Jayne Murray

ODC1 Is a Critical Determinant of MYCN Oncogenesis and a Therapeutic Target in Neuroblastoma

Neuroblastoma is a frequently lethal childhood tumor in which MYC gene deregulation, commonly as MYCN amplification, portends poor outcome. Identifying the requisite biopathways downstream of MYC may provide therapeutic opportunities. We used transcriptome analyses to show that MYCN-amplified neuroblastomas have coordinately deregulated myriad polyamine enzymes (including ODC1, SRM, SMS, AMD1, OAZ2, and SMOX) to enhance polyamine biosynthesis. High-risk tumors without MYCN amplification also overexpress ODC1, the rate-limiting enzyme in polyamine biosynthesis, when compared with lower-risk tumors, suggesting that this pathway may be pivotal. Indeed, elevated ODC1 (independent of MYCN amplification) was associated with reduced survival in a large independent neuroblastoma cohort. As polyamines are essential for cell survival and linked to cancer progression, we studied polyamine antagonism to test for metabolic dependence on this pathway in neuroblastoma. The Odc inhibitor alpha-difluoromethylornithine (DFMO) inhibited neuroblast proliferation in vitro and suppressed oncogenesis in vivo. DFMO treatment of neuroblastoma-prone genetically engineered mice (TH-MYCN) extended tumor latency and survival in homozygous mice and prevented oncogenesis in hemizygous mice. In the latter, transient Odc ablation permanently prevented tumor onset consistent with a time-limited window for embryonal tumor initiation. Importantly, we show that DFMO augments antitumor efficacy of conventional cytotoxics in vivo. This work implicates polyamine biosynthesis as an arbiter of MYCN oncogenesis and shows initial efficacy for polyamine depletion strategies in neuroblastoma, a strategy that may have utility for this and other MYC-driven embryonal tumors.

ABCC Multidrug Transporters in Childhood Neuroblastoma: Clinical and Biological Effects Independent of Cytotoxic Drug Efflux

Background Although the prognostic value of the ATP-binding cassette, subfamily C (ABCC) transporters in childhood neuroblastoma is usually attributed to their role in cytotoxic drug efflux, certain observations have suggested that these multidrug transporters might contribute to the malignant phenotype independent of cytotoxic drug efflux. Methods A v-myc myelocytomatosis viral related oncogene, neuroblastoma derived (MYCN)–driven transgenic mouse neuroblastoma model was crossed with an Abcc1-deficient mouse strain (658 hMYCN1/−, 205 hMYCN+/1 mice) or, alternatively, treated with the ABCC1 inhibitor, Reversan (n = 20). ABCC genes were suppressed using short interfering RNA or overexpressed by stable transfection in neuroblastoma cell lines BE(2)-C, SH-EP, and SH-SY5Y, which were then assessed for wound closure ability, clonogenic capacity, morphological differentiation, and cell growth. Real-time quantitative polymerase chain reaction was used to examine the clinical significance of ABCC family gene expression in a large prospectively accrued cohort of patients (n = 209) with primary neuroblastomas. Kaplan–Meier survival analysis and Cox regression were used to test for associations with event-free and overall survival. Except where noted, all statistical tests were two-sided. Results Inhibition of ABCC1 statistically significantly inhibited neuroblastoma development in hMYCN transgenic mice (mean age for palpable tumor: treated mice, 47.2 days; control mice, 41.9 days; hazard ratio [HR] = 9.3, 95% confidence interval [CI] = 2.65 to 32; P < .001). Suppression of ABCC1 in vitro inhibited wound closure (P < .001) and clonogenicity (P = .006); suppression of ABCC4 enhanced morphological differentiation (P < .001) and inhibited cell growth (P < .001). Analysis of 209 neuroblastoma patient tumors revealed that, in contrast with ABCC1 and ABCC4, low rather than high ABCC3 expression was associated with reduced event-free survival (HR of recurrence or death = 2.4, 95% CI = 1.4 to 4.2; P = .001), with 23 of 53 patients with low ABCC3 expression experiencing recurrence or death compared with 31 of 155 patients with high ABCC3. Moreover, overexpression of ABCC3 in vitro inhibited neuroblastoma cell migration (P < .001) and clonogenicity (P = .03). The combined expression of ABCC1, ABCC3, and ABCC4 was associated with patients having an adverse event, such that of the 12 patients with the “poor prognosis” expression pattern, 10 experienced recurrence or death (HR of recurrence or death = 12.3, 95% CI = 6 to 27; P < .001). Conclusion ABCC transporters can affect neuroblastoma biology independently of their role in chemotherapeutic drug efflux, enhancing their potential as targets for therapeutic intervention.

