MYCN Research Articles

Abstract 5086: Chronic radiation induced uPA expression mediates neuroblastoma cells survival

Abstract Neuroblastoma accounts for more than 15% of cancer-related deaths of children in the United States. Despite aggressive treatment regimens, the long-term survival for these children remains less than 40%. The urokinase plasminogen activator (uPA) system plays a role in several critically important biology processes, such as cell adhesion, migration, proliferation and survival. This system includes uPA, a serine protease which converts a zymogen plasminogen into the active serine protease plasmin, its surface receptor uPAR/CD57, which binds uPA with high affinity, facilitates proteolytic activation/conversion of uPA precursor (single-chain uPA, scuPA) into the active two-chain uPA protease (tcuPA) and mediates uPA-induced activation of several intracellular signaling cascades, and protects against several inhibitors, e.g. PAI-1, among others. Recent findings demonstrate that dividing/proliferating cells translocate intact scuPA to the nucleus via the nucleocytoplasmic shuttle protein nucleolin. In this study we demonstrate that both SKNBE(2) and NB1691 neuroblastoma cells when exposed to chronic radiation suppresses nMYC gene copy number and induces the overexpression of cMYC and uPA expression levels without observable change in uPA gene copy number. Increased uPA expression was accompanied with increase in the mitochondrial mass (4 fold in both NB1691 and SKNBE (2) cells). Knockdown of uPA expression in chronic radiation cells using siRNA resulted in reduced cell survival. RT-PCR analysis shows that uPA knockdown resulted in reduced expression of cMYC and its target genes cyclin A and cyclin D1. Overexpression of uPA in both SKNBE(2) and NB1691 wildtype cells resulted in increased cMYC and cyclin D1 expression but not nMYC expression suggesting that cMYC may be specifically regulated by uPA. In conclusion, our study shows that uPA plays a vital role in survival of neuroblastoma cells treated with chronic radiation by associating with cMYC to compensate for loss of nMYC expression. Our study also implies that targeting uPA might be a viable option as a combination therapy in the treatment of high-risk neuroblastoma. Citation Format: Manu Gnanamony, Karen Fernández, Jaime Libes, Julian Lin, Pushpa A. Joseph, Christopher S. Gondi. Chronic radiation induced uPA expression mediates neuroblastoma cells survival [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 5086. doi:10.1158/1538-7445.AM2017-5086

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

Abstract 1711: Detection of circulating tumor cells for use as prognostic, predictive and pharmacodynamic biomarkers in neuroblastoma

Abstract Introduction: Neuroblastoma (NB) is the commonest extracranial childhood solid tumour. New treatments and a better understanding of drug resistance are needed to improve survival. Circulating tumour cells (CTCs) may provide a source of tumour cells for genetic biomarkers, give insights into tumour load and serve as pharmacodynamic (PD) biomarkers for new treatments. In clinical diagnostics, slide based FISH is used to detect MYCN amplification in neuroblastoma tumours. As it is not possible to conduct slide based FISH on an entire blood sample due to the infrequency of CTCs in peripheral blood (1 in 106 leucocytes), an alternative method FISH-IS (in suspension) was used in the present study to detect MYCN gene amplification using the Imagestream imaging flow cytometer (ISx). Aims: 1) To detect CTCs from blood and disseminated tumour cells (DTC) from bone marrow (BM) from NB patients using the (ISx) 2) To use these cells for genetic and PD biomarker studies for novel targeted therapies. Methods: A panel of NB cell lines (n=6) cloned into neuronal (N) and substrate-adherent (S) types were used to determine expression of NB cell surface markers (GD2 and NCAM) using the ISx. Western blotting and fluorescence activated cell sorting (FACS) were used to validate NCAM and GD2 expression respectively. 25 paired NB patient blood and BM samples were analysed for CTCs and DTCs, 4 unpaired blood samples were analysed for CTCs and one unpaired BM sample was for DTCs using the ISx to detect GD2 +ve and CD45-ve cells. Results: Using the ISx 80-90% of N-type cells immunostained positive for GD2 compared to 0.8 -10% of S-type cells confirmed by FACS. CTCs were detected in 21/29 patient samples and DTCs in 19/26 cases. The numbers of CTCs ranged from 10-522/ml blood and DTCs from 57-35,515/ml of bone marrow. After validating FISH-IS protocol on ISx using NGP and SHSY5Y cell lines, 4 NB patient DTC samples were analysed for MYCN amplification. 2/4 cases showed MYCN gene amplification consistent with results obtained on the primary tumour. Conclusion: This the first study to show that DTCs and CTCs are detectable in NB patient samples using the ISx. Future work will involve further genetic characterisation and development of PD biomarkers for Phase I clinical trials. Citation Format: Swathi Merugu, Deborah Tweddle. Detection of circulating tumor cells for use as prognostic, predictive and pharmacodynamic biomarkers in 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 1711. doi:10.1158/1538-7445.AM2017-1711

