Inflammatory response to retrotransposons drives tumor drug resistance that can be prevented by reverse transcriptase inhibitors.

Activation of endogenous retrotransposons frequently occurs in cancer cells and contributes to tumor genomic instability. To test whether inhibition of retrotranspositions has an anticancer effect, we used treatment with the nucleoside reverse transcriptase inhibitor (NRTI) stavudine (STV) in mouse cancer models, MMTV-HER2/Neu and Th-MYCN, that spontaneously develop breast cancer and neuroblastoma, respectively. In both cases, STV in drinking water did not affect tumor incidence nor demonstrate direct antitumor effects. However, STV dramatically extended progression-free survival in both models following an initial complete response to chemotherapy. To approach the mechanism underlying this phenomenon, we analyzed the effect of NRTI on the selection of treatment-resistant variants in tumor cells in culture. Cultivation of mouse breast carcinoma 4T1 in the presence of STV dramatically reduced the frequency of cells capable of surviving treatment with anticancer drugs. Global transcriptome analysis demonstrated that the acquisition of drug resistance by 4T1 cells was accompanied by an increase in the constitutive activity of interferon type I and NF-κB pathways and an elevated expression of LINE-1 elements, which are known to induce inflammatory responses via their products of reverse transcription. Treatment with NRTI reduced NF-κB activity and reverted drug resistance. Furthermore, the inducible expression of LINE-1 stimulated inflammatory response and increased the frequency of drug-resistant variants in a tumor cell population. These results indicate a mechanism by which retrotransposon desilencing can stimulate tumor cell survival during treatment and suggest reverse transcriptase inhibition as a potential therapeutic approach for targeting the development of drug-resistant cancers.

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

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.

The transcriptional co-repressor Runx1t1 is essential for MYCN-driven neuroblastoma tumorigenesis.

MYCN oncogene amplification is frequently observed in aggressive childhood neuroblastoma. Using an unbiased large-scale mutagenesis screen in neuroblastoma-prone transgenic mice, we identify a single germline point mutation in the transcriptional corepressor Runx1t1, which abolishes MYCN-driven tumorigenesis. This loss-of-function mutation disrupts a highly conserved zinc finger domain within Runx1t1. Deletion of one Runx1t1 allele in an independent Runx1t1 knockout mouse model is also sufficient to prevent MYCN-driven neuroblastoma development, and reverse ganglia hyperplasia, a known pre-requisite for tumorigenesis. Silencing RUNX1T1 in human neuroblastoma cells decreases colony formation in vitro, and inhibits tumor growth in vivo. Moreover, RUNX1T1 knockdown inhibits the viability of PAX3-FOXO1 fusion-driven rhabdomyosarcoma and MYC-driven small cell lung cancer cells. Despite the role of Runx1t1 in MYCN-driven tumorigenesis neither gene directly regulates the other. We show RUNX1T1 forms part of a transcriptional LSD1-CoREST3-HDAC repressive complex recruited by HAND2 to enhancer regions to regulate chromatin accessibility and cell-fate pathway genes.

ABCC1 G2012T single nucleotide polymorphism is associated with patient outcome in primary neuroblastoma and altered stability of the ABCC1 gene transcript.

OBJECTIVE: Multidrug resistance is a major cause of treatment failure in neuroblastoma. Multidrug resistance protein 1 has been previously implicated in the development of drug resistance, particularly with regard to influencing clinical outcomes in neuroblastoma. Numerous single nucleotide polymorphisms (SNPs) in the gene encoding multidrug resistance protein 1, namely ATP-binding cassette sub-family C member 1 (ABCC1), have been identified, however, less is known about their potential association with patient outcome. Our aim was to determine the prognostic implications of ABCC1 polymorphisms in neuroblastoma. METHODS: We assessed the frequency of four nonsynonymous ABCC1 SNPs, namely G128C, G1299T, G2168A, and G2012T in both neuroblastoma samples and normal cord blood cells. Polymorphism rates and associations with clinicopathologic characteristics were assessed. RESULTS: The initial three examined polymorphisms were found to be present at very low levels in the Australian population. Of 195 neuroblastoma samples and 158 cord blood samples, none carried the G2012T homozygous variant allele, whereas 13% were heterozygous. The presence of the variant allele was associated with an improved outcome in patients with aggressive neuroblastoma, particularly in older children (P<0.05) and those with nonmetastatic disease (P<0.005). Tumor cell lines that were heterozygous for this SNP, expressed the variant ABCC1 gene transcript at significantly lower levels than the wild-type ABCC1 transcript, and this was associated with the reduced mRNA stability of the variant transcript. CONCLUSION: Collectively, our findings indicated a potential prognostic role of the G2012T ABCC1 polymorphism in clinically relevant subsets of patients with neuroblastoma, and provided further evidence for the ABCC1 gene being a major determinant in neuroblastoma biology.

