MYCN-amplified Neuroblastoma Cells Research Articles

MYCN is amplified during S phase, and c‑myb is involved in controlling MYCN expression and amplification in MYCN‑amplified neuroblastoma cell lines.

Neuroblastoma derived from primitive sympathetic neural precursors is a common type of solid tumor in infants. MYCN proto-oncogene bHLH transcription factor (MYCN) amplification and 1p36 deletion are important factors associated with the poor prognosis of neuroblastoma. Expression levels of MYCN and c-MYB proto-oncogene transcription factor (c-myb) decline during the differentiation of neuroblastoma cells; E2F transcription factor 1 (E2F1) activates the MYCN promoter. However, the underlying mechanism of MYCN overexpression and amplification requires further investigation. In the present study, potential c-Myb target genes, and the effect of c-myb RNA interference (RNAi) on MYCN expression and amplification were investigated in MYCN-amplified neuroblastoma cell lines. The mRNA expression levels and MYCN gene copy number in five neuroblastoma cell lines were determined by quantitative polymerase chain reaction. In addition, variations in potential target gene expression and MYCN gene copy number between pre- and post-c-myb RNAi treatment groups in MYCN-amplified Kelly, IMR32, SIMA and MHH-NB-11 cell lines, normalized to those of non-MYCN-amplified SH-SY5Y, were examined. To determine the associations between gene expression levels and chromosomal aberrations, MYCN amplification and 1p36 alterations in interphases/metaphases were analyzed using fluorescence in situ hybridization. Statistical analyses revealed correlations between 1p36 alterations and the expression of c-myb, MYB proto-oncogene like 2 (B-myb) and cyclin dependent kinase inhibitor 1A (p21). Additionally, the results of the present study also demonstrated that c-myb may be associated with E2F1 and L3MBTL1 histone methyl-lysine binding protein (L3MBTL1) expression, and that E2F1 may contribute to MYCN, B-myb, p21 and chromatin licensing and DNA replication factor 1 (hCdt1) expression, but to the repression of geminin (GMNN). On c-myb RNAi treatment, L3MBTL1 expression was silenced, while GMNN was upregulated, indicating G2/M arrest. In addition, MYCN gene copy number increased following treatment with c-myb RNAi. Notably, the present study also reported a 43.545% sequence identity between upstream of MYCN and Drosophila melanogaster amplification control element 3, suggesting that expression and/or amplification mechanisms of developmentally-regulated genes may be evolutionarily conserved. In conclusion, c-myb may be associated with regulating MYCN expression and amplification. c-myb, B-myb and p21 may also serve a role against chromosome 1p aberrations. Together, it was concluded that MYCN gene is amplified during S phase, potentially via a replication-based mechanism.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Abstract A17: Metronomic topotecan causes MYCN inactivation and impedes tumor growth selectively in MYCN-amplified neuroblastoma cells in vitro and in vivo by therapy-induced senescence

Abstract Background: Poor prognosis and frequent relapses are major challenges for patients with high-risk neuroblastoma (NB), especially when tumors show MYCN amplification. High-dose chemotherapy triggers apoptosis, necrosis, and senescence, a cellular stress response leading to permanent proliferative arrest and a typical senescence-associated secretome (SASP). SASP components reinforce growth arrest and act in an immune-stimulatory fashion, while others are tumor promoting. Studies on low-dose long-term, i.e., metronomic, treatment schedules have demonstrated effects on the tumor microenvironment, but have not comprehensively analyzed cellular responses in tumor cells. Methods and Results: We evaluated efficacy and mode of action of low-dose metronomic treatment schedules in in vitro and in vivo NB models. Importantly, by using the secretome as a discriminator for beneficial versus adverse effects of senescence, drugs with a tumor-inhibiting SASP were identified. Our preclinical work has demonstrated that low-dose long-term topotecan (TPT) treatment (0.1 mg/kg/d, i.p., daily over 15 weeks) improves survival in mouse NB xenografts and leads to long-term cure in 40% of mice. TPT induces a tumor-inhibiting type of senescence, accompanied by changes in the tumor transcriptome and secretome, specifically in MYCN-amplified NB cells. As senescent NB cells showed a reduced MYCN copy number and strongly downregulated MYCN expression, we investigated the mechanism of MYCN inactivation. This showed that upon senescence MYCN is epigenetically silenced and gene copies are recruited to the nuclear periphery where changes in the nuclear lamina composition take place. Conclusion: This new mode of action of metronomic TPT treatment, i.e., promoting a tumor-inhibiting type of senescence selectively in MYCN-amplified neuroblastoma, is clinically relevant as metronomic regimens are increasingly implemented in therapy protocols of various cancer entities and are considered as a feasible maintenance treatment option with moderate adverse event profiles. Citation Format: Sabine Taschner-Mandl, Teresa Gerber, Magdalena Schwarz, Johanna Blaha, Maximilian Kauer, Florian Kromp, Nelli Frank, Fikret Rifatbegovic, Tamara Weiss, Ruth Ladenstein, Martin Hohenegger, Inge M. Ambros, Peter F. Ambros. Metronomic topotecan causes MYCN inactivation and impedes tumor growth selectively in MYCN-amplified neuroblastoma cells in vitro and in vivo by therapy-induced senescence [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr A17.

