MYCN Research Articles

Targeting of MYCN by means of DNA vaccination is effective against neuroblastoma in mice.

The MYCN oncogene is a strong genetic marker associated with poor prognosis in neuroblastoma (NB). Therefore, MYCN gene amplification and subsequent overexpression provide a possible target for new treatment approaches in NB. We first identified an inverse correlation of MYCN expression with CD45 mRNA in 101 NB tumor samples. KEGG mapping further revealed that MYCN expression was associated with immune-suppressive pathways characterized by a down-regulation of T cell activation and up-regulation of T cell inhibitory gene transcripts. We then aimed to investigate whether DNA vaccination against MYCN is effective to induce an antigen-specific and T cell-mediated immune response. For this purpose, we generated a MYCN-expressing syngeneic mouse model by MYCN gene transfer to NXS2 cells. MYCN-DNA vaccines were engineered based on the pCMV-F3Ub plasmid backbone to drive ubiquitinated full-length MYCN-cDNA and minigene expression. Vaccines were delivered orally with attenuated S. typhimurium strain SL7207 as a carrier. Immunization with both MYCN-DNA vaccines significantly reduced primary tumor growth of MYCN-expressing NB cells in contrast to negative controls. The immune response was mediated by tumor-infiltrating T cells in vivo, which revealed MYCN-specific and MHC class I-restricted lysis of inducible MYCN-expressing NB target cells in vitro. Finally, these antigen-specific T cells also killed MYCN-negative mammary carcinoma cells pulsed with MYCN peptides in contrast to controls. In summary, we demonstrate proof of concept that MYCN can be targeted by DNA vaccination, which may provide an approach to overcoming MYCN immune-suppressive activities in patients with MYCN-amplified disease.

NDRG1 suppresses TGF-β-induced epithelial-mesenchymal transition in lung cancer A549 cells

Objective To explore the effect and molecular mechanism of N-myc downstream regulated gene 1 (NDRG1) on transforming growth factor-beta (TGF-β)-induced epithelial-mesenchymal transition (EMT) in human lung cancer cells. Methods Lung cancer A549 cells were transfected with NDRG1 overexpressed vector, and then treated with 5 μg/L TGF-β1. The abilities of invasion were detected by Transwell assay. The expressions of NDRG1 mRNA and protein were analyzed by teal-time reverse transcription polymerase chain reaction (RT-PCR) were examined with Western blot. The expressions of EMT-associated markers E-cadherin and Vimentin, and EMT-associated signaling molecules Snail, AKT and Smad were detected with Western blot. Results We found that TGF-β1 treatment could induce morphological alteration of A549 cells from epithelial morphology to mesenchymal morphology. TGF-β1 significantly increased the migration of A549 cells, and increased the expression of mesenchymal maker vimentin and decreased epithelial marker E-cadherin. More importantly, overexpression of NDRG1 significantly reversed the effects of TGF-β1 on A549 cells. Moreover, NDRG1 significantly decreased the levels of phospho-AKT, and suppressed the expression of EMT-related transcription factor Snail. Conclusions NDRG1 could reverse the effects of TGF-β1 on EMT in A549 cells, by which mechanism is related to reduction of the expressions of phospho-AKT and Snail. Key words: Intercellular signaling peptides and proteins/PD/ME; Transforming growth factor beta/AE; Lung neoplasms/PA; Cell transformation, neoplastic/DE; Epithelial cells/DE/PA; Stromal cells/DE/PA

Functional interplay between MYCN, NCYM, and OCT4 promotes aggressiveness of human neuroblastomas

