associates with NB cell survival, high chromatin accessibility and poor patient survival. (A) 1148 Mitochondrial Associated Genes (MAGs) were derived from the integration of 28 mitochondrial gene sets from the MSigDB. Hierarchical clustering of gene expression (1148 MAGs) in the SEQC NB Cohort (RNA‐Seq; GSE62564) are represented as a heatmap with clinical annotations (Age [<18 Months vs >18 Months], Stage [1,2,3,4 s vs 4], MYCN Status [Non‐Amp vs Amp], EFS Event, OS Event & MAG scores for each patient). β‐coefficients from univariate Cox Proportional Hazards (CoxPH) models, using the median expression of each MAG (n = 1148) as a cut‐off, are shown as a bidirectional bar graph. (B) Patients from the SEQC NB cohort (n = 498) were subdivided based on cluster expression (RNA‐Seq) and their overall survival probability was plotted on a Kaplan‐Meier curve. Comparisons are made between C5 vs C1,2,3,4,6 and C6 vs C1,23,4. (C) Mean hazard ratios (HR = e[β]) of each cluster are shown with the error bars representing the SEM of HR's within a cluster. (D) The proportion of genes with accessible promoters in each cluster with error bars representing the SEM across all 7 NB cell lines (where an accessible promoter had significant enrichment of ATAC signal, FDR q‐value <0.05, −1000/+100 bp from TSS). (E) The mean gene dependency probability scores for each gene cluster derived from Project Achilles. Error bars represent the SE of the mean in each MAG cluster. (F) Gene set enrichment of C6 genes using KEGG, GO and ReactomePA databases, the top 10 genesets enriched for C6 genes are shown, with gensets being ranked by ‐log10[Adjusted P value]. Each gene set is also categorised by broad process involvement (ETC, electron transport chain; TCA, tricarboxylic acid cycle, Oxphos, oxidative phosphorylation). Error bars represent the SE of the mean in each MAG cluster

FIGURE 2

High SLC25A5 gene expression is predictive of poor NB patient prognosis. (A) The median gene expression (RNA‐Seq) of each MAG (n = 1148) was considered as a prognostic variable alongside classical predictors of NB patient outcome (MYCN Amplification Status [amplified vs non‐amplified], Stage of Disease [Stage 1,2,3,4 s vs Stage 4] & Age [>18 months vs < 18 months]) in iterative multivariate CoxPH models using the SEQC NB patient cohort (n = 498). The dot plot represents the multivariate hazards ratios from the CoxPH models for each gene with regard to event free survival (EFS)/overall survival (OS) probability of patients and cluster identity. (B) Kaplan‐Meier survival curves showing the OS probability of patients in the SEQC NB cohort (n = 498) when dichotomised by median SLC25A5 gene expression (RNA‐Seq). Hazard Ratio's (HR) and log‐rank P values are presented from a univariate CoxPH model. (C) Summary of the OS multivariate CoxPH models with SLC25A5 gene expression against classical prognostic predictors, dots represent the median HR whilst lines represent the 95% confidence interval. (D) Box plots of SLC25A5 gene expression subdivided using established prognostic predictors of poor NB patient outcome in the SEQC cohort (n = 498), patients are stratified by either; MYCN amplification status (MYCN non‐amplified; MNA, MYCN amplified; MA), stage of disease according to the International Neuroblastoma Staging System (INSS) (Stage 1,2,3,4 s vs 4) and age at diagnosis (<18 vs >18 months of age). (E) Cell viability of BE(2)‐C, CHP‐134 and SK‐N‐AS cell lines following siRNA mediated knockdown of SLC25A5 for 48‐72 h. SLC25A5 siRNA (#5 & #9) are compared with the non‐targeted siControl using a two‐way ANOVA with multiple comparison, reported P values are adjusted for multiple comparisons. (F) Representative western blot of SK‐N‐BE(2)‐C, CHP‐134 and SK‐N‐AS cells transfected with control or SLC25A5 siRNA for 72 h to confirm sufficient knockdown of SLC25A5 for cell viability assays

FIGURE 3

Direct targeting of SLC25A5 with the selective inhibitor PENAO significantly reduces NB cell viability. (A) Cell viability curves of NB and normal lung fibroblast cell lines upon PENAO treatment (0‐20 μM) for 72 h, cell lines are ranked according to IC₅₀. (B) Average IC₅₀'s of cell lines subcategorised by mutations in the TP53 pathway (TP53, CDKN2A). (C) PENAO cytotoxicity (IC₅₀) in neuroblastoma cell lines stably transduced with an shRNA construct targeting TP53 mRNA. (D) PENAO cytotoxicity (IC₅₀) in SH‐EP clones stably expressing TP53 cDNA constructs with point mutations in the DNA‐binding domain (p.C135R & p.C135P mutants) or empty vector controls (EV1/EV2). Error bars represent the SE of the mean of at least three independent biological replicates