Abstract IA18: MYCN amplification and ATRX mutations are incompatible in neuroblastoma

Abstract Age and stage of disease at diagnosis are associated with outcome in neuroblastoma. A previous study identified ATRX point mutations and in-frame deletions in older patients with stage 4 disease. To validate the frequency of ATRX mutations and assess the relation of mutations and known prognostic variables, we collaborated with the Children’s Oncology Group and sequenced the entire ATRX genomic locus in 475 neuroblastoma tumor samples to validate the frequency of ATRX mutations and assess the relation of mutations and known prognostic variables. Older age at diagnosis, stage 4 disease, and unfavorable histology were significantly associated with mutations in the ATRX gene; MYCN amplification was mutually exclusive from ATRX mutations. To directly test if ATRX mutations and MYCN amplification are incompatible in neuroblastoma, we mutated the ATRX gene in MYCN amplified neuroblastoma cell lines and ectopically expressed MYCN in ATRX mutant neuroblastoma cell lines. Both approaches showed that ATRX and MYCN amplification are incompatible in neuroblastoma in culture and in vivo. To gain a better understanding of the underlying molecular and cellular mechanisms of this incompatibility, we performed a series of experiments including telomere analysis, epigenetic profiling, electron microscopic analysis of dying cells, and metabolic profiling. We discovered that induction of MYCN expression in ATRX mutant neuroblastoma cells leads to metabolic reprogramming, mitochondrial dysfunction, increased reactive oxygen species, and replicative stress. We propose that this leads to synthetic lethality in ATRX mutant neuroblastomas because of the underlying replicative stress in those cells as a result of dysfunction of this essential histone chaperone complex. Citation Format: Maged Zeineldin, Sara Federico, Xiang Chen, Beisi Xu, Elizabeth Stewart, Arlene Naranjo, Michael D. Hogarty, Michael A. Dyer. MYCN amplification and ATRX mutations are incompatible in neuroblastoma [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr IA18.

Aberrant O-glycosylation modulates aggressiveness in neuroblastoma

Neuroblastoma (NB) is the most common pediatric malignancy diagnosed before the first birthday in which MYCN oncogene amplification is associated with poor prognosis. Although aberrant glycosylation is an important actor in cell biology, little is known about its role in pediatric cancers such as NB. In this work we characterized the glycophenotype and the enzyme expression involved in glycans biosynthesis in five established human NB cell lines and in patient-derived primary tumors with different MYCN status. Our results show a high expression of Lewis glycan family both in MYCN-amplified cell lines and patient samples. Additionally, we report that MYCN-amplified cells overexpressed Core 2-initiating glycosyltransferase C2GNT1 in association with specific ST3Gals and FUTs, and showed increased binding to E- and P- selectins. Silencing of C2GNT1 expression in NB cells diminished expression of Lewis glycans, decreased the E- and P-selectin binding, and reduced cell adhesion, migration and proliferation in vitro. Treatment of MYCN-non-amplified cells with Trichostatin A (TSA), an histone deacetylase inhibitor, increased the expression of Lewis glycans and the enzymes involved in their biosynthesis. Our results demonstrate that MYCN-amplified NB cells overexpress Lewis family glycans, which belong to the Core 2 O-glycans group. Their expression plays a key role in the malignant behaviour of the NB cells and it is modulated by epigenetic mechanisms.

Etoposide-resistance in a neuroblastoma model cell line is associated with 13q14.3 mono-allelic deletion and miRNA-15a/16-1 down-regulation

Drug resistance is the major obstacle in successfully treating high-risk neuroblastoma. The aim of this study was to investigate the basis of etoposide-resistance in neuroblastoma. To this end, a MYCN-amplified neuroblastoma cell line (HTLA-230) was treated with increasing etoposide concentrations and an etoposide-resistant cell line (HTLA-ER) was obtained. HTLA-ER cells, following etoposide exposure, evaded apoptosis by altering Bax/Bcl2 ratio. While both cell populations shared a homozygous TP53 mutation encoding a partially-functioning protein, a mono-allelic deletion of 13q14.3 locus, where the P53 inducible miRNAs 15a/16-1 are located, and the consequent miRNA down-regulation were detected only in HTLA-ER cells. This event correlated with BMI-1 oncoprotein up-regulation which caused a decrease in p16 tumor suppressor content and a metabolic adaptation of HTLA-ER cells. These results, taken collectively, highlight the role of miRNAs 15a/16-1 as markers of chemoresistance.