Neuroblastoma is a pediatric solid tumor that originates from embryonic neural crest cells. The MYCN gene locus is frequently amplified in unfavorable neuroblastomas, and the gene product promotes the progression of neuroblastomas. However, the molecular mechanisms by which MYCN amplification contributes to stem cell-like states of neuroblastoma remain elusive. In this study, we show that MYCN and its cis-antisense gene, NCYM, form a positive feedback loop with OCT4, a core regulatory gene maintaining a multipotent state of neural stem cells. We previously reported that NCYM is co-amplified with the MYCN gene in primary human neuroblastomas and that the gene product promotes aggressiveness of neuroblastoma by stabilization of MYCN. In 36 MYCN-amplified primary human neuroblastomas, OCT4 mRNA expression was associated with unfavorable prognosis and was correlated with that of NCYM. The OCT4 protein induced both NCYM and MYCN in human neuroblastoma cells, whereas NCYM stabilized MYCN to induce OCT4 and stem cell-related genes, including NANOG, SOX2, and LIN28. In sharp contrast to MYCN, enforced expression of c-MYC did not enhance OCT4 expression in human neuroblastoma cells. All-trans retinoic acid treatment reduced MYCN, NCYM, and OCT4 expression, accompanied by the decreased amount of OCT4 recruited onto the intron 1 region of MYCN. Knockdown of NCYM or OCT4 inhibited formation of spheres of neuroblastoma cells and promoted asymmetric cell division in MYCN-amplified human neuroblastoma cells. These results suggest that the functional interplay between MYCN, NCYM, and OCT4 contributes to aggressiveness of MYCN-amplified human neuroblastomas.

Editorial: Targeting MYCN in Pediatric Cancers.

MYCN is the product of a gene frequently deregulated in childhood tumors that belongs to a small but very famous family of transcription factors whose prototype member is c-MYC. The other member of the family is L-MYC, identified as a gene amplified in a subset of lung cancers. c-MYC is widely expressed in normal tissues, is the most deregulated protooncogene in human cancer, and not surprisingly, it is the subject of intensive investigations in many laboratories worldwide. Clearly, decoding its function in tumorigenesis and finding ways of inhibiting its oncogenic activity would have a very large impact in terms of human health. In contrast to the broad significance of c-MYC, the expression of MYCN is temporally and spatially restricted during embryonal development, being detected mostly in cells of the developing nervous system. This more limited function is also reflected in human pathology, with only a few types of tumors presenting alterations of MYCN. These cancers arise in the nervous system, both central and peripheral, manifesting as medulloblastomas, gliomas, and neuroblastomas. Despite the fact that MYCN-positive tumors are relatively rare, their very aggressive nature and the pediatric setting make therapeutic treatments a clinical challenge.

Molecular and in vivo characterization of cancer-propagating cells derived from MYCN-dependent medulloblastoma.

Medulloblastoma (MB) is the most common malignant pediatric brain tumor. While the pathways that are deregulated in MB remain to be fully characterized, amplification and/or overexpression of the MYCN gene, which is has a critical role in cerebellar development as a regulator of neural progenitor cell fate, has been identified in several MB subgroups. Phenotypically, aberrant expression of MYCN is associated with the large-cell/anaplastic MB variant, which accounts for 5-15% of cases and is associated with aggressive disease and poor clinical outcome. To better understand the role of MYCN in MB in vitro and in vivo and to aid the development of MYCN-targeted therapeutics we established tumor-derived neurosphere cell lines from the GTML (Glt1-tTA/TRE-MYCN-Luc) genetically engineered mouse model. A fraction of GTML neurospheres were found to be growth factor independent, expressed CD133 (a marker of neural stem cells), failed to differentiate upon MYCN withdrawal and were highly tumorigenic when orthotopically implanted into the cerebellum. Principal component analyzes using single cell RNA assay data suggested that the clinical candidate aurora-A kinase inhibitor MLN8237 converts GTML neurospheres to resemble non-MYCN expressors. Correlating with this, MLN8237 significantly extended the survival of mice bearing GTML MB allografts. In summary, our results demonstrate that MYCN plays a critical role in expansion and survival of aggressive MB-propagating cells, and establish GTML neurospheres as an important resource for the development of novel therapeutic strategies.

IGF2BP1 harbors prognostic significance by gene gain and diverse expression in neuroblastoma.