A Primer for Assessing the Pathology in Mouse Models of Neuroblastoma.

Neuroblastoma, the most common extracranial solid tumor in young children, arises from the sympathetic nervous system. Our understanding of neuroblastoma has been improved by the development of both genetically engineered and xenograft mouse models of the disease. Anatomical pathology is an essential component of the phenotyping of mouse models of cancer, characterizing the morphologic effects of genetic manipulation and drug treatment. The Th-MYCN model, the most widely used of several genetically engineered mouse models of neuroblastoma, was established by targeted expression of the human MYCN gene to murine neural crest cells under the control of the rat tyrosine hydroxylase promoter. Neuroblastoma development in Th-MYCN mice is preceded by neuroblast hyperplasia-the persistence and proliferation of neural crest-derived neuroblasts within the sympathetic autonomic ganglia. The neuroblastomas that subsequently develop morphologically resemble human neuroblastoma and carry chromosomal gains and losses in regions syntenic with those observed in human tumors. In this overview, we describe the essential pathologic features for investigators when assessing mouse models of neuroblastoma. We outline human neuroblastoma as the foundation for understanding the murine disease, followed by details of the murine sympathetic ganglia from which neuroblastoma arises. Sympathetic ganglia, both with and without neuroblast hyperplasia, are described. The macroscopic and microscopic features of murine neuroblastoma are explained, including assessment of xenografts and tumors following drug treatment. An approach to experimental design is also detailed. Increased understanding of the pathology of murine neuroblastoma should improve reproducibility and comparability of research findings and assist investigators working with mouse models of neuroblastoma. © 2021 Wiley Periodicals LLC.

A G316A Polymorphism in the Ornithine Decarboxylase Gene Promoter Modulates MYCN-Driven Childhood Neuroblastoma.

Ornithine decarboxylase (ODC1), a critical regulatory enzyme in polyamine biosynthesis, is a direct transcriptional target of MYCN, amplification of which is a powerful marker of aggressive neuroblastoma. A single nucleotide polymorphism (SNP), G316A, within the first intron of ODC1, results in genotypes wildtype GG, and variants AG/AA. CRISPR-cas9 technology was used to investigate the effects of AG clones from wildtype MYCN-amplified SK-N-BE(2)-C cells and the effect of the SNP on MYCN binding, and promoter activity was investigated using EMSA and luciferase assays. AG clones exhibited decreased ODC1 expression, growth rates, and histone acetylation and increased sensitivity to ODC1 inhibition. MYCN was a stronger transcriptional regulator of the ODC1 promoter containing the G allele, and preferentially bound the G allele over the A. Two neuroblastoma cohorts were used to investigate the clinical impact of the SNP. In the study cohort, the minor AA genotype was associated with improved survival, while poor prognosis was associated with the GG genotype and AG/GG genotypes in MYCN-amplified and non-amplified patients, respectively. These effects were lost in the GWAS cohort. We have demonstrated that the ODC1 G316A polymorphism has functional significance in neuroblastoma and is subject to allele-specific regulation by the MYCN oncoprotein.

Dual Targeting of Chromatin Stability By The Curaxin CBL0137 and Histone Deacetylase Inhibitor Panobinostat Shows Significant Preclinical Efficacy in Neuroblastoma.

