Advanced Stage Neuroblastoma Research Articles

Extracellular vesicles in neuroblastoma: role in progression, resistance to therapy and diagnostics.

Neuroblastoma (NB) is the most common extracranial solid pediatric cancer, and is one of the leading causes of cancer-related deaths in children. Despite the current multi-modal treatment regimens, majority of patients with advanced-stage NBs develop therapeutic resistance and relapse, leading to poor disease outcomes. There is a large body of knowledge on pathophysiological role of small extracellular vesicles (EVs) in progression and metastasis of multiple cancer types, however, the importance of EVs in NB was until recently not well understood. Studies emerging in the last few years have demonstrated the involvement of EVs in various aspects of NB pathogenesis. In this review we summarize these recent findings and advances on the role EVs play in NB progression, such as tumor growth, metastasis and therapeutic resistance, that could be helpful for future investigations in NB EV research. We also discuss different strategies for therapeutic targeting of NB-EVs as well as utilization of NB-EVs as potential biomarkers.

Gene utility recapitulates chromosomal aberrancies in advanced stage neuroblastoma

Neuroblastoma (NB) is the most common extracranial solid tumor in children. Although only a few recurrent somatic mutations have been identified, chromosomal abnormalities, including the loss of heterozygosity (LOH) at the chromosome 1p and gains of chromosome 17q, are often seen in the high-risk cases. The biological basis and evolutionary forces that drive such genetic abnormalities remain enigmatic. Here, we conceptualize the Gene Utility Model (GUM) that seeks to identify genes driving biological signaling via their collective gene utilities and apply it to understand the impact of those differentially utilized genes on constraining the evolution of NB karyotypes. By employing a computational process-guided flow algorithm to model gene utility in protein–protein networks that built based on transcriptomic data, we conducted several pairwise comparative analyses to uncover genes with differential utilities in stage 4 NBs with distinct classification. We then constructed a utility karyotype by mapping these differentially utilized genes to their respective chromosomal loci. Intriguingly, hotspots of the utility karyotype, to certain extent, can consistently recapitulate the major chromosomal abnormalities of NBs and also provides clues to yet identified predisposition sites. Hence, our study not only provides a new look, from a gene utility perspective, into the known chromosomal abnormalities detected by integrative genomic sequencing efforts, but also offers new insights into the etiology of NB and provides a framework to facilitate the identification of novel therapeutic targets for this devastating childhood cancer.

A Phase 3, Randomized, Adaptive Study of Defibrotide (DF) Vs Best Supportive Care (BSC) for the Prevention of Hepatic Veno-Occlusive Disease/Sinusoidal Obstruction Syndrome (VOD/SOS) in Patients (pts) Undergoing Hematopoietic Cell Transplantation (HCT): Preliminary Results

A Phase 3, Randomized, Adaptive Study of Defibrotide (DF) Vs Best Supportive Care (BSC) for the Prevention of Hepatic Veno-Occlusive Disease/Sinusoidal Obstruction Syndrome (VOD/SOS) in Patients (pts) Undergoing Hematopoietic Cell Transplantation (HCT): Preliminary Results

Late mortality in survivors of childhood cancer in Hungary

The Hungarian Pediatric Oncology Network provides centralized treatment and population-based registration for cases of childhood cancer since 1973. We collected and analized data on late mortality, secondary malignancies and cardiac diseases in survivors (> 5 years) of childhood cancer to evaluate long-term risks. We extracted all solid tumour cases (3,650 followed up for 5–39.3 years, diagnosis: 1973–2008) from the database of the Hungarian Childhood Cancer Registry and checked against the Population Registry. Among the 301 patients who died after 5 years (8.2%) the most common causes of death were progression of primary cancer (52.5%), secondary malignancies (16%) and cardiovascular diseases (8%). Late mortality rates (SMR, total: 35,006 pyrs) showed highly elevated risk of death (SMR: 10.7 95% CI 9–12.4) for the second 5 years of follow up and moderately elevated risk for 10-year survivors (SMR: 3.5 95% CI 3–4.1). Marked differences were detected in the pattern of causes of death between diagnostic groups of primary cancer; with highest risks beyond 10 years for CNS tumours, Hodgkin disease, osteosarcoma and advanced stage neuroblastoma. The longstanding mortality risk for 5-year survivors underlines the need for tailored long-term follow-up and monitoring of late consequences according to the context of different primary diseases of childhood cancer.