Chromosomal 17q21-ter gain in neuroblastoma is both a common and prognostically significant event. The insulin-like growth factor-2 mRNA-binding protein 1 (IGF2BP1) gene is located near the proximal edge of this region. Here, its prognostic value is evaluated in neuroblastoma. The mRNA expression of IGF2BP family members was first evaluated by microarray data sets. In addition, in a separate cohort of 69 tumors, IGF2BP1 gene copy number, mRNA, and protein abundance were determined and compared with clinical parameters. In two independent microarray data sets, 77% to 100% of tumors had substantial IGF2BP1 mRNA levels measured. High IGF2BP1 transcript abundance was significantly associated with stage 4 tumors (P < .001) and decreased patient survival (P < .001). IGF2BP1 was also associated with MYCN gene amplification and MYCN mRNA abundance. In the 69 neuroblastoma samples, IGF2BP1 DNA copy number (increased in 84% of tumors), mRNA, and protein abundance were significantly higher in stage 4 compared with stage 1 tumors. Importantly, IGF2BP1 protein levels were associated with lower overall patient survival (P = .012) and positively correlated with MYCN mRNA, even when excluding MYCN-amplified tumors. Moreover, IGF2BP1 clearly affected MYCN expression and neuroblastoma cell survival in vitro. In neuroblastoma, IGF2BP1 was expressed in the majority of neuroblastoma specimens analyzed and was associated with lower overall patient survival and MYCN abundance. These data demonstrate that IGF2BP1 is a potential oncogene and an independent negative prognostic factor in neuroblastoma.

Oncolytic adenovirus-mediated short hairpin RNA targeting MYCN gene induces apoptosis by upregulating RKIP in neuroblastoma.

The amplification of MYCN is a typical characteristic of aggressive neuroblastomas, whereas acquired mutations of p53 lead to refractory and relapsed cases. We had previously examined the applicability of the replication-competent oncolytic adenovirus, ZD55-shMYCN, to deliver a short hairpin RNA targeting MYCN gene for p53-null and MYCN-amplified neuroblastoma cell line LA1-55N. Our data have shown that ZD55-shMYCN has an additive tumor growth inhibitory response through shRNA-mediated MYCN knockdown and ZD55-mediated cancer cell lysis. In this regard, ZD55-shMYCN can downregulate MYCN and perform anticancer effects, thereby acquiring significance in the administration of MYCN-amplified and p53-null neuroblastomas. Hence, we further investigated the anticancer properties of ZD55-shMYCN in neuroblastomas. Our data showed that ZD55-shMYCN induced G2/M arrest via decreasing the levels of cyclin D1 and cyclin B1 irrespective of p53 status. ZD55-shMYCN effectively induced apoptosis in neuroblastomas through activation of caspase-3 and enhancing PARP cleavage. Furthermore, ZD55-shMYCN could downregulate phosphoinositide 3-kinase and pAkt and upregulate RKIP levels. Similarly, pro-apoptosis was revealed by the histopathologic examination of paraffin-embedded section of resected tumors of mice xenograft. In vitro and in vivo studies, we elucidate the apoptosis properties and mechanisms of action of ZD55-shMYCN, which provide a promising approach for further clinical development.

Prognostic significance of MYCN amplification in children neuroblastic tumors

To summarize the clinicopathologic features of neuroblastic tumors (NT), and to explore the prognostic significance of MYCN amplification in NT. The clinicopathologic data of 267 NT were reviewed. MYCN gene amplification was detected by fluorescence in situ hybridization (FISH) in 119 cases and the relationship with pathological characteristics and prognostic significance were analyzed. The study included 267 cases of children NT from patients aged from 1 day to 13 years (median 27 months). The male to female ratio was 1.43. There were 38 cases (14.2%), 43 cases (16.1%), 71 cases (26.6%), and 115 cases (43.1%) of INSS stages I, II, III and IV respectively.Favorable histology group had 157 cases (59.9%); unfavorable histology group had 110 cases (40.1%).Of the 119 NT cases with MYCN FISH performed, 18 cases (15.1%) showed amplification and the signal ratio of MYCN to CEP2 was 4.08-43.29. One hundred and one cases of non-amplified MYCN included MYCN gain in 79 cases (66.3%) and MYCN negative in 22 cases (18.5%). MYCN expression showed significant difference (P = 0.000) between ages, gender, NT type and MKI, but not INPC and clinical stage (P > 0.05).Of the 18 cases with MYCN amplification, 3 were undifferentiated, and 15 poorly differentiated; 17 had high MKI and one moderate MKI. All 18 cases were in unfavorable histology group; the overall survival rate was 3/18, with an average survival time of (17.9 ± 2.4) months.Of the 101 MYCN non-amplification cases, the overall survival rate was 68.3% (69/101), with an average survival time of (29.8 ± 1.3) months. Survival analysis showed the cases with MYCN amplification had worse prognosis (P < 0.05). NT were commonly diagnosed in early ages and easily to metastasize. Most of cases with favorable histology. The cases of MYCN amplification showed unfavorable histology, and the majority cases with high MKI; The patients with MYCN gene amplification had poor prognosis.