PURPOSE: We investigated whether targeting chromatin stability through a combination of the curaxin CBL0137 with the histone deacetylase (HDAC) inhibitor, panobinostat, constitutes an effective multimodal treatment for high-risk neuroblastoma. EXPERIMENTAL DESIGN: The effects of the drug combination on cancer growth were examined in vitro and in animal models of MYCN-amplified neuroblastoma. The molecular mechanisms of action were analyzed by multiple techniques including whole transcriptome profiling, immune deconvolution analysis, immunofluorescence, flow cytometry, pulsed-field gel electrophoresis, assays to assess cell growth and apoptosis, and a range of cell-based reporter systems to examine histone eviction, heterochromatin transcription, and chromatin compaction. RESULTS: The combination of CBL0137 and panobinostat enhanced nucleosome destabilization, induced an IFN response, inhibited DNA damage repair, and synergistically suppressed cancer cell growth. Similar synergistic effects were observed when combining CBL0137 with other HDAC inhibitors. The CBL0137/panobinostat combination significantly delayed cancer progression in xenograft models of poor outcome high-risk neuroblastoma. Complete tumor regression was achieved in the transgenic Th-MYCN neuroblastoma model which was accompanied by induction of a type I IFN and immune response. Tumor transplantation experiments further confirmed that the presence of a competent adaptive immune system component allowed the exploitation of the full potential of the drug combination. CONCLUSIONS: The combination of CBL0137 and panobinostat is effective and well-tolerated in preclinical models of aggressive high-risk neuroblastoma, warranting further preclinical and clinical investigation in other pediatric cancers. On the basis of its potential to boost IFN and immune responses in cancer models, the drug combination holds promising potential for addition to immunotherapies.

Preclinical small molecule WEHI-7326 overcomes drug resistance and elicits response in patient-derived xenograft models of human treatment-refractory tumors.

Targeting cell division by chemotherapy is a highly effective strategy to treat a wide range of cancers. However, there are limitations of many standard-of-care chemotherapies: undesirable drug toxicity, side-effects, resistance and high cost. New small molecules which kill a wide range of cancer subtypes, with good therapeutic window in vivo, have the potential to complement the current arsenal of anti-cancer agents and deliver improved safety profiles for cancer patients. We describe results with a new anti-cancer small molecule, WEHI-7326, which causes cell cycle arrest in G2/M, cell death in vitro, and displays efficacious anti-tumor activity in vivo. WEHI-7326 induces cell death in a broad range of cancer cell lines, including taxane-resistant cells, and inhibits growth of human colon, brain, lung, prostate and breast tumors in mice xenografts. Importantly, the compound elicits tumor responses as a single agent in patient-derived xenografts of clinically aggressive, treatment-refractory neuroblastoma, breast, lung and ovarian cancer. In combination with standard-of-care, WEHI-7326 induces a remarkable complete response in a mouse model of high-risk neuroblastoma. WEHI-7326 is mechanistically distinct from known microtubule-targeting agents and blocks cells early in mitosis to inhibit cell division, ultimately leading to apoptotic cell death. The compound is simple to produce and possesses favorable pharmacokinetic and toxicity profiles in rodents. It represents a novel class of anti-cancer therapeutics with excellent potential for further development due to the ease of synthesis, simple formulation, moderate side effects and potent in vivo activity. WEHI-7326 has the potential to complement current frontline anti-cancer drugs and to overcome drug resistance in a wide range of cancers.

Targeting metabolic activity in high-risk neuroblastoma through Monocarboxylate Transporter 1 (MCT1) inhibition.

Amplification of the MYCN oncogene occurs in ~25% of primary neuroblastomas and is the single most powerful biological marker of poor prognosis in this disease. MYCN transcriptionally regulates a range of biological processes important for cancer, including cell metabolism. The MYCN-regulated metabolic gene SLC16A1, encoding the lactate transporter monocarboxylate transporter 1 (MCT1), is a potential therapeutic target. Treatment of neuroblastoma cells with the MCT1 inhibitor SR13800 increased intracellular lactate levels, disrupted the nicotinamide adenine dinucleotide (NADH/NAD+) ratio, and decreased intracellular glutathione levels. Metabolite tracing with 13C-glucose and 13C-glutamine following MCT1 inhibitor treatment revealed increased quantities of tricarboxylic acid (TCA) cycle intermediates and increased oxygen consumption rate. MCT1 inhibition was highly synergistic with vincristine and LDHA inhibition under cell culture conditions, but this combination was ineffective against neuroblastoma xenografts. Posttreatment xenograft tumors had increased synthesis of the MCT1 homolog MCT4/SLC16A, a known resistance factor to MCT1 inhibition. We found that MCT4 was negatively regulated by MYCN in luciferase reporter assays and its synthesis in neuroblastoma cells was increased under hypoxic conditions and following hypoxia-inducible factor (HIF1) induction, suggesting that MCT4 may contribute to resistance to MCT1 inhibitor treatment in hypoxic neuroblastoma tumors. Co-treatment of neuroblastoma cells with inhibitors of MCT1 and LDHA, the enzyme responsible for lactate production, resulted in a large increase in intracellular pyruvate and was highly synergistic in decreasing neuroblastoma cell viability. These results highlight the potential of targeting MCT1 in neuroblastoma in conjunction with strategies that involve disruption of pyruvate homeostasis and indicate possible resistance mechanisms.