Diagnostic accuracy of circulating-free DNA for the determination of MYCN amplification status in advanced-stage neuroblastoma: a systematic review and meta-analysis

BackgroundMYCN amplification (MNA) is the strongest indicator of poor prognosis in neuroblastoma (NB). This meta-analysis aims to determine the diagnostic accuracy of MNA analysis in circulating-free DNA (cfDNA) from advanced-stage NB patients.MethodsA systematic review of electronic databases was conducted to identify studies exploring the detection of MNA in plasma/serum cfDNA from NB patients at diagnosis using PCR methodology. Pooled estimates for sensitivity, specificity and diagnostic odds ratio (DOR) were calculated by conducting a bivariate/HSROC random-effects meta-analysis.ResultsSeven studies, with a total of 529 advanced-stage patients, were eligible. The pooled sensitivity of cfDNA-based MNA analysis was 0.908 (95% CI, 0.818–0.956), the pooled specificity was 0.976 (0.940–0.991) and the DOR was 410.0 (−103.6 to 923.7). Sub-grouped by INSS stage, the sensitivity for stage 3 and 4 patients was 0.832 (0.677–0.921) and 0.930 (0.834–0.972), respectively. The specificity was 0.999 (0.109–1.000) and 0.974 (0.937–0.990), respectively, and the DOR was 7855.2 (−66267.0 to 81977.4) and 508.7 (−85.8 to 1103.2), respectively.ConclusionsMNA analysis in cfDNA using PCR methodology represents a non-invasive approach to rapidly and accurately determine MNA status in patients with advanced-stage NB. Standardised methodology must be developed before this diagnostic test can enter the clinic.

Binding of N8-Acetylspermidine Analogues to Histone Deacetylase 10 Reveals Molecular Strategies for Blocking Polyamine Deacetylation.

Eukaryotic histone deacetylase 10 (HDAC10) is a Zn2+-dependent hydrolase that exhibits catalytic specificity for the hydrolysis of the polyamine N8-acetylspermidine. The recently determined crystal structure of HDAC10 from Danio rerio (zebrafish) reveals a narrow active site cleft and a negatively charged "gatekeeper" (E274) that favors the binding of the slender cationic substrate. Because HDAC10 expression is upregulated in advanced-stage neuroblastoma and induces autophagy, the selective inhibition of HDAC10 suppresses the autophagic response and renders cancer cells more susceptible to cytotoxic chemotherapeutic drugs. Here, we describe X-ray crystal structures of zebrafish HDAC10 complexed with eight different analogues of N8-acetylspermidine. These analogues contain different Zn2+-binding groups, such as hydroxamate, thiolate, and the tetrahedral gem-diolate resulting from the addition of a Zn2+-bound water molecule to a ketone carbonyl group. Notably, the chemistry that accompanies the binding of ketonic substrate analogues is identical to the chemistry involved in the first step of catalysis, i.e., nucleophilic attack of a Zn2+-bound water molecule at the scissile carbonyl group of N8-acetylspermidine. The most potent inhibitor studied contains a thiolate Zn2+-binding group. These structures reveal interesting geometric changes in the metal coordination polyhedron that accommodate inhibitor binding. Additional interactions in the active site highlight features contributing to substrate specificity. These interactions are likely to contribute to inhibitor binding selectivity and will inform the future design of compounds selective for HDAC10 inhibition.

Regulation of neuroblastoma migration, invasion, and in vivo metastasis by genetic and pharmacological manipulation of MDA-9/Syntenin.

Despite multi-modality treatments, prognosis for advanced stage neuroblastoma (NB) remains challenging with residual long-term disabilities in survivors. Advanced stage NB is metastatic, which is a principal cause of cancer-related deaths. We presently document a primary role of MDA-9 in NB progression and define the molecular mechanisms by which MDA-9 promotes transformed phenotypes. NB cell lines and clinical samples display elevated MDA-9 expression and bioinformatic analysis supports an association between elevated MDA-9 and bone metastasis and poor prognosis. Genetic (shmda-9, mda-9 siRNA) or pharmacological (small molecule inhibitor of protein-protein interactions; PDZ1i) blockade of MDA-9 decreases NB migration, invasion and metastasis. Blocking mda-9 expression or disrupting MDA-9 partner protein interactions downregulates integrin α6 and β4, diminishing Src activity and suppressing Rho-Rac-Cdc42 activity. These signaling changes inhibit cofilin and matrix metalloproteinases reducing in vitro and in vivo NB cell migration. Overexpression of integrin α6 and β4 rescues the invasion phenotype and increases Src activity, supporting integrins as essential regulators of MDA-9-mediated NB migration and invasion. We identify MDA-9 as a key contributor to NB pathogenesis and show that genetic or pharmacological inhibition suppresses NB pathogenesis by an integrin-mediated Src-disruption pathway.