Impact of laparoscopy on the biological behavior and gene expression of endometrial adenocarcinoma cells

The current study investigated the effect of laparoscopy on the biological behavior and gene expression of endometrial adenocarcinoma cells. Totally, 40 patients with stage I endometrial adenocarcinoma and 20 patients with benign uterine diseases were enrolled in this study. For patients with endometrial adenocarcinoma, laparoscopy was performed in 20 cases and laparotomy was carried out in the other 20 cases. Total laparoscopic hysterectomy was performed in patients with benign diseases. Cell apoptotic rate and the gene expression of N-myc, Fas, metastasis-associated protein 1 (MTA1), and nm23-H1 were determined in the normal and cancerous endometrial tissues both preoperatively and postoperatively. For endometrial adenocarcinoma cells, laparoscopy, instead of laparotomy, promoted the apoptosis of endometrial adenocarcinoma cells, down-regulated the expression of apoptosis suppressor gene N-myc and metastasis-promoting gene MTA1, up-regulated the expression of apoptosis-promoting gene Fas and metastasis suppressor gene nm23-H1. However, laparoscopy did not affect the apoptotic rate and gene expression in normal endometrial cells. Laparoscopy may be used as a safe and effective intervention for endometrial cancer.

The Histone Methyltransferase DOT1L Promotes Neuroblastoma by Regulating Gene Transcription.

Myc oncoproteins exert tumorigenic effects by regulating expression of target oncogenes. Histone H3 lysine 79 (H3K79) methylation at Myc-responsive elements of target gene promoters is a strict prerequisite for Myc-induced transcriptional activation, and DOT1L is the only known histone methyltransferase that catalyzes H3K79 methylation. Here, we show that N-Myc upregulates DOT1L mRNA and protein expression by binding to the DOT1L gene promoter. shRNA-mediated depletion of DOT1L reduced mRNA and protein expression of N-Myc target genes ODC1 and E2F2 DOT1L bound to the Myc Box II domain of N-Myc protein, and knockdown of DOT1L reduced histone H3K79 methylation and N-Myc protein binding at the ODC1 and E2F2 gene promoters and reduced neuroblastoma cell proliferation. Treatment with the small-molecule DOT1L inhibitor SGC0946 reduced H3K79 methylation and proliferation of MYCN gene-amplified neuroblastoma cells. In mice xenografts of neuroblastoma cells stably expressing doxycycline-inducible DOT1L shRNA, ablating DOT1L expression with doxycycline significantly reduced ODC1 and E2F2 expression, reduced tumor progression, and improved overall survival. In addition, high levels of DOT1L gene expression in human neuroblastoma tissues correlated with high levels of MYCN, ODC1, and E2F2 gene expression and independently correlated with poor patient survival. Taken together, our results identify DOT1L as a novel cofactor in N-Myc-mediated transcriptional activation of target genes and neuroblastoma oncogenesis. Furthermore, they characterize DOT1L inhibitors as novel anticancer agents against MYCN-amplified neuroblastoma. Cancer Res; 77(9); 2522-33. ©2017 AACR.