Selective gene dependencies in MYCN-amplified neuroblastoma include the core transcriptional regulatory circuitry.

Childhood high-risk neuroblastomas with MYCN gene amplification are difficult to treat effectively1. This has focused attention on tumor-specific gene dependencies that underlie tumorigenesis and thus provide valuable targets for the development of novel therapeutics. Using unbiased genome-scale CRISPR-Cas9 approaches to detect genes involved in tumor cell growth and survival2–6, we identified 147 candidate gene dependencies selective for MYCN-amplified neuroblastoma cell lines, compared to over 300 other human cancer cell lines. We then used genome-wide ChIP-seq analysis to demonstrate that a small number of essential transcription factors: MYCN, HAND2, ISL1, PHOX2B, GATA3, and TBX2, are members of the transcriptional core regulatory circuitry (CRC) that maintains cell state in MYCN-amplified neuroblastoma. To disable the CRC, we tested a combination of BRD4 and CDK7 inhibitors, which act synergistically, in vitro and in vivo, with rapid downregulation of CRC transcription factor gene expression. This study defines a set of critical dependency genes in MYCN-amplified neuroblastoma that are essential for cell state and survival in this tumor.

Abstract 2062: Selective gene dependencies in MYCN-amplified neuroblastoma include the core transcriptional regulatory circuitry

Abstract Childhood neuroblastomas with MYCN gene amplification form a particularly high-risk subset of this disease and are difficult to treat effectively. This has focused attention on tumor-specific gene dependencies that reflect important pathways in tumorigenesis, and thus could provide valuable targets for the development of novel therapeutics. Using genome-scale CRISPR-Cas9 approaches that allow unbiased detection of genes critically involved in tumor cell growth and survival, we identified 147 candidate genes associated with selective vulnerabilities in nine MYCN-amplified neuroblastoma cell lines, compared to findings in over 300 other human cancer cell lines representing multiple tumor cell types. We then used genome-wide ChIP-seq analysis to test the hypothesis that a small number of transcription factors - MYCN, HAND2, ISL1, PHOX2B, GATA3, and TBX2, all represented in the selective dependency group - are members of the transcriptional core regulatory circuitry (CRC) that underlies cell state in MYCN-amplified neuroblastoma. We show that these transcription factors bind as dense clusters at defined epicentres within the enhancers of their own genes, as well as those of the other CRC transcription factor genes, creating a positive feed-forward autoregulatory loop that establishes and maintains high levels of gene expression. To disable the CRC, we tested a combination of BRD4 and CDK7 inhibitors, which we postulated would act synergistically by targeting both transcriptional initiation and elongation required to synthesize regulatory transcription factors. MYCN-amplified neuroblastoma cells treated with both drugs were killed synergistically, in vitro and in vivo, and accompanied by rapid downregulation of CRC transcription factor gene expression. This study defines a set of critical dependency genes in MYCN-amplified neuroblastoma, a subset of which comprises the oncogenic transcriptional regulatory circuitry that underlies cell state and survival in this tumor. Citation Format: Adam D. Durbin, Mark W. Zimmerman, Neekesh V. Dharia, Brian J. Abraham, Brian J. Abraham, Amanda Balboni-Iniguez, Nina Weichert-Leahey, Shuning He, John M. Krill-Burger, David E. Root, Francisca Vazquez, Aviad Tsherniak, William C. Hahn, Todd R. Golub, Richard A. Young, A. Thomas Look, Kimberly Stegmaier. Selective gene dependencies in MYCN-amplified neuroblastoma include the core transcriptional regulatory circuitry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2062.

Abstract 3521: Glutamine metabolic inhibition synergizes with L-asparaginase in MYCN-amplified neuroblastoma