CCI52 sensitizes tumors to 6-mercaptopurine and inhibits MYCN-amplified tumor growth.

The antimetabolite 6-mercaptopurine (6-MP) is an important component in the treatment of specific cancer subtypes, however, the development of drug resistance and dose-limiting toxicities can limit its effectiveness. The therapeutic activity of 6-MP requires cellular uptake, enzymatic conversion to thio-GMP and incorporation of thio-GTP into RNA and DNA, as well as inhibition of de novo purine synthesis by methyl-thio-IMP. Mechanisms that prevent 6-MP entry into the cell, prevent 6-MP metabolism or deplete thiopurine intermediates, can all lead to 6-MP resistance. We previously conducted a high-throughput screen for inhibitors of the multidrug transporter MRP4 using 6-MP sensitivity as the readout. In addition to MRP4-specific inhibitors, we identified a compound, CCI52, that sensitized cell lines to 6-MP independent of this transporter. CCI52 and its more stable analogue CCI52-14 also function as effective chemosensitizers in vivo, substantially extending survival in a transgenic mouse cancer model treated with 6-MP. Chemosensitization was associated with an increase in thio-IMP, suggesting that CCI52 functions directly on 6-MP uptake or metabolism. In addition to its chemosensitizing effects, CCI52 and CCI52-14 inhibited the growth of MYCN-amplified high-risk neuroblastoma cell lines and delayed tumor progression in a MYCN-driven, transgenic mouse model of neuroblastoma. These multifunctional inhibitors may be useful for the further development of anticancer agents and as tools to better understand 6-MP metabolism.

Intratumoral Copper Modulates PD-L1 Expression and Influences Tumor Immune Evasion.

Therapeutic checkpoint antibodies blocking programmed death receptor 1/programmed death ligand 1 (PD-L1) signaling have radically improved clinical outcomes in cancer. However, the regulation of PD-L1 expression on tumor cells is still poorly understood. Here we show that intratumoral copper levels influence PD-L1 expression in cancer cells. Deep analysis of the The Cancer Genome Atlas database and tissue microarrays showed strong correlation between the major copper influx transporter copper transporter 1 (CTR-1) and PD-L1 expression across many cancers but not in corresponding normal tissues. Copper supplementation enhanced PD-L1 expression at mRNA and protein levels in cancer cells and RNA sequencing revealed that copper regulates key signaling pathways mediating PD-L1-driven cancer immune evasion. Conversely, copper chelators inhibited phosphorylation of STAT3 and EGFR and promoted ubiquitin-mediated degradation of PD-L1. Copper-chelating drugs also significantly increased the number of tumor-infiltrating CD8+ T and natural killer cells, slowed tumor growth, and improved mouse survival. Overall, this study reveals an important role for copper in regulating PD-L1 and suggests that anticancer immunotherapy might be enhanced by pharmacologically reducing intratumor copper levels. SIGNIFICANCE: These findings characterize the role of copper in modulating PD-L1 expression and contributing to cancer immune evasion, highlighting the potential for repurposing copper chelators as enhancers of antitumor immunity. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/19/4129/F1.large.jpg.

Macrophage-Derived IL1ß and TNFα Regulate Arginine Metabolism in Neuroblastoma.