Abstract 3669: Defining an ELAVL1-Musashi 2 signaling cascade in neuroblastoma tumorigenesis

Abstract Background: Patients with high-risk neuroblastoma endure an extremely intense, multidrug treatment regimen and yet, approximately half of them die, thus mandating a better approach to this cancer. Our laboratory is investigating the overarching hypothesis that RNA binding proteins (RBPs), which play key roles in balancing self-renewal, differentiation, and cell proliferation, are deregulated in neuroblastoma, and drive oncogenic signaling networks. Therefore, RBPs may constitute novel targets for therapeutic development. In this work, we focus on Musashi 2 (MSI2), an RBP that has been shown to play a critical role in the maintenance of stem cell populations and in the formation of aggressive tumors, notably in acute myeloid leukemia and in colon cancer. The role of MSI2 in neuroblastoma, however, has not been assessed and is the focus of this investigation. Methods: To test this hypothesis, we used in silico analysis of neuroblastoma gene expression datasets annotated with clinical parameters. Additionally, we used shRNAs to manipulate transcripts of interest in neuroblastoma cells and measured effects on cell proliferation, cell cycle status, and apoptosis. Results: Gene expression profiling revealed that MSI2 is robustly expressed in neuroblastoma cell lines, tumors, and patient derived xenografts, within both the MYCN and non-MYCN amplified context. Immunoblotting in a representative panel of lines confirmed the expression of MSI2 at the level of protein. High MSI2 expression was associated with more advanced stages of neuroblastoma across multiple independent datasets and was correlated with worse survival. Depletion of MSI2 using four independent shRNAs led to 2 to 3-fold decreased proliferation across four cell lines that was due in part to increased apoptosis. Another RBP, ELAVL1, is also expressed within neural lineages, and has been shown to regulate MSI1 expression. We hypothesized that ELAVL1 might similarly promote MSI2 expression and we demonstrated that, ELAVL1 depletion led to decreased MSI2 expression. Conclusions: MSI2 is robustly expressed in multiple models of neuroblastoma and appears to drive increased proliferation and survival. Currently, we are investigating the functional interplay between ELAVL1 and MSI2 providing a foundation for designing strategies to target the ELAVL1-MSI2 interaction for therapeutic effect. Citation Format: Selma M. Cuya, Adeiye Pilgrim, Komal Rathi, DongDong Chen, Robert Schnepp. Defining an ELAVL1-Musashi 2 signaling cascade in neuroblastoma tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3669.

Nedd4-Binding Protein 1 and TNFAIP3-Interacting Protein 1 Control MHC-1 Display in Neuroblastoma.

: Neuroblastoma is the second most common tumor in children. The cause of neuroblastoma is thought to lie in aberrant development of embryonic neural crest cells and is accompanied by low MHC-1 expression and suppression of the NF-κB transcription factor, thereby gearing cells toward escape from immunosurveillance. Here, we assess regulation of the MHC-1 gene in neuroblastoma to enhance its immunogenic potential for therapeutic T-cell targeting. A genome-wide CRISPR screen identified N4BP1 and TNIP1 as inhibitory factors of NF-κB-mediated MHC-1 expression in neuroblastoma. Patients with advanced stage neuroblastoma who expressed high levels of TNIP1 and N4BP1 exhibited worse overall survival. Depletion of N4BP1 or TNIP1 increased NF-κB and MHC-1 expression and stimulated recognition by antigen-specific CD8+ T cells. We confirmed that TNIP1 inhibited canonical NF-κB member RelA by preventing activation of the RelA/p50 NF-κB dimer. Furthermore, N4BP1 inhibited both canonical and noncanonical NF-κB through binding of deubiquitinating enzyme CEZANNE, resulting in stabilization of TRAF3 and degradation of NF-κB-inducing kinase NIK. These data suggest that N4BP1/CEZANNE or TNIP1 may be candidate targets for immunotherapy in neuroblastoma tumors and should lift NF-κB suppression, thereby triggering increased peptide/MHC1-mediated tumor reactivity to enhance therapeutic T-cell targeting. SIGNIFICANCE: Aberrant regulation of NF-κB and MHC-1 in neuroblastoma tumors provides new targets for immunotherapeutic approaches against neuroblastoma.