MYCN amplification predicts poor prognosis based on interphase fluorescence in situ hybridization analysis of bone marrow cells in bone marrow metastases of neuroblastoma

BackgroundMYCN gene amplification is related to risk stratification. Therefore it is important to identify accurately the level of the MYCN gene as early as possible in neuroblastoma (NB); however, for patients with bone marrow (BM) metastasis who need chemotherapy before surgery, timely detection of the MYCN gene is not possible due to the unavailability of primary tumors.MethodsMYCN gene status was evaluated in 81 BM metastases of NB by interphase fluorescence in situ hybridization (FISH) analysis of BM cells. The clinicobiological characteristics and prognostic impact of MYCN amplification in NB metastatic to BM were analyzed.ResultsMYCN amplification was found in 16% of patients with metastases, and the results were consistent with the primary tumors detected by pathological tissue FISH. MYCN amplification was associated with age, lactate dehydrogenase (LDH) levels and prognosis (P = 0.038, P < 0.001, P = 0.026). Clinical outcome was poorer in patients with MYCN amplification than in those without amplification (3-year EFS 28.8 ± 13.1 vs. 69.7 ± 5.7%, P = 0.005; 3-year OS 41.5 ± 14.7 vs. 76.7 ± 5.5%, P = 0.005).ConclusionsMYCN amplification predicts a poor outcome in NB metastatic to BM, and interphase FISH of bone marrow cells provides a timely direct and valid method to evaluate the MYCN gene status.

Modeling and Targeting MYC Genes in Childhood Brain Tumors.

Brain tumors are the second most common group of childhood cancers, accounting for about 20%–25% of all pediatric tumors. Deregulated expression of the MYC family of transcription factors, particularly c-MYC and MYCN genes, has been found in many of these neoplasms, and their expression levels are often correlated with poor prognosis. Elevated c-MYC/MYCN initiates and drives tumorigenesis in many in vivo model systems of pediatric brain tumors. Therefore, inhibition of their oncogenic function is an attractive therapeutic target. In this review, we explore the roles of MYC oncoproteins and their molecular targets during the formation, maintenance, and recurrence of childhood brain tumors. We also briefly summarize recent progress in the development of therapeutic approaches for pharmacological inhibition of MYC activity in these tumors.

Polyphyllin D, a steroidal saponin in Paris polyphylla, induces apoptosis and necroptosis cell death of neuroblastoma cells.

Neuroblastoma is a refractory pediatric malignant solid tumor. The previous studies demonstrated that Polyphyllin D, the main constituent of Paris polyphylla, a traditional Chinese medicine, exerts an anti-tumor effect on many tumors. However, its effects against neuroblastomas are unclear. We examined the anti-tumor effect of polyphyllin D in human neuroblastoma using IMR-32 and LA-N-2 cells, which exhibit MYCN gene amplification, and NB-69 cells, which do not exhibit MYCN gene amplification. All cell lines showed reduced cell viability in response to polyphyllin D treatment. No caspase-3/-7, -8, and -9 activity was observed in IMR-32 and LA-N-2 cells treated with polyphyllin D. In contrast, activation of caspase-3/-7, and -8 activity was observed in NB-69 cells. When polyphyllin D and specific inhibitors of RIPK3 involved in necroptosis were added to IMR-32 and LA-N-2 cell lines, polyphyllin D-induced cell death was inhibited. Together, this indicates that the underlying mechanism of polyphyllin D-induced cell death in NB-69 cells is apoptosis, whereas the cell death of IMR-32 and LA-N-2 cells occurs by necroptosis. We continue research on this topic and look forward the discovery of a new therapeutic agent for neuroblastoma.

Phosphorylation of pRb: mechanism for RB pathway inactivation in MYCN-amplified retinoblastoma.

A small, but unique subgroup of retinoblastoma has been identified with no detectable mutation in the retinoblastoma gene (RB1) and with high levels of MYCN gene amplification. This manuscript investigated alternate pathways of inactivating pRb, the encoded protein in these tumors. We analyzed the mutation status of the RB1 gene and MYCN copy number in a series of 245 unilateral retinoblastomas, and the phosphorylation status of pRb in a subset of five tumors using immunohistochemistry. There were 203 tumors with two mutations in RB1 (RB1 −/−, 83%), 29 with one (RB1 +/−, 12%) and 13 with no detectable mutations (RB1 +/+, 5%). Eighteen tumors carried MYCN amplification between 29 and 110 copies: 12 had two (RB1 −/−) or one RB1 (RB1 +/−) mutations, while six had no mutations (RB1 +/+). Immunohistochemical staining of tumor sections with antibodies against pRb and phosphorylated Rb (ppRb) displayed high levels of pRb and ppRb in both RB1 +/+ and RB1 +/− tumors with MYCN amplification compared to no expression of these proteins in a classic RB1 −/−, MYCN‐low tumor. These results establish that high MYCN amplification can be present in retinoblastoma with or without coding sequence mutations in the RB1 gene. The functional state of pRb is inferred to be inactive due to phosphorylation of pRb in the MYCN‐amplified retinoblastoma without coding sequence mutations. This makes inactivation of RB1 by gene mutation or its protein product, pRb, by protein phosphorylation, a necessary condition for initiating retinoblastoma tumorigenesis, independent of MYCN amplification.