Mxi1 and Mxi1-0 Antagonize N-Myc Function and Independently Mediate Apoptosis in Neuroblastoma

Neuroblastoma (NB) is the third most common malignancy of childhood, and outcomes for children with advanced disease remain poor; amplification of the MYCN gene portends a particularly poor prognosis. Mxi1 antagonizes N-Myc by competing for binding to Max and E-boxes. Unlike N-Myc, Mxi1 mediates transcriptional repression and suppresses cell proliferation. Mxi1 and Mxi1-0 (an alternatively transcribed Mxi1 isoform) share identical Max and DNA binding domains but differ in amino-terminal sequences. Because of the conservation of these critical binding domains, we hypothesized that Mxi1-0 antagonizes N-Myc activity similar to Mxi1. SHEP NB cells and SHEP cells stably transfected with MYCN (SHEP/MYCN) were transiently transfected with vectors containing full-length Mxi1, full-length Mxi1-0, or the common Mxi domain encoded by exons 2 to 6 (ex2-6). After incubation in low serum, parental SHEP/MYCN cell numbers were reduced compared with SHEP cells. Activated caspase-3 staining and DNA fragmentation ELISA confirmed that SHEP/MYCN cells undergo apoptosis in low serum, while SHEP/MYCN cells transfected with Mxi1 or Mxi1-0 do not. However, SHEP/MYCN cells transfected with Mxi1 or Mxi1-0 and grown in normal serum showed proliferation rates similar to SHEP cells. Mxi ex2-6 did not affect cell number in low or normal serum, suggesting that amino terminal domains of Mxi1 and Mxi1-0 are critical for antagonism. In the absence of N-Myc, Mxi1 and Mxi1-0 induce apoptosis independently through the caspase-8–dependent extrinsic pathway, while N-Myc activates the caspase-9–dependent intrinsic pathway. Together, these data indicate that Mxi1 and Mxi1-0 antagonize N-Myc but also independently impact NB cell survival.

Multiple mechanisms of MYCN dysregulation in Wilms tumour.

Genomic gain of the proto-oncogene transcription factor gene MYCN is associated with poor prognosis in several childhood cancers. Here we present a comprehensive copy number analysis of MYCN in Wilms tumour (WT), demonstrating that gain of this gene is associated with anaplasia and with poorer relapse-free and overall survival, independent of histology. Using whole exome and gene-specific sequencing, together with methylation and expression profiling, we show that MYCN is targeted by other mechanisms, including a recurrent somatic mutation, P44L, and specific DNA hypomethylation events associated with MYCN overexpression in tumours with high risk histologies. We describe parallel evolution of genomic copy number gain and point mutation of MYCN in the contralateral tumours of a remarkable bilateral case in which independent contralateral mutations of TP53 also evolve over time. We report a second bilateral case in which MYCN gain is a germline aberration. Our results suggest a significant role for MYCN dysregulation in the molecular biology of Wilms tumour. We conclude that MYCN gain is prognostically significant, and suggest that the novel P44L somatic variant is likely to be an activating mutation.

PD-L1 expression in metastatic neuroblastoma as an additional mechanism for limiting immune surveillance