9-ING-41, a small-molecule glycogen synthase kinase-3 inhibitor, is active in neuroblastoma.

Advanced stage neuroblastoma is a very aggressive pediatric cancer with limited treatment options and a high mortality rate. Glycogen synthase kinase-3β (GSK-3β) is a potential therapeutic target in neuroblastoma. Using immunohistochemical staining, we observed positive GSK-3β expression in 67% of human neuroblastomas (34 of 51 cases). Chemically distinct GSK-3 inhibitors (AR-A014418, TDZD-8, and 9-ING-41) suppressed the growth of neuroblastoma cells, whereas 9-ING-41, a clinically relevant small-molecule GSK-3β inhibitor with broad-spectrum preclinical antitumor activity, being the most potent. Inhibition of GSK-3 resulted in a decreased expression of the antiapoptotic molecule XIAP and an increase in neuroblastoma cell apoptosis. Mouse xenograft studies showed that the combination of clinically relevant doses of CPT-11 and 9-ING-41 led to greater antitumor effect than was observed with either agent alone. These data support the inclusion of patients with advanced neuroblastoma in clinical studies of 9-ING-41, especially in combination with CPT-11.

A kinome-wide RNAi screen identifies ALK as a target to sensitize neuroblastoma cells for HDAC8-inhibitor treatment

The prognosis of advanced stage neuroblastoma patients remains poor and, despite intensive therapy, the 5-year survival rate remains less than 50%. We previously identified histone deacetylase (HDAC) 8 as an indicator of poor clinical outcome and a selective drug target for differentiation therapy in vitro and in vivo. Here, we performed kinome-wide RNAi screening to identify genes that are synthetically lethal with HDAC8 inhibitors. These experiments identified the neuroblastoma predisposition gene ALK as a candidate gene. Accordingly, the combination of the ALK/MET inhibitor crizotinib and selective HDAC8 inhibitors (3–6 µM PCI-34051 or 10 µM 20a) efficiently killed neuroblastoma cell lines carrying wildtype ALK (SK-N-BE(2)-C, IMR5/75), amplified ALK (NB-1), and those carrying the activating ALK F1174L mutation (Kelly), and, in cells carrying the activating R1275Q mutation (LAN-5), combination treatment decreased viable cell count. The effective dose of crizotinib in neuroblastoma cell lines ranged from 0.05 µM (ALK-amplified) to 0.8 µM (wildtype ALK). The combinatorial inhibition of ALK and HDAC8 also decreased tumor growth in an in vivo zebrafish xenograft model. Bioinformatic analyses revealed that the mRNA expression level of HDAC8 was significantly correlated with that of ALK in two independent patient cohorts, the Academic Medical Center cohort (n = 88) and the German Neuroblastoma Trial cohort (n = 649), and co-expression of both target genes identified patients with very poor outcome. Mechanistically, HDAC8 and ALK converge at the level of receptor tyrosine kinase (RTK) signaling and their downstream survival pathways, such as ERK signaling. Combination treatment of HDAC8 inhibitor with crizotinib efficiently blocked the activation of growth receptor survival signaling and shifted the cell cycle arrest and differentiation phenotype toward effective cell death of neuroblastoma cell lines, including sensitization of resistant models, but not of normal cells. These findings reveal combined targeting of ALK and HDAC8 as a novel strategy for the treatment of neuroblastoma.