Neuroblastoma is the most common childhood solid tumor, yet the prognosis for high-risk disease remains poor. We demonstrate here that arginase 2 (ARG2) drives neuroblastoma cell proliferation via regulation of arginine metabolism. Targeting arginine metabolism, either by blocking cationic amino acid transporter 1 (CAT-1)-dependent arginine uptake in vitro or therapeutic depletion of arginine by pegylated recombinant arginase BCT-100, significantly delayed tumor development and prolonged murine survival. Tumor cells polarized infiltrating monocytes to an M1-macrophage phenotype, which released IL1ß and TNFα in a RAC-alpha serine/threonine-protein kinase (AKT)-dependent manner. IL1ß and TNFα established a feedback loop to upregulate ARG2 expression via p38 and extracellular regulated kinases 1/2 (ERK1/2) signaling in neuroblastoma and neural crest-derived cells. Proteomic analysis revealed that enrichment of IL1ß and TNFα in stage IV human tumor microenvironments was associated with a worse prognosis. These data thus describe an immune-metabolic regulatory loop between tumor cells and infiltrating myeloid cells regulating ARG2, which can be clinically exploited. SIGNIFICANCE: These findings illustrate that cross-talk between myeloid cells and tumor cells creates a metabolic regulatory loop that promotes neuroblastoma progression.

Inhibition of polyamine synthesis and uptake reduces tumor progression and prolongs survival in mouse models of neuroblastoma.

Amplification of the MYCN oncogene is associated with an aggressive phenotype and poor outcome in childhood neuroblastoma. Polyamines are highly regulated essential cations that are frequently elevated in cancer cells, and the rate-limiting enzyme in polyamine synthesis, ornithine decarboxylase 1 (ODC1), is a direct transcriptional target of MYCN. Treatment of neuroblastoma cells with the ODC1 inhibitor difluoromethylornithine (DFMO), although a promising therapeutic strategy, is only partially effective at impeding neuroblastoma cell growth due to activation of compensatory mechanisms resulting in increased polyamine uptake from the surrounding microenvironment. In this study, we identified solute carrier family 3 member 2 (SLC3A2) as the key transporter involved in polyamine uptake in neuroblastoma. Knockdown of SLC3A2 in neuroblastoma cells reduced the uptake of the radiolabeled polyamine spermidine, and DFMO treatment increased SLC3A2 protein. In addition, MYCN directly increased polyamine synthesis and promoted neuroblastoma cell proliferation by regulating SLC3A2 and other regulatory components of the polyamine pathway. Inhibiting polyamine uptake with the small-molecule drug AMXT 1501, in combination with DFMO, prevented or delayed tumor development in neuroblastoma-prone mice and extended survival in rodent models of established tumors. Our findings suggest that combining AMXT 1501 and DFMO with standard chemotherapy might be an effective strategy for treating neuroblastoma.

Drugging MYCN Oncogenic Signaling through the MYCN-PA2G4 Binding Interface.

MYCN is a major driver for the childhood cancer, neuroblastoma, however, there are no inhibitors of this target. Enhanced MYCN protein stability is a key component of MYCN oncogenesis and is maintained by multiple feedforward expression loops involving MYCN transactivation target genes. Here, we reveal the oncogenic role of a novel MYCN target and binding protein, proliferation-associated 2AG4 (PA2G4). Chromatin immunoprecipitation studies demonstrated that MYCN occupies the PA2G4 gene promoter, stimulating transcription. Direct binding of PA2G4 to MYCN protein blocked proteolysis of MYCN and enhanced colony formation in a MYCN-dependent manner. Using molecular modeling, surface plasmon resonance, and mutagenesis studies, we mapped the MYCN-PA2G4 interaction site to a 14 amino acid MYCN sequence and a surface crevice of PA2G4. Competitive chemical inhibition of the MYCN-PA2G4 protein-protein interface had potent inhibitory effects on neuroblastoma tumorigenesis in vivo. Treated tumors showed reduced levels of both MYCN and PA2G4. Our findings demonstrate a critical role for PA2G4 as a cofactor in MYCN-driven neuroblastoma and highlight competitive inhibition of the PA2G4-MYCN protein binding as a novel therapeutic strategy in the disease. SIGNIFICANCE: Competitive chemical inhibition of the PA2G4-MYCN protein interface provides a basis for drug design of small molecules targeting MYC and MYCN-binding partners in malignancies driven by MYC family oncoproteins.