Neuroblastomas (NB) are neuroectodermal tumors that account for 15% of all childhood cancer deaths. Different prognostic factors including age, stage and amplification of MYCN ( MYCN ampl ), are critical for defining NB risk and for guiding therapeutic choices 1 . In this context, although different strategies have been approached, the prognosis of high-risk neuroblastoma (NB) remains poor due the high frequency of relapse which not only occurs at the primary tumor site but frequently arises in the bone marrow. Thus, in vivo, neuroblastoma exploits different mechanisms to escape the immune surveillance 2 . These might include the so called “adaptive immune resistance” characterized by the de-novo expression of Programmed Death Ligands (PD-Ls) 3 induced by IFN-γ whose storm occurs after different immunotherapeutic approaches. In the present study we analyzed the constitutive and the inducible surface expression of PD-Ls in NB cells. We showed that PD-L1 is constitutively express by virtually all HLA class I pos NB cell lines, whereas PD-L2 is rarely detected. Moreover PD-L1 could be acquired/upregulated in both NB cell lines and NB engrafted nude/nude mice. Importantly, after IFN-γ stimulation, PD-L1 can be expressed by metastatic neuroblasts isolated from bone marrow aspirates of high-risk NB patients, characterized by different MYCN amplification status. Interesting - ly, in one case, metastatic neuroblasts were poorly responsive to IFN-γ stimulation, suggesting that responsiveness to IFN-γ might represent a further element of heterogeneity in metastatic neuroblasts. Finally, we documented the presence of PD-1-positive lymphocytes in NB-infiltrated bone marrow of patients. These cells are mainly represented by aβ T cells but also include small populations of gδ T cells and NK cells. Moreover, PD-1 pos T cells expressed higher levels of activation markers as com- pared to the negative counterpart. Overall our data show that a PD-L1-mediated immune resistance mechanism might occur in metastatic neuroblasts and provide a biological rationale for blocking the PD-1/PD-Ls axis in future combined immunotherapeutic approaches.

The efficacy of delayed surgery in children with high-risk neuroblastoma.

Surgery is an important part of treatment in children with neuroblastoma; however, exact timing is unclear. Both initial and delayed surgery was suggested as the best by numerous studies. Thus, we aimed to investigate the role of delayed surgery on 31 children with high-risk neuroblastoma. Thirty-one children with high-risk neuroblastoma were enrolled into the study. Statistical analysis was performed using Statistical Package for the Social Sciences (SPSS) for windows 10.0. There 'were 15 male and 16 female patients with a median age of 3.0 ± 3.2 years. Primary tumor site was adrenal in 27, non-adrenal in two, pelvic in one, and mediastinal in one patient. MYCN gene was amplified in four and non-amplified in 11 children on totally 15 children with available data. Lactate dehydrogenase was elevated in 30 children. The tumor volumes at diagnosis and before surgery in the whole group were 154.3 and 12.5 mL, respectively. The decline in tumor volume was statistically significant (P < 0.0001). Initial surgery was performed in three and delayed in 20 children, and eight children were inoperable. Surgical complication rate was 66.6% (two out of three patients) in initial surgery group; however, the rate was 15% (3 out of 20 patients) in delayed surgery group. The 5-year event-free survival and overall survival rates in the whole group were 44.8% and 50.8%, respectively. Primary tumor area control rate was 95% In conclusion, the delayed surgery with intensive chemotherapy and radiotherapy has been successful for primary control in high-risk neuroblastoma patients.

Frequency of the ALK gene and its prognostic value in neuroblastoma by FISH

Introduction The anaplastic lymphoma kinase (ALK) gene has been identified as a major neuroblastoma (NB) predisposition gene. Furthermore, there are controversies on the correlation between ALK gene aberration and clinical outcome in neuroblastoma (NBL). Materials and methods We evaluated N-MYC/ALK gene copy number by fluorescence in situ hybridization and analyzed 32 bone marrow samples infiltrated by NB and analyzed their association with the clinical outcome of the patients. Results Although an increase in ALK gene aberration is a recurrent genetic abnormality of NB (50%, 16/32), ALK amplification was only present in one NB (3.2%, 1/32). In addition, ALK positivity also significantly correlates with N-MYC gene copy number ( P P Conclusion ALK aberrations in NB were correlated with advanced tumor types and an increase in both N-MYC/ALK gene aberrations. ALK aberrations predict an inferior prognosis, which can be used as a prognostic factor in NB in clinical practice.