Combined Stereotactic Body Radiation Therapy and Immune Checkpoint Inhibition Slows Tumor Growth in a Novel Syngeneic Model of Neuroblastoma

The majority of pediatric patients diagnosed with advanced-stage neuroblastoma die from their disease despite intensive multimodality therapy. Combination stereotactic body radiation therapy (SBRT) and immune checkpoint inhibition (ICI) represents a promising therapeutic avenue. We sought to develop a highly reproducible, syngeneic mouse model of advanced-stage neuroblastoma, in order to investigate the effects of combination SBRT/ICI on local control and the tumor immune microenvironment. We prepared an intrarenal tumorgraft model using the 9464D cell line, derived from the TH-MYCN transgenic neuroblastoma mouse. Following tumor implantation, mice were randomized to SBRT versus sham RT, as well as concurrently dosed anti-PD-1 antibody (αPD1) versus isotype control. SBRT fractions were delivered using the Small Animal Radiation Research Platform in 360 degree axial arcs to minimize normal tissue toxicity. Given increased radioresistance of 9464D relative to human neuroblastoma cell lines, three 8-Gy fractions were delivered. Tumor volume was monitored via CT and 3D ultrasound. Quantitative multiplex immunohistochemistry (qmIHC) staining was performed for immune markers including CD3 (T lymphocytes), F4/80 (macrophages) and PD-L1. All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Columbia University Medical Center. 100% of 47 mice developed tumors, with 0% surgical mortality. Tumor grafts grew more uniformly than tumors derived from intrarenal injection of 9464D cell suspension (P < 0.05). Tumors were clearly delineated within the kidney following injection of iohexol contrast agent, allowing precise radiation targeting. RT induced intratumoral PD-L1 expression. Combined SBRT/αPD1 was more effective than either SBRT or αPD1 alone in reducing the rate of tumor growth (P < 0.001) as well as tumor weight at 35 days post-implant (P < 0.05). Combination therapy, however, was associated with toxicity, with 40% of mice dying within one week following SBRT/αPD1 administration. Tumors treated with combined SBRT/αPD1 demonstrated dense infiltration by macrophages and T lymphocytes. Our intrarenal syngeneic tumor graft model of advanced-stage neuroblastoma produces uniform tumor growth and is amenable to image-guided radiation therapy. SBRT/ICI promotes tumor infiltration by immune cells and slows tumor growth, and further mechanistic studies are warranted.

PlGF and VEGF-A Regulate Growth of High-Risk MYCN-Single Copy Neuroblastoma Xenografts via Different Mechanisms.

Neuroblastoma (NB) is the most common extracranial solid tumor of childhood and is a rapidly growing, highly-vascularized cancer. NBs frequently express angiogenic factors and high tumor angiogenesis has been associated with poor outcomes. Placental growth factor (PlGF) is an angiogenic protein belonging to the vascular endothelial growth factor (VEGF) family and is up-regulated mainly in pathologic conditions. Recently, PlGF was identified as a member of a gene expression signature characterizing highly malignant NB stem cells drawing attention as a potential therapeutic target in NB. In the present study, we sought to investigate the expression of PlGF in NB patients and the effect of PlGF inhibition on high-risk MYCN-non-amplified SK-N-AS NB xenografts. Human SK-N-AS cells, which are poorly differentiated and express PlGF and VEGF-A, were implanted subcutaneously in athymic nude mice. Treatment was done by intratumoral injection of replication-incompetent adenoviruses (Ad) expressing PlGF- or VEGF-specific short hairpin (sh)RNA, or soluble (s)VEGF receptor 2 (VEGFR2). The effect on tumor growth and angiogenesis was analyzed. High PlGF expression levels were observed in human advanced-stage NBs. Down-regulating PlGF significantly reduced NB growth in established NB xenografts by reducing cancer cell proliferation but did not suppress angiogenesis. In contrast, blocking VEGF by administration of Ad(sh)VEGF and Ad(s)VEGFR2 reduced tumor growth associated with decreased tumor vasculature. These findings suggest that PlGF and VEGF-A modulate MYCN-non-amplified NB tumors by different mechanisms and support a role for PlGF in NB biology.

BRG1/SMARCA4 is essential for neuroblastoma cell viability through modulation of cell death and survival pathways.

Neuroblastoma (NB) is a neoplasm of the sympathetic nervous system, and is the most common solid tumor of infancy. NBs are very heterogeneous, with a clinical course ranging from spontaneous regression to resistance to all current forms of treatment. High-risk patients need intense chemotherapy, and only 30-40% will be cured. Relapsed or metastatic tumors acquire multi-drug resistance, raising the need for alternative treatments. Owing to the diverse mechanisms that are responsible of NB chemoresistance, we aimed to target epigenetic factors that control multiple pathways to bypass therapy resistance. We found that the SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a, member 4 (SMARCA4/BRG1) was consistently upregulated in advanced stages of NB, with high BRG1 levels being indicative of poor outcome. Loss-of-function experiments in vitro and in vivo showed that BRG1 is essential for the proliferation of NB cells. Furthermore, whole-genome transcriptome analysis revealed that BRG1 controls the expression of key elements of oncogenic pathways such as PI3K/AKT and BCL2, which offers a promising new combination therapy for high-risk NB.