Cell lines from MYCN transgenic murine tumours reflect the molecular and biological characteristics of human neuroblastoma.

Overexpression of the human MYCN oncogene driven by a tyrosine hydroxylase promoter causes tumours in transgenic mice that recapitulate the childhood cancer neuroblastoma. To establish an in vitro model to study this process, a series of isogenic cell lines were developed from these MYCN-driven murine tumours. Lines were established from tumours arising in homozygous and hemizygous MYCN transgenic mice. Hemizygous tumours gave rise to cell lines growing only in suspension. Homozygous tumours gave rise to similar suspension lines as well as morphologically distinct substrate-adherent lines characteristic of human S-type neuroblastoma cells. FISH analysis demonstrated selective MYCN transgene amplification in cell lines derived from hemizygous mice. Comparative genomic hybridisation (CGH) and fluorescence in situ hybridisation (FISH) analysis confirmed a range of neuroblastoma-associated genetic changes in the various lines, in particular, gain of regions syntenic with human 17q. These isogenic lines together with the transgenic mice thus represent valuable models for investigating the biological characteristics of aggressive neuroblastoma.

Extensive Proliferation of Human Cancer Cells with Ever-Shorter Telomeres.

Acquisition of replicative immortality is currently regarded as essential for malignant transformation. This is achieved by activating a telomere lengthening mechanism (TLM), either telomerase or alternative lengthening of telomeres, to counter normal telomere attrition. However, a substantial proportion of some cancer types, including glioblastomas, liposarcomas, retinoblastomas, and osteosarcomas, are reportedly TLM-negative. As serial samples of human tumors cannot usually be obtained to monitor telomere length changes, it has previously been impossible to determine whether tumors are truly TLM-deficient, there is a previously unrecognized TLM, or the assay results are false-negative. Here, we show that a subset of high-risk neuroblastomas (with ∼50% 5-year mortality) lacked significant TLM activity. Cancer cells derived from these highly aggressive tumors initially had long telomeres and proliferated for >200 population doublings with ever-shorter telomeres. This indicates that prevention of telomere shortening is not always required for oncogenesis, which has implications for inhibiting TLMs for cancer therapy.

Suppression of the ATP-binding cassette transporter ABCC4 impairs neuroblastoma tumour growth and sensitises to irinotecan in vivo.

The ATP-binding cassette transporter ABCC4 (multidrug resistance protein 4, MRP4) mRNA level is a strong predictor of poor clinical outcome in neuroblastoma which may relate to its export of endogenous signalling molecules and chemotherapeutic agents. We sought to determine whether ABCC4 contributes to development, growth and drug response in neuroblastoma in vivo. In neuroblastoma patients, high ABCC4 protein levels were associated with reduced overall survival. Inducible knockdown of ABCC4 strongly inhibited the growth of human neuroblastoma cells in vitro and impaired the growth of neuroblastoma xenografts. Loss of Abcc4 in the Th-MYCN transgenic neuroblastoma mouse model did not impact tumour formation; however, Abcc4-null neuroblastomas were strongly sensitised to the ABCC4 substrate drug irinotecan. Our findings demonstrate a role for ABCC4 in neuroblastoma cell proliferation and chemoresistance and provide rationale for a strategy where inhibition of ABCC4 should both attenuate the growth of neuroblastoma and sensitise tumours to ABCC4 chemotherapeutic substrates.

Glutathione biosynthesis is upregulated at the initiation of MYCN-driven neuroblastoma tumorigenesis.

The MYCN gene is amplified and overexpressed in a large proportion of high stage neuroblastoma patients and has been identified as a key driver of tumorigenesis. However, the mechanism by which MYCN promotes tumor initiation is poorly understood. Here we conducted metabolic profiling of pre-malignant sympathetic ganglia and tumors derived from the TH-MYCN mouse model of neuroblastoma, compared to non-malignant ganglia from wildtype littermates. We found that metabolites involved in the biosynthesis of glutathione, the most abundant cellular antioxidant, were the most significantly upregulated metabolic pathway at tumor initiation, and progressively increased to meet the demands of tumorigenesis. A corresponding increase in the expression of genes involved in ribosomal biogenesis suggested that MYCN-driven transactivation of the protein biosynthetic machinery generated the necessary substrates to drive glutathione biosynthesis. Pre-malignant sympathetic ganglia from TH-MYCN mice had higher antioxidant capacity and required glutathione upregulation for cell survival, when compared to wildtype ganglia. Moreover, in vivo administration of inhibitors of glutathione biosynthesis significantly delayed tumorigenesis when administered prophylactically and potentiated the anticancer activity of cytotoxic chemotherapy against established tumors. Together these results identify enhanced glutathione biosynthesis as a selective metabolic adaptation required for initiation of MYCN-driven neuroblastoma, and suggest that glutathione-targeted agents may be used as a potential preventative strategy, or as an adjuvant to existing chemotherapies in established disease.