Identification and Pharmacological Inactivation of the MYCN Gene Network as a Therapeutic Strategy for Neuroblastic Tumor Cells

The MYC family of transcription factors consists of three well characterized members, c-MYC, L-MYC, and MYCN, deregulated in the majority of human cancers. In neuronal tumors such as neuroblastoma, MYCN is frequently activated by gene amplification, and reducing its expression by RNA interference has been shown to promote growth arrest and apoptosis of tumor cells. From a clinical perspective, RNA interference is not yet a viable option, and small molecule inhibitors of transcription factors are difficult to develop. We therefore planned to identify, at the global level, the genes interacting functionally with MYCN required to promote fitness of tumor cells facing oncogenic stress. To find genes whose inactivation is synthetically lethal to MYCN, we implemented a genome-wide approach in which we carried out a drop-out shRNA screen using a whole genome library that was delivered into isogenic neuroblastoma cell lines expressing or not expressing MYCN. After the screen, we selected for in-depth analysis four shRNAs targeting AHCY, BLM, PKMYT1, and CKS1B. These genes were chosen because they are directly regulated by MYC proteins, associated with poor prognosis of neuroblastoma patients, and inhibited by small molecule compounds. Mechanistically, we found that BLM and PKMYT1 are required to limit oncogenic stress and promote stabilization of the MYCN protein. Cocktails of small molecule inhibitors of CKS1B, AHCY, BLM, and PKMYT1 profoundly affected the growth of all neuroblastoma cell lines but selectively caused death of MYCN-amplified cells. Our findings suggest that drugging the MYCN network is a promising avenue for the treatment of high risk, neuroblastic cancers.

Analysis of genes involved in response to doxorubicin and a GD2 ganglioside-specific 14G2a monoclonal antibody in IMR-32 human neuroblastoma cells.

Neuroblastoma is the most common extra-cranial solid tumor of childhood and it is characterized by the presence of a glycosphingolipid, GD2 ganglioside. Monoclonal antibodies targeting the antigen are currently tested in clinical trials. Additionally, several research groups reported results revealing that ganglioside-specific antibodies can affect cellular signaling and cause direct cytotoxicity against tumor cells. To shed more light on gene expression signatures of tumor cells, we used microarrays to analyze changes of transcriptome in IMR-32 human neuroblastoma cell cultures treated with doxorubicin (DOX) or a mouse monoclonal antibody binding to GD2 ganglioside 14G2a (mAb) for 24 h. The obtained results highlight that disparate cellular pathways are regulated by doxorubicin and 14G2a. Next, we used RT-PCR to verify mRNA levels of selected DOX-responsive genes such as RPS27L, PPM1D, SESN1, CDKN1A, TNFSF10B, and 14G2a-responsive genes such as SVIL, JUN, RASSF6, TLX2, ID1. Then, we applied western blot and analyzed levels of RPS27L, PPM1D, sestrin 1 proteins after DOX-treatment. Additionally, we aimed to measure effects of doxorubicin and topotecan (TPT) and 14G2a on expression of a novel human NDUFAF2 gene encoding for mimitin protein (MYC-induced mitochondrial protein) and correlate it with expression of the MYCN gene. We showed that expression of both genes was concomitantly decreased in the 14G2a-treated IMR-32 cells after 24 h and 48 h. Our results extend knowledge on gene expression profiles after application of DOX and 14G2a in our model and reveal promising candidates for further research aimed at finding novel anti-neuroblastoma targets.

Exploration of the selective recognition of the G-quadruplex in the N-myc oncogene by electrospray ionization mass spectrometry.

The N-myc gene is a member of the MYC family and its amplification is highly correlated with the pathophysiology of cancers. The G-rich sequence, d(AG3CG3AG3AG3A), in the first intron of N-myc can form a G-quadruplex structure. Small molecules binding to it with high affinity and selectivity may provide a potential approach to modulate the expression of the N-myc gene. Electrospray ionization (ESI) mass spectrometry was used to analyze the G-quadruplex formation of the d(AG3CG3AG3AG3A) sequence, and to evaluate the binding affinities and selectivities of natural small molecules with the N-myc G-quadruplex. Enniatin B was found to have the highest binding affinity with this G-quadruplex within the 12 small molecules. Moreover, it also showed a biased selectivity toward the N-myc G-quadruplex compared with the other five G-quadruplexes derived from C-myc, Bcl2, Chl1, c-kit promoters and telomere G-rich sequences. In this study, we found a natural small molecule, enniatin B, which could bind to the G-quadruplex of the d(AG3CG3AG3AG3A) sequence from the first intron of the N-myc gene with high affinity and selectivity, which may lead to a potential modulation of the N-myc gene.