Segmental Chromosomal Aberrations in Localized Neuroblastoma Can be Detected in Formalin-Fixed Paraffin-Embedded Tissue Samples and Are Associated With Recurrence.

Array comparative genomic hybridization (CGH) analyses of frozen tumors have shown strong associations between the pattern of chromosomal aberrations and outcome in patients with advanced-stage neuroblastoma. New platforms for analyzing chromosomal aberrations using formalin-fixed paraffin-embedded (FFPE) tissue have recently been developed. We sought to determine whether chromosomal microarray analysis (CMA) using FFPE tumors is feasible and if segmental chromosomal aberrations were prognostic of recurrence in localized neuroblastoma. Patients with MYCN nonamplified International Neuroblastoma Staging System stage 1 and 2 disease who recurred were identified. CMA was performed with diagnostic FFPE samples using OncoScan™ FFPE Express 2.0. The prognostic significance of chromosomal pattern was validated in 105 patients with available CGH results. In 26 evaluable patients, 11 recurred locally, nine had metastatic relapse, and six remained progression free >3 years from diagnosis. No chromosomal aberrations were identified in four tumors. Numerical chromosomal aberrations (NCAs) without segmental chromosomal aberration (SCA) were identified in 11 patients: six progressed locally, two had metastatic progression and 3 remained progression-free. Eleven patients had SCAs: four progressed locally, six developed metastatic progression and one remained progression-free. Five or more SCAs were only detected in tumors from patients who developed metastases (P = 0.0004). In the validation cohort, SCAs were associated with inferior event-free survival (EFS) compared to NCA (5-year EFS 68% ± 8.3% vs. 91% ± 3.6%, respectively; P = 0.0083). It is feasible to evaluate chromosomal aberrations using FFPE neuroblastoma tissue. SCA is associated with inferior EFS in localized neuroblastoma patients, and multiple SCAs may be predictive of metastatic relapse.

WT1 expression is inversely correlated with MYCN amplification or expression and associated with poor survival in non-MYCN-amplified neuroblastoma

Neuroblastoma (NB) is the most common extra cranial solid tumor in childhood and the most frequently diagnosed neoplasm during infancy. A striking feature of this tumor is its clinical heterogeneity. Several tumor progression markers have been delineated so far, among which MYCN amplification, which occurs in about 25% of total NB cases, with the percentage increasing to 30% in advanced stage NB. Although MYCN amplification is strongly correlated with NB of poor outcome, the MYCN status cannot alone predict all cases of poor survival in NB. Indeed NB without MYCN amplification (about 70–80% of NB) are not always favorable. WT1 was initially identified as a tumor suppressor gene involved in the development of a pediatric renal tumor (Wilms' tumor). Here, we describe an inverse correlation between WT1 expression and MYCN amplification and expression. However and most notably, our results show that WT1 gene expression is associated with a poor outcome for patients showing non-MYCN-amplified tumors. Thus WT1 expression is clinically significant in NB and may be a prognostic marker for better risk stratification and for an optimized therapeutic management of NB.

Abstract 158: The p16/ARF intron 2 imbedded long non-coding RNA CAI2 is highly expressed in tumor cells and modulates chromatin structure