Polyamine Antagonist Therapies Inhibit Neuroblastoma Initiation and Progression.

PURPOSE: Deregulated MYC drives oncogenesis in many tissues yet direct pharmacologic inhibition has proven difficult. MYC coordinately regulates polyamine homeostasis as these essential cations support MYC functions, and drugs that antagonize polyamine sufficiency have synthetic-lethal interactions with MYC Neuroblastoma is a lethal tumor in which the MYC homologue MYCN, and ODC1, the rate-limiting enzyme in polyamine synthesis, are frequently deregulated so we tested optimized polyamine depletion regimens for activity against neuroblastoma. EXPERIMENTAL DESIGN: We used complementary transgenic and xenograft-bearing neuroblastoma models to assess polyamine antagonists. We investigated difluoromethylornithine (DFMO; an inhibitor of Odc, the rate-limiting enzyme in polyamine synthesis), SAM486 (an inhibitor of Amd1, the second rate-limiting enzyme), and celecoxib (an inducer of Sat1 and polyamine catabolism) in both the preemptive setting and in the treatment of established tumors. In vitro assays were performed to identify mechanisms of activity. RESULTS: An optimized polyamine antagonist regimen using DFMO and SAM486 to inhibit both rate-limiting enzymes in polyamine synthesis potently blocked neuroblastoma initiation in transgenic mice, underscoring the requirement for polyamines in MYC-driven oncogenesis. Furthermore, the combination of DFMO with celecoxib was found to be highly active, alone, and combined with numerous chemotherapy regimens, in regressing established tumors in both models, including tumors harboring highest risk genetic lesions such as MYCN amplification, ALK mutation, and TP53 mutation with multidrug resistance. CONCLUSIONS: Given the broad preclinical activity demonstrated by polyamine antagonist regimens across diverse in vivo models, clinical investigation of such approaches in neuroblastoma and potentially other MYC-driven tumors is warranted. Clin Cancer Res; 22(17); 4391-404. ©2016 AACR.

Methyl-CpG-binding domain sequencing reveals a prognostic methylation signature in neuroblastoma.

Accurate assessment of neuroblastoma outcome prediction remains challenging. Therefore, this study aims at establishing novel prognostic tumor DNA methylation biomarkers. In total, 396 low- and high-risk primary tumors were analyzed, of which 87 were profiled using methyl-CpG-binding domain (MBD) sequencing for differential methylation analysis between prognostic patient groups. Subsequently, methylation-specific PCR (MSP) assays were developed for 78 top-ranking differentially methylated regions and tested on two independent cohorts of 132 and 177 samples, respectively. Further, a new statistical framework was used to identify a robust set of MSP assays of which the methylation score (i.e. the percentage of methylated assays) allows accurate outcome prediction. Survival analyses were performed on the individual target level, as well as on the combined multimarker signature. As a result of the differential DNA methylation assessment by MBD sequencing, 58 of the 78 MSP assays were designed in regions previously unexplored in neuroblastoma, and 36 are located in non-promoter or non-coding regions. In total, 5 individual MSP assays (located in CCDC177, NXPH1, lnc-MRPL3-2, lnc-TREX1-1 and one on a region from chromosome 8 with no further annotation) predict event-free survival and 4 additional assays (located in SPRED3, TNFAIP2, NPM2 and CYYR1) also predict overall survival. Furthermore, a robust 58-marker methylation signature predicting overall and event-free survival was established. In conclusion, this study encompasses the largest DNA methylation biomarker study in neuroblastoma so far. We identified and independently validated several novel prognostic biomarkers, as well as a prognostic 58-marker methylation signature.