A Myc Activity Signature Predicts Poor Clinical Outcomes in Myc-Associated Cancers.

Myc transcriptional activity is frequently deregulated in human cancers, but a Myc-driven gene signature with prognostic ability across multiple tumor types remains lacking. Here, we selected 18 Myc-regulated genes from published studies of Myc family targets in epithelial ovarian cancer (EOC) and neuroblastoma. A Myc family activity score derived from the 18 genes was correlated to MYC/MYCN/MYCL1 expression in a panel of 35 cancer cell lines. The prognostic ability of this signature was evaluated in neuroblastoma, medulloblastoma, diffuse large B-cell lymphoma (DLBCL), and EOC microarray gene expression datasets using Kaplan-Meier and multivariate Cox regression analyses and was further validated in 42 primary neuroblastomas using qPCR. Cell lines with high MYC, MYCN, and/or MYCL1 gene expression exhibited elevated expression of the signature genes. Survival analysis showed that the signature was associated with poor outcome independently of well-defined prognostic factors in neuroblastoma, breast cancer, DLBCL, and medulloblastoma. In EOC, the 18-gene Myc activity signature was capable of identifying a group of patients with poor prognosis in a "high-MYCN" molecular subtype but not in the overall cohort. The predictive ability of this signature was reproduced using qPCR analysis of an independent cohort of neuroblastomas, including a subset of tumors without MYCN amplification. These data reveal an 18-gene Myc activity signature that is highly predictive of poor prognosis in diverse Myc-associated malignancies and suggest its potential clinical application in the identification of Myc-driven tumors that might respond to Myc-targeted therapies. Cancer Res; 77(4); 971-81. ©2016 AACR.

Clinical features, treatment and prognosis of neuroblastoma with bone metastasis in children

Objective To summarize the clinical features of neuroblastoma (NB) with bone metastasis, and preliminarily to analyze the relationship between clinical features, treatment effects and prognosis of NB with bone metastasis and further studying why children with NB would have bone metastasis happened.As such, this paper was seek for more effective therapy to improve patients′survival rates. Methods The retrospective research was carried out to analyze the medical records of 130 patients with bone metastasis of NB from March 2007 to July 2013 who were diagnosed and systematically treated in Beijing Children′s Hospital(BCH) Affiliated to Capital Medical University accor-ding to BCH-NB-2007 protocol.The data were reviewed for the medical history, clinical features and laboratory examination results before treatment.The research evaluated the treatment outcome and overall survival rate of the 88 cases treated and followed up all along at BCH.The deadline for follow-up was December 31, 2014. Results One hundred and thirty children of NB with bone metastasis were included in the research.There were 87 boys and 43 girls; the age was between 8-147 months, the median age was 41 months, 94(72.3%) children′s age was less than 5 years old.The main primary site was retroperitoneal and adrenal region(106 cases, 81.5%) and mediastinum(17 cases, 13.1%). The main clinical symptoms were fever(81 cases, 62.3%), bone pain(51 cases, 39.2%), abdominal pain(29 cases, 22.3%), and abdominal mass(14 cases, 10.8%). A total of 88 cases got systemic treatment and follow-up at BCH.Only 1 infant with NB, at 8 months old with a retroperitoneal mass, with bone marrow and bone metastasis, but no MYCN gene amplification, was given carboplatin plus etoposide and cyclophosvnamide, vincristine and doxorubicin according to European neuroblastoma risk grouping for alternating 8 times, he received tumor resection after 4 courses of chemotherapy and was followed up for 20 months, and the condition was now in stable.The remaining 87 patients as high risk(HR)-NB, were treated regularly by BCH-NB-2007 protocol, with the median follow-up time of 23 months, 52 cases(59.1%) turned out to have tumor progression or relapse.Forty cases died of tumor related diseases and 1 case died of measles infection.The expected overall survival rates were 49.5% in 3 years and 41.3% in 5 years. Conclusions NB with bone metastasis was very common in children over 18 months, and their common clinical symptoms are fever, bone pain and anemia.More than 80% of primary tumor was located in retroperitoneal and adrenal gland area, most of the patients with bone marrow metastasis.Since the overall prognosis is poor in these children, continuous clinical and further study is needed to investigate the causes of bone metastasis in children with NB, and to provide a basis for more effective treatment. Key words: Neuroblastoma; Bone metastasis; Clinical features; Prognosis