Abstract Over the last several years it has become apparent that the majority of the genome is transcribed with the number of non-coding RNA (ncRNA) genes significantly higher than the number of protein-encoding genes. The 9p21 locus harbors a number of tumor suppressor genes including p15, p10, ARF, chARF, p16, p16γ, p12 and p16-ACT, and the long ncRNAs ANRIL and p15AS. The abundance of regulatory genes in such a short span perhaps accounts for why this region is frequently inactivated in many cancers. We report on the novel lncRNA CAI2, a non-conserved gene imbedded in p16/ARF intron 2 with homology that is limited to the upper primates. CAI2 is a single exon, RNA pol II regulated gene with a poly A signal localized in p16/ARF exon 3, yet independent of the p16/ARF poly A signal. The expression levels of CAI2, p16 and ARF genes are highly correlated, being low in normal tissue but high in most tumor cell lines with an intact 9p21 locus. Notably, high levels of CAI2 expression are associated with advanced-stage of neuroblastoma, one of the few tumor types where 9p21 alterations are infrequent and where p16 is paradoxically overexpressed. CAI2 expression was associated with a poor outcome in neuroblastoma, suggesting value as a biomarker of tumor cell stage and patient outcome. Despite the strong correlation between CAI2 and p16 expression, CAI2 is independently regulated and is expressed in cell lines deleted or epigenetically silenced for p16, ARF and even p15. In response to serum deprivation, CAI2 levels rise rapidly in normal mononuclear cells, but slowly in tumor cell lines, suggesting that CAI2 is also a stress-response gene. CAI2 levels also rise in response to chemotherapeutic insult such as etoposide treatment. The NMB7 neuroblastoma cell line is one of very few neuroblastoma cell lines to express CAI2 at low levels. During passage, this cell line undergoes morphological changes, proliferates faster and exhibits an increase of more than 100-fold in CAI2 expression, though a concomitant decrease in Tyrosine Hydroxylase expression suggests the changes are not due to differentiation. Consistent with this, Retinoic Acid treatment, part of standard neuroblastoma therapeutic regimen known to induce neuroblastoma cell differentiation, slightly decreased CAI2 expression. As many lncRNAs regulate chromatin states, we considered epigenetic roles for CAI2 in gene regulation. RNA Immunoprecipitation revealed that CAI2 interacts with H3K27me3, a recruiter of Polycomb Repressive Complex 1 (PRC1) that modulates chromatin structure and gene expression. As many advanced stage neuroblastomas and neuroblastoma cell lines highly express p16, we speculate that CAI2 binding to H3K27me3 may modulate chromatin structure and regulate expression at the 9p21 locus. Citation Format: Olga Cohen, Alice Yu, Mitchell B. Diccianni. The p16/ARF intron 2 imbedded long non-coding RNA CAI2 is highly expressed in tumor cells and modulates chromatin structure. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 158. doi:10.1158/1538-7445.AM2015-158

Abstract 191: The role of microRNAs in the epigenetic silencing of CHD5, a tumor suppressor in neuroblastoma

Abstract Background: Neuroblastoma (NB) is a tumor of the sympathetic nervous system that is the most common extracranial solid tumor of childhood. NBs show remarkable clinical heterogeneity, and we, along with others, have identified different patterns of genomic change that underlie these diverse clinical behaviors. We first identified 1p deletion as a characteristic change in advanced stage NBs. CHD5, a tumor suppressor gene, was first identified because of its location on 1p36. However, mutation of the remaining allele of CHD5 is rare in these tumors. Therefore, it is likely that epigenetic mechanisms play important roles in CHD5 downregulation. MicroRNAs (miRNAs) are small RNAs that bind to the 3′ UTR to mediate downregulation of target gene expression by translational repression or cleavage of the target gene message. Dysregulation of CHD5 via miR-211 has been reported in colon cancer. In order to understand the role of miRNA regulation of CHD5 in NBs, we wanted to assess the function of all miRNAs that are predicted to target CHD5. Method/approach: We used TargetScan, miRanda, MirTarget2 and other prediction algorithms to identify miRNAs that were predicted to bind to the CHD5-3′UTR. We identified 18 miRNAs that were predicted by 2 or more programs: miR-204-5p, -211, -216b, -17, -19ab, -20ab, -93, -106ab, -130ab, -301ab, -454, -519d, -3666. We used a renilla-luciferase reporter plasmid that contains 103 bp of the 3′UTR of CHD5 targeted by all miRNAs except miR-204, -211, - 219b, and -3666. We also created control plasmids with no CHD5 3′UTR insert and a mutated CHD5 3′UTR with mutated miRNA binding sites. Allstar siRNA served as an additional negative control. We performed transient transfections in two NB cell lines, NLF (a MYCN amplified cell line) and SY5Y (a MYCN nonamplified cell line), with the reporter plasmid and miRNA mimic. We measured luciferase (internal control) and renilla (reporter) activity. Results: Our results from the luciferase reporter assay indicate that four miRNAs are strong candidates for CHD5 downregulation in NB: miR-17, -93, -20b, and miR-106b. Each exhibited a significant decrease of fluorescence activity in cells transfected with CHD5 3′UTR in comparison to cells transfected with either of the control plasmids. Interestingly, MYCN upregulates three of the four miRNA candidates: miR-17, -93, &amp; -20b. We further confirmed consistent downregulation of the CHD5 by all four miRNAs. Conclusion: CHD5 expression is downregulated by miR-17, -93, -2b, and 106b. MYCN amplification and overexpression leads to upregulation of miR-17, -93, and -20b, and this may lead to down regulation of CHD5. Citation Format: Koumudi Naraparaju, Venkatadri Kolla, Tiangang Zhuang, Mayumi Higashi, Gerd A. Blobel, Garrett M. Brodeur. The role of microRNAs in the epigenetic silencing of CHD5, a tumor suppressor in neuroblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 191. doi:10.1158/1538-7445.AM2015-191