Protein arginine methyltransferase 5 is a key regulator of the MYCN oncoprotein in neuroblastoma cells

Approximately half of poor prognosis neuroblastomas (NBs) are characterized by pathognomonic MYCN gene amplification and MYCN over-expression. Here we present data showing that short-interfering RNA mediated depletion of the protein arginine methyltransferase 5 (PRMT5) in cell-lines representative of NBs with MYCN gene amplification leads to greatly impaired growth and apoptosis. Growth suppression is not apparent in the MYCN-negative SH-SY5Y NB cell-line, or in two immortalized human fibroblast cell-lines. Immunoblotting of NB cell-lines shows that high PRMT5 expression is strongly associated with MYCN-amplification (P < 0.004, Mann–Whitney U-test) and immunohistochemical analysis of primary NBs reveals that whilst PRMT5 protein is ubiquitously expressed in the cytoplasm of most cells, MYCN-amplified tumours exhibit pronounced nuclear PRMT5 staining. PRMT5 knockdown in MYCN-overexpressing cells, including the SHEP-21N cell-line with inducible MYCN expression leads to a dramatic decrease in MYCN protein and MYCN-associated cell-death in SHEP-21N cells. Quantitative gene expression analysis and cycloheximide chase experiments suggest that PRMT5 regulates MYCN at a post-transcriptional level. Reciprocal co-immunoprecipitation experiments demonstrated that endogenous PRMT5 and MYCN interact in both SK-N-BE(2)C and NGP cell lines. By using liquid chromatography – tandem mass spectrometry (LC-MS/MS) analysis of immunoprecipitated MYCN protein, we identified several potential sites of arginine dimethylation on the MYCN protein. Together our studies implicate PRMT5 in a novel mode of MYCN post-translational regulation and suggest PRMT5 plays a major role in NB tumorigenesis. Small-molecule inhibitors of PRMT5 may therefore represent a novel therapeutic strategy for neuroblastoma and other cancers driven by the MYCN oncogene.

ET-25 * THERAPEUTIC MODULATION OF MDM2 IN NEUROBLASTOMA

Neuroblastoma (NB) is the most common extracranial solid tumor of childhood. In high-risk NB, the MYCN gene is amplified in approximately 25% of all cases, and is considered to be a negative prognostic factor. MYCN can transcriptionally activate the murine double minute-2 (MDM2) gene, leading to increased expression of MDM2, downregulation of the tumor suppressor p53, and increased tumorigenesis. To improve treatment outcome in NB, inhibition of MDM2 function with front-line chemotherapy is being explored. The E3 ubiquitin ligase, MDM2, which ubiquitinates and targets p53 for proteosomal degradation, is a multifunctional protein that regulates the p53 signaling network and the DNA damage response. Cell growth assays indicate that inhibition of MDM2 function by the small molecule antagonist nutlin3a is synergistic to additive in combination with frontline chemotherapeutics in IMR32 NB cells (wt p53, MYCN amplified). To determine the effects of the combination of nutlin3a and cisplatin on a cell line without MYCN amplification, cell growth assays using SK-N-SH NB cells (wt p53, single MYCN copy) were performed. Results demonstrated that combined treatment with nutlin3a and cisplatin is synergistic in SK-N-SH NB cells. To delineate the mechanism of action in IMR32 cells exposed to cisplatin and nutlin3a, the impact of the two drugs, alone and in combination, was investigated in the context of MDM2 expression, modulation of the cell cycle, and DNA damage response. Western blot analyses were performed to monitor expression of cell cycle proteins and DNA damage/repair responses that we hypothesized are regulated by nutlin3a-mediated MDM2 blockade. Results demonstrated that combination treatment in IMR32 NB cells enhanced the DNA damage response and inhibited cell growth. The data provide further support for the hypothesis that MDM2 is a target that can be therapeutically exploited in combination with front-line chemotherapy to improve treatment outcome in MYCN non-amplified and MYCN amplified neuroblastoma.