CD133 and MYCN Amplification Induce Chemo-Resistance and Reduce Average Survival Time in Pediatric Neuroblastoma

Objectives: Neuroblastoma (NB) is the most common pediatric solid tumor derived from the sympathetic nervous system. MYCN gene exists in nearly half of the NB patient and its association with rapid disease progression and poor outcome is controversial. Cancer Stem Cells (CSCs) characterization in NB has been rarely studied. This study is to figure out whether the MYCN gene and CSCs are associated with chemotherapy resistance and survival time in the NB patients. Methods: Based on unequivocal pathological diagnosis, 50 NB patients are recruited. MYSN amplification is measured before any therapy. The CSCs are derived and their multi-potencies are tested by directed differentiation. Response to chemotherapy and average survival time of these patients are gathered and compared with following groups: CD133+, CD133-, MYCN ≥ 5, MYCN<5, CD133+ plus MYCN ≥ 5, CD133- plus MYCN<5. Results: CD133+ CSCs differentiate into neuron like cells; CD133+ patients have significant poorer response to chemotherapy comparing with the CD133- patients (P<0.01); CD133+ and MYCN ≥ 5 patients have significantly shorter average survival time than the CD133- and MYCN<5 patients (P<0.01). Conclusions: CD133+ CSCs produce chemo-resistance. CD133 and MYCN amplification can be used together as a prognostic value for predicting disease outcome.

培養神経芽腫細胞 TGW における癌遺伝子 N-myc の Amplification と Rearrangement

培養神経芽腫細胞 TGW における癌遺伝子 N-myc の Amplification と Rearrangement

Results of neuroblastoma treatment in Lithuania: a single centre experience.

Background.Neuroblastoma (NB) is the most common extracranial solid tumour in children. This is a very rare disease with heterogeneous biology varying from complete spontaneous regression to a highly aggressive tumour responsible for 15% of malignancy-related death in early childhood. Analyses of survival rates in Europe have shown a considerable difference between Northern/Western and Eastern European countries. Treatment results of NB in Lithuania have never been analyzed.Aim.To assess the survival rate of children with NB according to initial spread of the disease, age at diagnosis, the MYCN amplification, risk group, and treatment period.Patients and methods.A retrospective single-centre analysis of patients’ records was performed. Children diagnosed and treated for NB between 2000 and 2015 at the Centre of Paediatric Oncology and Haematology of the Children’s Hospital, Affiliate of Vilnius University Hospital Santaros Klinikos were included. The patients were divided into three groups according to the spread of the disease: group 1 – patients with local NB older than 12 years of age; group 2 – stage IV patients, also called the M stage; group 3 – infants with stages 4S and MS. The patients were stratified into three risk groups – low, intermediate and high risk. Estimates of five-year overall survival (OS5y) were calculated using the Kaplan-Meier method comparing survival probability according to spread of the disease, age at diagnosis, the MYCN amplification, risk group and treatment period (2000–2007 vs 2008–2015).Results.Overall 60 children (31 girls and 29 boys) with NB were included. The median age at diagnosis was 1.87 years (ranged from 4 days to 15 years). Seventy-eight percent of cases were found to be differentiated or undifferentiated NB, 22% – ganglioneuroblastoma. The local form of the disease was predominant: 57% (34/60) of patients were allocated to the group 1, 37% (22/60) with initial metastatic disease were assigned to group 2, and infants with 4S or MS stage comprising 7% (4/60) allocated to group 3, respectively. The probability of OS5y for the entire cohort was 71% with the median follow-up of 8.8 ± 4.8 years. The probability of OS5y for local disease (group 1) was significantly higher compared to metastatic disease (group 2) (94% vs. 34%, p = 0.001, respectively) as well as for infants compared to children older than 12 months at the time of diagnosis (90% vs 60%, p = 0.009, respectively). The MYCN gene amplification had a negative influence on OS5y, with 78% of MYCN-negative patients surviving in comparison to 40% of MYCN-positive patients who did not survive (p = 0.153). The high-risk patients had significantly worse OS5y than children with intermediated or low risk (35% vs. 82% vs. 100%, respectively, p = 0.001). Comparison of OS5y between two treatment periods in the entire patient population revealed a non-significant increase in survival from 66% in the 2000–2007 period to 82% in the 2008–2015 period (p = 0.291), mostly due to a dramatic improvement achieved for high-risk patients whose survival rate increased from 9% in the 2000–2007 period to 70% in the 2008–2015 period (p = 0.009).Conclusions.There was a slight predominance of low-risk patients, probably due to a higher number of infants. A better probability of OS5y was confirmed in infants with local disease and in MYCN-negative patients. The OS5y for children treated for NB at our institution over 16 years increased from 66% in the 2000–2007 period to 82% in the 2008–2015 period with the most significant improvement achieved for high risk patients. The current survival rate of children treated for NB at our institution is in line with the reported numbers in Northern and Western European countries.