Small-Molecule Multidrug Resistance–Associated Protein 1 Inhibitor Reversan Increases the Therapeutic Index of Chemotherapy in Mouse Models of Neuroblastoma

The multidrug resistance-associated protein 1 (MRP1) has been closely linked to poor treatment response in several cancers, most notably neuroblastoma. Homozygous deletion of the MRP1 gene in primary murine neuroblastoma tumors resulted in increased sensitivity to MRP1 substrate drugs (vincristine, etoposide, and doxorubicin) compared with tumors containing both copies of wild-type MRP1, indicating that MRP1 plays a significant role in the drug resistance in this tumor type and defining this multidrug transporter as a target for pharmacologic suppression. A cell-based readout system was created to functionally determine intracellular accumulation of MRP1 substrates using a p53-responsive reporter as an indicator of drug-induced DNA damage. Screening of small-molecule libraries in this readout system revealed pyrazolopyrimidines as a prominent structural class of potent MRP1 inhibitors. Reversan, the lead compound of this class, increased the efficacy of both vincristine and etoposide in murine models of neuroblastoma (syngeneic and human xenografts). As opposed to the majority of inhibitors of multidrug transporters, Reversan was not toxic by itself nor did it increase the toxicity of chemotherapeutic drug exposure in mice. Therefore, Reversan represents a new class of nontoxic MRP1 inhibitor, which may be clinically useful for the treatment of neuroblastoma and other MRP1-overexpressing drug-refractory tumors by increasing their sensitivity to conventional chemotherapy.

Therapeutic targeting of the MYC signal by inhibition of histone chaperone FACT in neuroblastoma

Histone chaperone FACT acts in a positive feedback loop with MYCN and is a therapeutic target in neuroblastoma. Uncovering the FACTs in neuroblastoma Neuroblastoma is a common pediatric cancer of the nervous system. It is often difficult to treat, and tumors with amplifications of the MYC oncogene are particularly aggressive. Carter et al. have identified a histone chaperone called FACT as a mediator of MYC signaling in neuroblastoma and demonstrated its role in a feedback loop that allows tumor cells to maintain a high expression of both MYC and FACT. The authors then used curaxins, which are drugs that inhibit FACT, to break the vicious cycle. They demonstrated that curaxins work in synergy with standard genotoxic chemotherapy to kill cancer cells and treat neuroblastoma in mouse models. Amplification of the MYCN oncogene predicts treatment resistance in childhood neuroblastoma. We used a MYC target gene signature that predicts poor neuroblastoma prognosis to identify the histone chaperone FACT (facilitates chromatin transcription) as a crucial mediator of the MYC signal and a therapeutic target in the disease. FACT and MYCN expression created a forward feedback loop in neuroblastoma cells that was essential for maintaining mutual high expression. FACT inhibition by the small-molecule curaxin compound CBL0137 markedly reduced tumor initiation and progression in vivo. CBL0137 exhibited strong synergy with standard chemotherapy by blocking repair of DNA damage caused by genotoxic drugs, thus creating a synthetic lethal environment in MYCN-amplified neuroblastoma cells and suggesting a treatment strategy for MYCN-driven neuroblastoma.

Polymorphisms in genes encoding drug metabolizing enzymes and their influence on the outcome of children with neuroblastoma