Abstract Neuroblastoma is the most common extracranial solid tumor in children. Though it accounts for about 10% of pediatric cancers, it is disproportionately responsible for 15% of pediatric cancer deaths. MYCN is amplified in 20% of neuroblastomas and correlates with adverse outcome. MYCN is involved in the maintenance of cancer stem cells, as well as in driving tumor cell growth, proliferation and tumorigenesis. Attempts to inhibit MYCN directly have been largely unsuccessful. MYCN is known to drive tumor cell reliance on glutamine for cellular metabolism. 6-Diazo-5-oxo-L-norleucine (DON) is a well-characterized glutamine analogue that inhibits glutamine metabolism by irreversibly inactivating multiple glutamine-utilizing enzymes. DON was well tolerated in a previous phase I clinical trial in pediatric patients, but it has never been systematically tested in neuroblastoma patients. We show that MYCN-amplification confers sensitivity to DON therapy in in vitro models of neuroblastoma, and that DON administered by intraperitoneal injection twice weekly significantly reduces flank tumor volume in orthotopic mouse models of MYCN-amplified neuroblastoma (mean tumor volume 1715 mm3 vs. 207 mm3 in control animals, p-value = 0.00017 by t-test). We have also developed an orally bioavailable DON prodrug, JHU083, and we found that this drug administered orally three times weekly was similarly able to suppress neuroblastoma tumor growth in mice (mean tumor volume 1,115 mm3 vs. 217 mm3 in control animals, p-value = 0.0000088 by t-test). In metabolic flux experiments, tracing glutamine and glucose donation of 13C and 15N via liquid chromatography/mass spectrometry, DON prevents asparagine synthesis, depleting intracellular asparagine levels by 40% compared to control treated cells (p-value = 0.006). DON combined with L-asparaginase synergistically inhibits growth of MYCN-amplified neuroblastoma cell lines (CI = 0.25 by the Chou-Talalay method, indicating strong synergy; p-value = 0.00011). We conclude that DON depletes cellular pools of asparagine, and combination therapy with DON and L-asparaginase synergistically inhibits the growth of MYCN-amplified neuroblastoma. These studies provide the preclinical justification for potential clinical trials for the use of DON or DON prodrugs in combination with L-asparaginase as new therapeutic options for patients with MYCN-amplified neuroblastoma. Citation Format: Micah J. Maxwell, Brad Poore, Allison Hanaford, Jesse Alt, Rana Rais, Barbara S. Slusher, Charles G. Eberhart, Eric H. Raabe. Glutamine metabolic inhibition synergizes with L-asparaginase in MYCN-amplified neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3521.

Transcription factor activating protein 4 is synthetically lethal and a master regulator of MYCN-amplified neuroblastoma

Despite the identification of MYCN amplification as an adverse prognostic marker in neuroblastoma, MYCN inhibitors have yet to be developed. Here, by integrating evidence from a whole-genome shRNA library screen and the computational inference of master regulator proteins, we identify transcription factor activating protein 4 (TFAP4) as a critical effector of MYCN amplification in neuroblastoma, providing a novel synthetic lethal target. We demonstrate that TFAP4 is a direct target of MYCN in neuroblastoma cells, and that its expression and activity strongly negatively correlate with neuroblastoma patient survival. Silencing TFAP4 selectively inhibits MYCN-amplified neuroblastoma cell growth both in vitro and in vivo, in xenograft mouse models. Mechanistically, silencing TFAP4 induces neuroblastoma differentiation, as evidenced by increased neurite outgrowth and upregulation of neuronal markers. Taken together, our results demonstrate that TFAP4 is a key regulator of MYCN-amplified neuroblastoma and may represent a valuable novel therapeutic target.

Tumorigenic and Antiproliferative Properties of the TALE-Transcription Factors MEIS2D and MEIS2A in Neuroblastoma.

Neuroblastoma is one of only a few human cancers that can spontaneously regress even after extensive dissemination, a poorly understood phenomenon that occurs in as many as 10% of patients. In this study, we identify the TALE-homeodomain transcription factor MEIS2 as a key contributor to this phenomenon. We identified MEIS2 as a MYCN-independent factor in neuroblastoma and showed that in this setting the alternatively spliced isoforms MEIS2A and MEIS2D exert antagonistic functions. Specifically, expression of MEIS2A was low in aggressive stage 4 neuroblastoma but high in spontaneously regressing stage 4S neuroblastoma. Moderate elevation of MEIS2A expression reduced proliferation of MYCN-amplified human neuroblastoma cells, induced neuronal differentiation and impaired the ability of these cells to form tumors in mice. In contrast, MEIS2A silencing or MEIS2D upregulation enhanced the aggressiveness of the tumor phenotype. Mechanistically, MEIS2A uncoupled a negative feedback loop that restricts accumulation of cellular retinoic acid, an effective agent in neuroblastoma treatment. Overall, our results illuminate the basis for spontaneous regression in neuroblastoma and identify an MEIS2A-specific signaling network as a potential therapeutic target in this common pediatric malignancy.Significance: This study illuminates the basis for spontaneous regressions that can occur in a common pediatric tumor, with implications for the development of new treatment strategies. Cancer Res; 78(8); 1935-47. ©2018 AACR.