A sketch of known and novel MYCN-associated miRNA networks in neuroblastoma.

Neuroblastoma (NB) originates from neural crest-derived precursors and represents the most common childhood extracranial solid tumour. MicroRNAs (miRNAs), a class of small non-coding RNAs that participate in a wide variety of biological processes by regulating gene expression, appear to play an essential role within the NB context. High-throughput next generation sequencing (NGS) was applied to study the miRNA transcriptome in a cohort of NB tumours with and without MYCN-amplification (MNA and MNnA, respectively) and in dorsal root ganglia (DRG), as a control. Out of the 128 miRNAs differentially expressed in the NB vs. DRG comparison, 47 were expressed at higher levels, while 81 were expressed at lower levels in the NB tumours. We also found that 23 miRNAs were differentially expressed in NB with or without MYCN-amplification, with 17 miRNAs being upregulated and 6 being downregulated in the MNA subtypes. Functional annotation analysis of the target genes of these differentially expressed miRNAs demonstrated that many mRNAs were involved in cancer-related pathways, such as DNA-repair and apoptosis as well as FGFR and EGFR signalling. In particular, we found that miR-628-3p negatively affects MYCN gene expression. Furthermore, we identified a novel miRNA candidate with variable expression in MNA vs. MNnA tumours, whose putative target genes are implicated in the mTOR pathway. The present study provides further insight into the molecular mechanisms that correlate miRNA dysregulation to NB development and progression.

Upregulation of N - myc downstram regulated gene 1 expression by silencing DNA methyltransferases influences the biological behaviors of prostate cancer DU145 cells