The P2X7 receptor is a key modulator of the PI3K/GSK3β/VEGF signaling network: evidence in experimental neuroblastoma.

Neuroblastoma (NB) is an aggressive pediatric tumor, responsible for 15% of cancer-related deaths in childhood, lacking an effective treatment in its advanced stages. The P2X7 receptor for extracellular ATP was associated to NB cell proliferation and recently emerged as a promoter of tumor engraftment, growth and vascularization. In an effort to identify new therapeutic options for neuroblastoma, we studied the role of P2X7 receptor in NB biology. We first analyzed the effect of P2X7 activation or down-modulation of the main biochemical ways involved in NB progression: the PI3K/Akt/GSK3β/MYCN and the HIF1α/VEGF pathways. In ACN human NB cells, P2X7 stimulation enhanced PI3K/Akt, while decreasing GSK3β activity. In the same model, P2X7 silencing or antagonist administration reduced the activity of PI3K/Akt and increased that of GSK3β, leading to a decrease in cellular glycogen stores. Similarly, P2X7 downmodulation caused a reduction in HIF1α levels and vascular endothelial growth factor (VEGF) secretion. Systemic administration of two different P2X7 antagonists (AZ10606120 or A740003) in nude/nude mice reduced ACN-derived tumor growth. An even stronger effect of P2X7 blockade was obtained in a syngeneic immune-competent neuroblastoma model: Neuro2A cells injected in AlbinoJ mice. Together with tumor regression, treatment with P2X7 antagonists caused downmodulation of the Akt/HIF1α axis, leading to reduced VEGF content and decreased vessel formation. Interestingly, in both experimental models, P2X7 antagonists strongly reduced the expression of the probably best-accepted oncogene in NB: MYCN. Finally, we associated P2X7 overexpression with poor prognosis in advanced-stage NB patients. Taken together, our data suggest that P2X7 receptor is an upstream regulator of the main signaling pathways involved in NB growth, metabolic activity and angiogenesis, and a promising therapeutic target for neuroblastoma treatment.

Radionuclide therapy of endocrine-related cancer

This article gives an overview of the established radionuclide therapies for endocrine-related cancer that already have market authorization or are currently under evaluation in clinical trials. Radioiodine therapy is still the gold standard for differentiated iodine-avid thyroid cancer. In patients with bone and lung metastases (near) total remission is seen in approximately 50% and the 15-year survival rate for these patients is approximately 90%. In contrast to the USA, meta-iodobenzylguanidine (MIBG) therapy has market approval in Europe. According to the current literature, in the setting of advanced stage neuroblastoma and malignant pheochromocytoma or paraganglioma, radiological remission can be achieved in >30% and symptom control in almost 80% of the treated patients. Somatostatin receptor targeted radionuclide therapies (e.g. with DOTATATE or DOTATOC) demonstrated promising results in phase 2 trials, reporting progression-free survival in the range of 24-36 months. A first phase 3 pivotal trial for intestinal carcinoids is currently recruiting and another trial for pancreatic neuroendocrine tumors is planned. Radiopharmaceuticals based on glucagon-like peptide 1 (GLP1) or minigastrins are in the early evaluation stage for application in the treatment of insulinomas and medullary thyroid cancer. In general, radiopharmaceutical therapy belongs to the group of so-called theranostics which means that therapy is tailored for individual patients based on molecular imaging diagnostics to stratify target positive or target negative tumor phenotypes.