The long non-coding RNA MYCNOS-01 regulates MYCN protein levels and affects growth of MYCN-amplified rhabdomyosarcoma and neuroblastoma cells

BackgroundMYCN is amplified in small cell lung cancers and several pediatric tumors, including alveolar rhabdomyosarcomas and neuroblastomas. MYCN protein is known to play a key oncogenic role in both alveolar rhabdomyosarcomas and neuroblastomas. MYCN opposite strand (MYCNOS) is a gene located on the antisense strand to MYCN that encodes alternatively spliced transcripts, two of which (MYCNOS-01 and MYCNOS-02) are known to be expressed in neuroblastoma and small cell lung cancer with reciprocal regulation between MYCNOS-02 and MYCN reported for neuroblastomas. We sought to determine a functional role for MYCNOS-01 in alveolar rhabdomyosarcoma and neuroblastoma cells and identify any associated regulatory effects between MYCN and MYCNOS-01.MethodsMYCNOS-01, MYCNOS-02 and MYCN expression levels were assessed in alveolar rhabdomyosarcoma and neuroblastoma cell lines and tumor samples from patients using Affymetrix microarray data and quantitative RT-PCR. Following MYCNOS-01 or MYCN siRNA knockdown and MYCNOS-01 overexpression, transcript levels were assayed by quantitative RT-PCR and MYCN protein expression assessed by Western blot and immunofluorescence. Additionally, effects on cell growth, apoptosis and cell cycle profiles were determined by a metabolic assay, caspase activity and flow cytometry, respectively.ResultsMYCNOS-01 transcript levels were generally higher in NB and RMS tumor samples and cell lines with MYCN genomic amplification. RNA interference of MYCNOS-01 expression did not alter MYCN transcript levels but decreased MYCN protein levels. Conversely, MYCN reduction increased MYCNOS-01 transcript levels, creating a negative feedback loop on MYCN protein levels. Reduction of MYCNOS-01 or MYCN expression decreased cell growth in MYCN-amplified alveolar rhabdomyosarcoma and neuroblastoma cell lines. This is consistent with MYCNOS-01-mediated regulation of MYCN contributing to the phenotype observed.ConclusionsAn alternative transcript of MYCNOS, MYCNOS-01, post-transcriptionally regulates MYCN levels and affects growth in MYCN-amplified rhabdomyosarcoma and neuroblastoma cells.

MYCN-amplified neuroblastoma maintains an aggressive and undifferentiated phenotype by deregulation of estrogen and NGF signaling

Neuroblastoma (NB) is a remarkably heterogenic childhood tumor of the sympathetic nervous system with clinical behavior ranging from spontaneous regression to poorly differentiated tumors and metastasis. MYCN is amplified in 20% of cases and correlates with an undifferentiated, aggressive phenotype and poor prognosis. Estrogen receptor alpha (ERα) and the nerve growth factor (NGF) receptors TrkA and p75NTR are involved in neuronal differentiation and survival. We have previously shown that MYCN, via miR-18a, targets ERα in NB cells. Here, we demonstrate that interference with miR-18a or overexpression of ERα is sufficient to induce NGF signaling and to modulate both basal and NGF-induced neuronal differentiation in MYCN-amplified NB cells. Proteomic analysis confirmed an increase of neuronal features and showed that processes linked to tumor initiation and progression were inhibited upon ERα overexpression. Indeed, ectopic ERα expression was sufficient to inhibit metabolic activity and tumorigenic processes, including glycolysis, oxidative phosphorylation, cell viability, migration, and anchorage independent growth. Importantly, ERα overexpression reduced tumor burden in NB mouse models and high ERα levels were linked to improved survival in patients. In addition to ERα, several other nuclear hormone receptors (NHRs), including the glucocorticoid and the retinoic acid receptors, correlated with clinical markers for favorable and low-stage NB disease. Our data suggest that MYCN targets ERα and thereby NGF signaling to maintain an undifferentiated and aggressive phenotype. Notably, we identified the estrogen-NGF crosstalk, as well as a set of other NHRs, as potential prognostic markers and targets for therapeutic strategies against NB.

The protective autophagy activated by GANT-61 in MYCN amplified neuroblastoma cells is mediated by PERK.

The proto-oncogene MYC can trigger the unfolded protein response (UPR). The double-stranded RNA-activated protein kinase-like endoplasmic reticulum kinase (PERK), one of three primary branches of the UPR, is a key regulator of autophagy, promoting tumorigenesis. Upon activation of PERK, there is an increase in phosphorylation of the eukaryotic initiation factor-2 alpha (eIF2α), which in turn, activates the transcription factor-4 (ATF4), responsible for an increased expression of LC3, a common autophagy marker. PERK is repressed upon GLI1 and GLI2 induction. GANT-61 is an inhibitor of GLI1 and GLI2, known to reduce autophagy in MYCN non-amplified, but not in MYCN amplified neuroblastoma (NB) cells. In our study, we tested the effect of the joint administration of a PERK inhibitor (GSK2606414) and the GLI inhibitor GANT-61 to MYCN amplified and MYCN non-amplified NB cells. Our results suggest that inhibition of PERK impairs GANT-61 induced autophagy in NB cells with MYCN amplification, but had no effect on the MYCN non-amplified NB cells. In summary, PERK seems to be a good therapeutic target for NB. Inhibition of PERK reduces autophagy in MYCN amplified NB cells, thus amplifying the efficacy of the GLI inhibitor GANT-61 in reducing proliferation of this type of cancer cells.