Objective To investigate the influence of the upregulation of N-myc downstram regulated gene 1 (NDRG1) expression by silencing DNA methyltransferases (DNMTs) using small interfering RNAs (siRNAs) on the biological behaviors of prostate cancer DU145 cells. Methods SiRNAs were used to inhibit the expression of DNMT1, DNMT3b of DU145 cells; Western blotting and quantitative real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) were used to detect the expression levels of DNMT1, DNMT3b and NDRG1 of DU145 cells; cell counting kit-8 (CCK-8) assay, muse cell analyzer, wound healing assay and Transwell migration assay were used to detect the proliferation, apoptosis, migration and invasion of DU145 cells; then we inhibited the expression of NDRG1 of DU145 cells which had the most significant changes of cell biological behaviors; and we detected the expression level of NDRG1 and the changes of cell biological behaviors. Results Western blotting and RT-qPCR showed that the mRNA (1.59±0.03, 1.23±0.01, 1.36±0.11 vs.0.90±0.15, 1.00±0.03) and protein (1.07±0.16, 0.49±0.01, 0.92±0.09 vs.0.23±0.04, 0.30±0.07) expression levels of NDRG1 of DNMT1 inhibition group, DNMT3b inhibition group and double inhibition group were significantly higher than these in control groups (P<0.05). CCK-8 assay showed that, at 24, 48 and 72 h, the optical density (A) values of DNMT1 inhibition group (0.189±0.030, 0.266±0.048, 0.419±0.058), DNMT3b inhibition group (0.221±0.023, 0.318±0.072, 0.498±0.064) and double inhibition group (0.199±0.021, 0.284±0.062, 0.488±0.091) were significantly lower than control groups (P<0.05). Apoptosis results showed that the apoptosis rates of DNMT1 inhibition group (22.40±1.36)%, DNMT3b inhibition group (14.20±0.80)% and double inhibition group (17.60±0.74)% were significantly higher than control groups (P<0.05). Wound healing assay showed that the healing rates of DNMT1 inhibition group (53.01±3.22)%, DNMT3b inhibition group (70.97±2.12)% and double inhibition group (63.93±4.42)% were significantly lower than control groups (P<0.05). Transwell migration assay showed that the amount of invasion cells in DNMT1 inhibition group [(16±3) cells], in DNMT3b inhibition group [(32±5) cells] and in double inhibition group [(20±4) cells] were significantly less than control groups (P<0.05). Among these groups, DNMT1 inhibition group had the most significant effect (P<0.05), but the co-transfection had no significant synergistic effect. Compared with control groups, NDRG1-siRNA could significantly reverse the changes of cell biological behaviors of DU145 cells (P<0.05). Conclusion Inhibition of DNMT1, DNMT3b of DU145 cells could partially restore NDRG1 expression, which could inhibit the proliferation, apoptosis, migration and invasion of cancer cells. Moreover, DNMT1 inhibition group had the most significant effect, but the co-transfection had no significant synergistic effect, suggesting that DNMT1 could become an effective target for demethylation treatment of prostate cancer. Key words: N-myc downstram regulated gene 1; DNA methylation; DNA methyltransferase; RNA interference; Prostate cancer

Uncovering Clonal and Subclonal Druggable Targets in Multiple Myeloma Using Omic Data

The Multiple Myeloma Research Foundation (MMRF) CoMMpass trial is the keystone program working towards personalized medicine in multiple myeloma (MM). CoMMpass has characterized over 730 samples from 669 patients, a subset having time-course data, using multiple platforms and has made these data publicly available. Screening for druggable somatic mutations and gene expression outliers revealed great potential for repurposing existing pharmacotherapies.We have developed and curated a Database of Evidence for Precision Oncology (DEPO) that includes 442 mutations and 49 genes with expression changes implicated as drug targets from 34 cancer types. We found 43.6% of CoMMpass samples have one of 26 somatic mutations that could be targeted by 9 different drugs, suggesting many drugs may be repurposed for use in MM. HotSpot3D, a protein-structure-guided analysis tool, showed 3.3% (24/730) of samples have mutations involving BRAF and KRAS, clustering with known drug targets, and another 2.1%(15/730) have mutations in clusters formed from a prior TCGA pan-cancer analysis involving 22 cancer types. This indicates additional potential drug targets and functional mutations in MM.Interestingly, 11 samples (including relapse), have subclonal mutations in both KRAS and BRAF with variable allelic fractions, implicating different treatment requirements for tumor subpopulations and disease stages. Additionally, druggable gene expression outlier analysis of 591 samples reveals an average of nearly 5 outlier genes per sample from among 49 known target genes, with 18.1% (107/591) of samples with MYCN gene outliers as compared to MYC 1.4% (8/591), despite MYC being a known driver of MM. Other top outliers are DLL3 10.2%, EGFR 10.5%, FGFR1 10.2%, and FGFR2 12.5%. Our study highlights candidate drug targets previously omitted in MM. Taken together, it suggests that heterogeneity analysis and pharmaceutical repurposing could lead to substantially improved outcomes. DisclosuresNo relevant conflicts of interest to declare.