Background Although several studies have shown that drug metabolizing enzyme gene polymorphisms may influence the impact of therapy in childhood leukemia, no comprehensive investigations have been carried out in children with neuroblastoma. The aim of this study was to identify polymorphisms in the genes encoding phase I and II drug metabolizing enzymes associated with the risk of relapse or death in a cohort of 209 children with neuroblastoma. Methods Real-time PCR allelic discrimination was used to characterize the presence of polymorphisms in DNA from children with neuroblastoma. Three broad gene categories were examined: cytochrome P450, glutathione-S-transferase and N-acetyltransferase. Cumulative event-free survival was computed by the Kaplan–Meier method. The influence of selected factors on event-free survival was tested using the Cox proportional hazards model. Results As previously reported, amplification of MYCN (hazards ratio=4.25, 95% confidence interval=2.76–6.56, P<0.001), unfavorable stage (hazard ratio=4.14, 95% confidence interval=2.3–7.47, P<0.001) or age more than 1 year at diagnosis (hazard ratio=1.86, 95% confidence interval=1.19–2.92, P=0.007) were all associated with an increased risk of relapse or death. Carriers of a NAT1*11 allele variant were significantly less likely to relapse or die compared with those with NAT1*10 or other NAT1 allele variants (P<0.001). In multivariate analysis, children who were GSTM1 null were more likely to relapse or die during follow-up after adjusting for MYCN amplification, stage and age at diagnosis (hazard ratio=1.6, 95% confidence interval=1.02–2.9, P=0.04). Conclusions These observations suggest that the NAT1*11 variant and the GSTM1 wild-type genotype contribute to a more favorable outcome in patients treated for neuroblastoma and are the first to demonstrate a relationship between NAT1 and GSTM1 genotypes in childhood neuroblastoma.

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.

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.

N-Myc Regulates Expression of the Detoxifying Enzyme Glutathione Transferase GSTP1, a Marker of Poor Outcome in Neuroblastoma

Amplification of the transcription factor MYCN is associated with poor outcome and a multidrug-resistant phenotype in neuroblastoma. N-Myc regulates the expression of several ATP-binding cassette (ABC) transporter genes, thus affecting global drug efflux. Because these transporters do not confer resistance to several important cytotoxic agents used to treat neuroblastoma, we explored the prognostic significance and transcriptional regulation of the phase II detoxifying enzyme, glutathione S-transferase P1 (GSTP1). Using quantitative real-time PCR, GSTP1 gene expression was assessed in a retrospective cohort of 51 patients and subsequently in a cohort of 207 prospectively accrued primary neuroblastomas. These data along with GSTP1 expression data from an independent microarray study of 251 neuroblastoma samples were correlated with established prognostic indicators and disease outcome. High levels of GSTP1 were associated with decreased event-free and overall survival in all three cohorts. Multivariable analyses, including age at diagnosis, tumor stage, and MYCN amplification status, were conducted on the two larger cohorts, independently showing the prognostic significance of GSTP1 expression levels in this setting. Mechanistic investigations revealed that GSTP1 is a direct transcriptional target of N-Myc in neuroblastoma cells. Together, our findings reveal that N-Myc regulates GSTP1 along with ABC transporters that act to control drug metabolism and efflux. Furthermore, they imply that strategies to jointly alter these key multidrug resistance mechanisms may have therapeutic implications to manage neuroblastomas and other malignancies driven by amplified Myc family genes.

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

The RNA-binding protein dyskerin, encoded by the DKC1 gene, functions as a core component of the telomerase holoenzyme as well as ribonuclear protein complexes involved in RNA processing and ribosome biogenesis. The diverse roles of dyskerin across many facets of RNA biology implicate its potential contribution to malignancy. In this study, we examined the expression and function of dyskerin in neuroblastoma. We show that DKC1 mRNA levels were elevated relative to normal cells across a panel of 15 neuroblastoma cell lines, where both N-Myc and c-Myc directly targeted the DKC1 promoter. Upregulation of MYCN was shown to dramatically increase DKC1 expression. In two independent neuroblastoma patient cohorts, high DKC1 expression correlated strongly with poor event-free and overall survival (P < 0.0001), independently of established prognostic factors. RNAi-mediated depletion of dyskerin inhibited neuroblastoma cell proliferation, including cells immortalized via the telomerase-independent ALT mechanism. Furthermore, dyskerin attenuation impaired anchorage-independent proliferation and tumor growth. Overexpression of the telomerase RNA component, hTR, demonstrated that this proliferative impairment was not a consequence of telomerase suppression. Instead, ribosomal stress, evidenced by depletion of small nucleolar RNAs and nuclear dispersal of ribosomal proteins, was the likely cause of the proliferative impairment in dyskerin-depleted cells. Accordingly, dyskerin suppression caused p53-dependent G1 cell-cycle arrest in p53 wild-type cells, and a p53-independent pathway impaired proliferation in cells with p53 dysfunction. Together, our findings highlight dyskerin as a new therapeutic target in neuroblastoma with crucial telomerase-independent functions and broader implications for the spectrum of malignancies driven by MYC family oncogenes. Cancer Res; 76(12); 3604-17. ©2016 AACR.

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.