Reversible LSD1 inhibition with HCI-2509 induces the p53 gene expression signature and disrupts the MYCN signature in high-risk neuroblastoma cells.

Lysine-Specific Demethylase 1 (LSD1) over-expression correlates with poorly differentiated neuroblastoma and predicts poor outcome despite multimodal therapy. We have studied the efficacy of reversible and specific LSD1 inhibition with HCI-2509 in neuroblastoma cell lines and particularly the effect of HCI-2509 on the transcriptomic profile in MYCN amplified NGP cells. Cell survival assays show that HCI-2509 is cytotoxic to poorly differentiated neuroblastoma cell lines in low micromole or lower doses. Transcriptional profiling of NGP cells treated with HCI-2509 shows a significant effect on p53, cell cycle, MYCN and hypoxia pathway gene sets. HCI-2509 results in increased histone methyl marks and p53 levels along with cell cycle arrest in the G2/M phase and inhibition of colony formation of NGP cells. Our findings indicate that LSD1 inhibition with HCI-2509 has a multi-target effect in neuroblastoma cell lines, mediated in part via p53. MYCN-amplified neuroblastoma cells have a targeted benefit as HCI-2509 downregulates the MYCN upregulated gene set.

Optimising the chick chorioallantoic membrane xenograft model of neuroblastoma for drug delivery

BackgroundNeuroblastoma is a paediatric cancer that despite multimodal therapy still has a poor outcome for many patients with high risk tumours. Retinoic acid (RA) promotes differentiation of some neuroblastoma tumours and cell lines, and is successfully used clinically, supporting the view that differentiation therapy is a promising strategy for treatment of neuroblastoma. To improve treatment of a wider range of tumour types, development and testing of novel differentiation agents is essential. New pre-clinical models are therefore required to test therapies in a rapid cost effective way in order to identify the most useful agents.MethodsAs a proof of principle, differentiation upon ATRA treatment of two MYCN-amplified neuroblastoma cell lines, IMR32 and BE2C, was measured both in cell cultures and in tumours formed on the chick chorioallantoic membrane (CAM). Differentiation was assessed by 1) change in cell morphology, 2) reduction in cell proliferation using Ki67 staining and 3) changes in differentiation markers (STMN4 and ROBO2) and stem cell marker (KLF4). Results were compared to MLN8237, a classical Aurora Kinase A inhibitor. For the in vivo experiments, cells were implanted on the CAM at embryonic day 7 (E7), ATRA treatment was between E11 and E13 and tumours were analysed at E14.ResultsTreatment of IMR32 and BE2C cells in vitro with 10 μM ATRA resulted in a change in cell morphology, a 65% decrease in cell proliferation, upregulation of STMN4 and ROBO2 and downregulation of KLF4. ATRA proved more effective than MLN8237 in these assays. In vivo, 100 μM ATRA repetitive treatment at E11, E12 and E13 promoted a change in expression of differentiation markers and reduced proliferation by 43% (p < 0.05). 40 μM ATRA treatment at E11 and E13 reduced proliferation by 37% (p < 0.05) and also changed cell morphology within the tumour.ConclusionDifferentiation of neuroblastoma tumours formed on the chick CAM can be analysed by changes in cell morphology, proliferation and gene expression. The well-described effects of ATRA on neuroblastoma differentiation were recapitulated within 3 days in the chick embryo model, which therefore offers a rapid, cost effective model compliant with the 3Rs to select promising drugs for further preclinical analysis.

Abstract A119: Nanocarriers with a camptothecin-mitocan co-drug suppress tumor growth and extend survival in models of chemosensitive and chemoresistant neuroblastoma