The Molecular Genetics of Retinoblastoma

Retinoblastoma is a rare, malignant, childhood tumor that is primarily initiated by the inactivation of both alleles of the retinoblastoma tumor susceptibility gene, RB1, in a developing human retinal cell. A rare subset of retinoblastoma is initiated by somatic amplification of the MYCN oncogene in a predisposing retinal cell. Surprisingly the retinoblastoma protein (pRB), encoded by RB1, is an important transcription factor. Cell-cycle control by pRB is mainly accomplished by transcriptional repression of the genes required for cell-cycle progression. Control of differentiation by pRB is achieved by the activation of transcription. Through extensive post-translational modifications and interactions with other proteins, pRB and family members also influence senescence, chromosomal stability, and apoptosis. Almost every type of tumor has disruption in the retinoblastoma pathway associated with tumor progression, but germline mutation of the RB1 gene predisposes children to a 95% specific risk of developing retinoblastoma and a significantly increased risk of second primary tumors, such as osteosarcoma and melanoma. However, retinoblastoma is also characterized by other genomic changes subsequent to RB1 mutation. Keywords: aneuploidy; apoptosis; chromosome instability (CIN); E2F ; LOH ; MYCN ; proband; RB1 ; retinoblast; retinoma; SV40 ; TAg ; tumor suppressor

Constitutional 560.49 kb chromosome 2p24.3 duplication including the MYCN gene identified by SNP chromosome microarray analysis in a child with multiple congenital anomalies and bilateral Wilms tumor

Fewer than 100 patients with partial chromosome 2p trisomy have been reported. Clinical features are variable and depend on the size of the duplicated segment, but generally include psychomotor delay, facial anomalies, congenital heart defect, and other abnormalities. We report a 560.49 kb duplication of chromosome 2p in a 13 month-old male with hydrocephaly, ventricular septal defect, partial agenesis of the corpus callosum, and bilateral Wilms tumor. After discovery of bilateral renal masses at four months of age, the child underwent neoadjuvant chemotherapy followed by right radical nephrectomy that revealed triphasic Wilms' tumor. A needle core biopsy on one of two lesions on the left kidney also revealed Wilms tumor. A partial left nephrectomy revealed focally positive margins that necessitated left flank radiotherapy. The tumor karyotype was 46,XY,t(7;8)(q36;p11)[8]/46,XY [12] while his constitutional karyotype was 46,XY, suggesting that the t(7;8)(q36;p11) was associated with the malignancy. Single nucleotide polymorphism (SNP) chromosome microarray analysis of peripheral blood identified a maternally-inherited 560.49 kb chromosome 2p24.3 duplication that involved four OMIM genes: NBAS, DDX1, MYCNOS, and MYCN. SNP array analysis of the tumor revealed the same 2p24.3 duplication. At present, the now 5-year-old boy continues to do well without clinical or radiographic evidence of recurrent disease. This case is instructive because the child's health insurer initially denied authorization for chromosome microarray analysis (CMA), and it took more than one year before such authorization was finally granted. That initial decision to deny coverage could have had untoward health implications for this child, as the identification of constitutional MYCN duplication necessitated surveillance imaging for a number of pediatric malignancies associated with MYCN overexpression/dysregulation.

NCYM is upregulated by lncUSMycN and modulates N-Myc expression.

Neuroblastoma is the most common solid tumor in early childhood. Patients with neuroblastoma due to the amplification of a 130-kb genomic DNA region containing the MYCN, MYCN antisense NCYM and lncUSMycN genes show poor prognosis. BET bromodomain inhibitors show anticancer efficacy against neuroblastoma partly by reducing MYCN gene transcription and N-Myc mRNA and protein expression. We have previously shown that the long nocoding RNA lncUSMycN upregulates N-Myc mRNA expression by binding to the RNA-binding protein NonO. In this study, we found that lncUSMycN upregulated NCYM expression, and knocking-down lncUSMycN reduced histone H3 lysine 4 trimethylation, a marker for active gene transcription, at the NCYM gene promoter. NCYM upregulated N-Myc mRNA expression, NCYM RNA formed a complex with NonO protein, and knocking down NCYM expression reduced neuroblastoma cell proliferation. Importantly, treatment with BET bromodomain inhibitors reduced NCYM expression. In human neuroblastoma patients, high levels of NCYM expression in tumor tissues correlated with high levels of N-Myc, NonO and lncUSMycN expression as well as poor patient prognosis. Taken together, our findings suggest that lncUSMycN upregulates NCYM expression by activating its gene transcription, and that NCYM RNA upregulates N-Myc mRNA expression by binding to NonO. Our findings also provide further evidence for the application of BET bromodomain inhibitors for the therapy of neuroblastoma characterized by MYCN/NCYM gene locus amplification.