FIGURE 4

SAHA combined with PENAO synergistically induces apoptotic cell death in mutant TP53 NB cell lines. (A) Heatmap of drug sensitivity data (IC₅₀) from the Genomics of Drug Sensitivity in Cancer database (GDSC) z‐scaled for 251 compounds across 32 NB cell lines, TP53 mutations identified by whole exome sequencing (WES) are annotated at the bottom. Black squares on the heatmap represent missing drug screening data. (ESS_Splice = mutation in the exonic splicing silencer site or at donor/acceptor splice sites, Inframe_Del = inframe deletion). (B) 251 compounds ranked by average drug sensitivity (z‐score) in mutant TP53 NB cell lines, the top five compounds are annotated. (C) Cell viability curves of SK‐N‐BE(2)‐C, Kelly, CHP134 & SH‐SY5Y neuroblastoma cells 72 h post treatment with SAHA (0‐2.0 μM) and/or PENAO (0‐3.0 μM) in a constant 1:1.5 ratio. Combination Index (CI) Values generated from CalcuSyn at the corresponding doses are also displayed. (D) Propidium iodide cell cycle assays represented in stacked column graphs indicating the proportion of cells in each cell cycle phase (including sub‐G₁) after 72 h of treatment. (E) BrdU cell proliferation assays 72 h posttreatment in NB cell lines. (F) Annexin‐V/7‐AAD apoptosis assays in the same cell lines 72 h post treatment, represented in stacked column graphs indicating the proportion of apoptotic/dead cells. (G) JC‐1 assays representing the proportion of cells with depolarised mitochondria in each of the four cell lines after 48 h of treatment. *P < .05, **P < .01, ***P < .001, ****P < .0001, Reported P‐values are from one way ANOVA's, error bars represent the SE of the mean of at least three independent biological replicates

FIGURE 5

SAHA combined with PENAO synergistically reduces mutant p53 protein stability. (A) The TP53 wild type cell line SH‐SY5Y was subject to either vehicle control, SAHA (1 μM), PENAO (1.5 μM) or combination treatments for 8 h, followed by mRNA microarray analysis. Differentially expressed genes between conditions were determined and ranked for a Gene Set Enrichment Analysis (GSEA) using the MSigDb hallmark database. Significant pathways (FDR < 0.05) are displayed along with their normalised enrichment score (NES). (B) Enrichment plot displaying the relative enrichment and rank of differentially expressed genes present in the HALLMARK_P53_PATHWAY gene set (indicated by black lines on the x‐axis). Statistics from the GSEA are reported inside the plot. (C) Heatmap of gene expression among treatment conditions in genes within the HALLMARK_P53_PATHWAY gene set, highly expressed genes in the combination which are clustered tightly are highlighted and annotated on the side. (D) Immunoblot of NB cell lysates after 24 h of the indicated treatments, probed with either; anti‐p53, anti‐p21, anti‐Bax or anti‐β‐Actin antibodies. Approximate band sizes are shown to the left of the blots, cell lines used are indicated the top, treatments are indicated below. Both a short and long exposure of anti‐p53 probed membrane are provided. (E) Immunoblot of cycloheximide chases spanning 3 h in KELLY cells treated with DMSO, SAHA 1 μM, PENAO 1.5 μM or the combination for 24 h, probed with anti‐p53 and anti‐β‐Actin. (F) Densitometry of the cycloheximide chase immunoblots are provided, p53 levels are normalised to loading controls and then to the 0‐min time point, error bars represent the stand error of the mean from three independent experiments. The half‐life (t _1/2) of p53 in each condition is reported in the legend

FIGURE 6

SAHA combined with PENAO significantly delays NB tumour growth and induces apoptosis in vivo. (A) Kaplan‐Meier survival curves of Balb/c nude mice xenografted with SK‐N‐BE(2)‐C or (B) KELLY cells, post tumour formation and during the administration of Saline, SAHA (17.5 mg/kg), PENAO (20 mg/kg I.P.) or a combination of both for up to 42 days on a 5 day on/2 day off treatment schedule. P values are from log‐rank statistic tests between the combination and all other treatment groups. (C) Representative ×20 images, with ×40 images inset (×10 objective lens), of treated SK‐N‐BE(2)‐C or (D) KELLY xenograft tumour sections stained with Haematoxylin & Eosin (H&E), anti‐Cleaved Caspase 3 (CC3) or anti‐Ki67. CC3 and Ki67 stained sections are counterstained with Haematoxylin. Scale bars represent 20 μM