Abstract Background: Neuroblastoma (NB) ranks as the most common solid tumor in infants and is responsible for 15% of all childhood cancer deaths. Chemotherapeutics used as a first-line treatment in high-risk NB patients are inherently toxic and ultimately ineffective. High-risk NB patients have &amp;lt;50% survival rate, and a particular challenge is posed by a group of “ultrahigh”-risk patients who show no response to therapy. With the goal of developing a clinically viable treatment strategy for high-risk NB, this study focused on an experimental approach integrating novel mutual prodrugs (co-drugs) and biodegradable nanocarriers. Nanocarrier-mediated co-drug delivery is designed to take advantage of enhanced accumulation and retention of sub-100 nm-sized nanoparticles (NP) in the tumor, followed by controlled release and in situ activation of the co-drug. Toward this goal, we developed a series of reversibly hydrophobized co-drugs exhibiting stable encapsulation in NP and activated hydrolytically to produce in one step 1) a potent camptothecin analog that inhibits topoisomerase I (SN-38) and 2) a pro-oxidant “helper” agent of a redox-silent tocol family of mitocans acting on the mitochondrial respiratory chain of cancerous cells. The effectiveness of co-drug loaded NP was evaluated in vitro and in orthotopic models of MYCN-amplified, chemosensitive, and drug-resistant NB. Methods and Results: A series of SN38-tocol conjugates constructed with progressively increasing hydrolytic activation rates were synthesized in high yields via direct coupling of the components. Co-drug loaded biodegradable NP (average size: 85±36 nm) were formulated using a nanoprecipitation approach adapted for producing sub-100 nm-sized nanocarriers. In comparative in vitro studies, a co-drug designed with a phenolic ester linkage was found to be most effective, achieving profound and sustained growth inhibition of MYCN-amplified NB cells, both chemo-naïve and chemoresistant (IMR-32 and BE(2)-C cells derived at diagnosis and during relapse after chemotherapy, respectively) under conditions modeling different levels of exposure experienced by NB cells within the tumor. Phenolic carbonate and aliphatic ester designs were notably less efficient. In an in vivo model of chemosensitive disease (IMR-32 orthotopic xenograft), nanocarriers with phenolic ester co-drug administered over 4 weeks (10 mg/kg, once a week) induced tumor regression and completely inhibited tumor growth over a 26-week period. This co-drug/nanocarrier formulation tested against chemoresistant NB (BE(2)-C orthotopic xenograft) potently suppressed tumor growth and extended animal survival up to 7 weeks, in comparison to irinotecan, which was only marginally effective (event-free survival of 3 weeks vs. 2 weeks in "no treatment" and drug-free NP groups). Conclusions: Camptothecin-mitocan co-drugs can be rationally designed and optimized to enhance their performance in the context of nanocarrier-based therapy of aggressive NB. The co-drug/nanocarrier combination strategy, whose feasibility and effectiveness against both chemosensitive and drug-resistant NB was demonstrated in the present preclinical studies, holds promise as a new therapeutic strategy for high-risk NB as well as for other solid tumors currently lacking effective treatment options. Citation Format: David Guerrero, Ivan S. Alferiev, Ferro Nguyen, Peng Guan, Venkatadri Kolla, Danielle Soberman, Ilia Fishbein, Robert J. Levy, Garrett M. Brodeur, Michael Chorny. Nanocarriers with a camptothecin-mitocan co-drug suppress tumor growth and extend survival in models of chemosensitive and chemoresistant neuroblastoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A119.

Isoxazole compound ML327 blocks MYC expression and tumor formation in neuroblastoma.

Neuroblastomas are the most common extracranial solid tumors in children and arise from the embryonic neural crest. MYCN-amplification is a feature of ∼30% of neuroblastoma tumors and portends a poor prognosis. Neural crest precursors undergo epithelial-to-mesenchymal transition (EMT) to gain migratory potential and populate the sympathoadrenal axis. Neuroblastomas are posited to arise due to a blockade of neural crest differentiation. We have recently reported effects of a novel MET inducing compound ML327 (N-(3-(2-hydroxynicotinamido) propyl)-5-phenylisoxazole-3-carboxamide) in colon cancer cells. Herein, we hypothesized that forced epithelial differentiation using ML327 would promote neuroblastoma differentiation. In this study, we demonstrate that ML327 in neuroblastoma cells induces a gene signature consistent with both epithelial and neuronal differentiation features with adaptation of an elongated phenotype. These features accompany induction of cell death and G1 cell cycle arrest with blockage of anchorage-independent growth and neurosphere formation. Furthermore, pretreatment with ML327 results in persistent defects in proliferative potential and tumor-initiating capacity, validating the pro-differentiating effects of our compound. Intriguingly, we have identified destabilization of MYC signaling as an early and consistent feature of ML327 treatment that is observed in both MYCN-amplified and MYCN-single copy neuroblastoma cell lines. Moreover, ML327 blocked MYCN mRNA levels and tumor progression in established MYCN-amplified xenografts. As such, ML327 may have potential efficacy, alone or in conjunction with existing therapeutic strategies against neuroblastoma. Future identification of the specific intracellular target of ML327 may inform future drug discovery efforts and enhance our understanding of MYC regulation.

Abstract 5816: Ablation of oncogenic MYCN expression by hTERT-driven oncolytic adenovirus induces cell death in MYCN-amplified neuroblastoma

Abstract Background: Neuroblastoma (NB) is a primary malignant tumor of the peripheral sympathetic nervous system. Amplification and overexpression of the MYCN proto-oncogene occur in approximately 20% of NB. Since MYCN activation is highly associated with poor prognosis, MYCN is an attractive target for the treatment of MYCN-amplified NB. MYCN is a transcriptional activator that induces the expression of many down-stream target genes including human telomerase reverse transcriptase (hTERT). We developed two types of hTERT-driven oncolytic adenoviruses, OBP-301 and OBP-702, in which the hTERT promoter drives the expression of the viral E1A and E1B genes for tumor-specific virus replication, and OBP-702 further expresses tumor suppressor p53 protein. Although hTERT-driven oncolytic viruses are expected to be effective against MYCN-amplified NB with hTERT activation, the therapeutic potential of these viruses in MYCN-amplified NB remains unclear. In this study, we investigated whether OBP-301 and OBP-702 induce cell death and modulate MYCN expression in MYCN-amplified NB cells. Methods: We used 3 human NB cell lines with MYCN amplification, including IMR-32, CHP-134 and LA-N-5. The antitumor effect of OBP-301 and OBP-702 was evaluated using XTT assay. Virus-mediated cell death and modulation of MYCN and E2F1 expression were analyzed by Western blot analysis. The mRNA expression of hTERT and MYCN was analyzed by real-time RT-PCR analysis. To explore the role of E2F1 in the virus-mediated MYCN modulation, Ad-E2F1, a replication-defective adenovirus expressing E2F1 gene, was further used. Results: All NB cell lines showed high hTERT mRNA expression. OBP-301 and OBP-702 showed profound antitumor effect through autophagy-related cell death in all NB cell lines. Both viruses induced E1A and its target mediator E2F1 expression in these cell lines. Expression of MYCN mRNA and protein was downregulated by these viruses. Replication-deficient Ad-E2F1 infection also downregulated the expression of MYCN. Conclusions: These results suggest that hTERT-driven oncolytic adenoviruses are promising antitumor agent for the treatment of MYCN-amplified NB. These viruses induce profound autophagy through hTERT-dependent viral replication. E2F1 upregulation by viral infection is supposed to be one of the causes for MYCN downregulation and autophagy-related cell death. In vivo experiments are under way to investigate the antitumor effect of these viruses against xenograft NB tumors. Citation Format: Terutaka Tanimoto, Hiroshi Tazawa, Hiroshi Noso, Takanori Oyama, Yasuo Urata, Shunsuke Kagawa, Takuo Noda, Toshiyoshi Fujiwara. Ablation of oncogenic MYCN expression by hTERT-driven oncolytic adenovirus induces cell death in MYCN-amplified neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5816. doi:10.1158/1538-7445.AM2017-5816

Abstract 3871: Modeling the chromatin and transcriptional landscape of MYC and MYCN driven neuroblastoma in zebrafish

Abstract Elevated expression levels of MYC family genes are frequently observed in human cancer cells and correlate with tumor aggressiveness and poor prognosis. In neuroblastoma 40% of all cases are high-risk, of which 20% harbor amplification of the MYCN proto-oncogene. In high-risk cases lacking MYCN gene amplification, high expression levels of c-MYC (MYC) are often present and are associated with unfavorable histology and a poor survival. Unlike MYCN amplification, which is frequently observed in the presence of segmental chromosomal aberrations, MYC overexpression is not associated with genetic abnormalities or somatic mutations. In order to study this newly defined subgroup, we have created a novel transgenic zebrafish model in which overexpression of MYC alone in the peripheral sympathetic nervous system (PSNS) drives early-onset neuroblastoma in nearly every fish by seven weeks of age. The tumors resulting from MYC overexpression arise in the interrenal gland, which is the fish counterpart of the adrenal medulla, and are histologically identical to human neuroblastoma. We next performed the Assay for Transposase Accessible Chromatin (ATAC) sequencing and RNA-seq to identify open chromatin regions that correlate with activation of gene transcription. Lineage specific genes essential for neuronal precursor cell identity, such as PHOX2B, HAND2, and TFAP2A are highly expressed in both MYC-expressing and MYCN-amplified human neuroblastoma cell lines and are actively transcribed in zebrafish models of MYC and MYCN driven neuroblastoma. Furthermore, these studies reveal shared and differential regulatory of effects of MYC relative to MYCN activity in maintaining the malignant phenotype of neuroblastoma in vivo. Additional insight into the mechanisms of aberrant transcriptional regulation will inform the future design and use of therapeutic strategies targeting transcription in this high-risk malignancy of childhood. Citation Format: Mark W. Zimmerman, Shuning He, Shizhen Zhu, Song Yang, Yi Zhou, Leonard I. Zon, A Thomas Look. Modeling the chromatin and transcriptional landscape of MYC and MYCN driven neuroblastoma in zebrafish [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3871. doi:10.1158/1538-7445.AM2017-3871