Abstract B49: Role of ubiquitin carboxyl-terminal esterase L1 (UCHL1) on neuroblastoma cancer stem-like cells

Abstract Neuroblastoma (NB) is a pediatric malignancy derived from the sympaticoadrenal lineage of neural crest. It is the most commonly occurring extra-cranial nervous system malignancy of infants and children under the age of 5. Long-term survival is less than 40% in patients with high risk NB and over half of the survivors relapse. Emerging evidence supports the notion that there exists within tumors a small population of cells exhibiting stem-like properties, termed cancer stem-like cells (CSC), that is responsible for clinical relapse; therefore, investigating CSC targets may lead to improved treatments. Recent studies have demonstrated that deubiquitinating enzymes (DUBs) of the ubiquitin proteasome system are important players in cancer-associated pathways. These enzymes could be potential effective therapeutic targets. Previous work has identified expression of ubiquitin carboxyl-terminal esterase L1 (UCHL1), a DUB, in the CSC pool of neuroblastoma cells. Our studies are focused on the potential pathways and mechanism by which UCHL1 is acting in the CSC pool. We performed in vitro analyses on NB cell lines SK-N-BE(2), SK-N-AS, and SK-N-DZ. These cells differ in their MYCN amplification and P53 status. The CSC pool was enriched using the sphere formation assay. The small molecule inhibitor, LDN-57444, was used to impair UCHL1 activity. Furthermore, site-directed mutagenesis was used to generate the C90S mutant, which also blocks UCHL1 activity. The effect of blocking UCHL1 activity on cellular proliferation and CSC self-renewal was determined by the MTS assay and sphere formation assay respectively. We further confirmed the effect of impairing UCHL1 activity on the CSC population using flow cytometry by assessing the CD133 and side population. The soft agar assay was used to study the anchorage independent growth of UCHL1 impaired cells. Droplet digital PCR was used to investigate the effect of impaired UCHL1 activity on genes in the Wnt signaling pathway. Immunoprecipitation assays were used to confirm presence of C90S mutant in transfected cells and to determine potential interaction between UCHL1 and Wnt signaling proteins such as β-catenin. UCHL1 inhibition with LDN-57444 demonstrated a dose-dependent decrease in cellular proliferation and impaired sphere formation capability (read-out for self-renewal). We observed differential gene expression levels of MYC and MYCN in cells with UCHL1 inhibited activity via LDN-57444 treatment. C90S mutant transfected cells also demonstrated impaired self-renewal. C90S mutant cells showed decreased gene expression of Wnt signaling proteins and differential expression of MYC and MYCN. Immunoprecipitation studies and CD133 and side population analyses for assessing the CSC population are ongoing. Our data indicates that UCHL1 is potentially an important player in the regulation of CSC self-renewal. Our results also suggest that UCHL1 may mediate self-renewal through the MYC family of transcription factors. The MYC family has been shown to be promising targets in NB. Reduced MYC or MYCN gene expression in NB leads to cell cycle arrest and/or apoptosis. Further investigation of the effect of UCHL1 impaired activity on Wnt signaling will clarify whether UCHL1 is affecting MYC transcription through this pathway. Our studies indicated that UCHL1 might potentially be an effective therapeutic target for the CSC population to supplement current treatments. Citation Format: Judy C. Chang, Jaclyn Y. Hung. Role of ubiquitin carboxyl-terminal esterase L1 (UCHL1) on neuroblastoma cancer stem-like cells. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B49.

ATF4 and N-Myc coordinate glutamine metabolism in MYCN-amplified neuroblastoma cells through ASCT2 activation.

Amplification of the MYCN gene in human neuroblastoma predicts poor prognosis and resistance to therapy. We previously showed that MYCN-amplified neuroblastoma cells constantly require large amounts of glutamine to support their unabated growth. However, the identity and regulation of the transporter(s) that capture glutamine in MYCN-amplified neuroblastoma cells and the clinical significance of the transporter(s) in neuroblastoma diagnosis remain largely unknown. Here, we performed a systemic glutamine influx analysis and identified that MYCN-amplified neuroblastoma cells predominantly rely on activation of ASCT2 (solute carrier family 1 member 5, SLC1A5) to maintain sufficient levels of glutamine essential for the TCA cycle anaplerosis. Consequently, ASCT2 depletion profoundly inhibited glutaminolysis, concomitant with a substantial decrease in cell proliferation and viability in vitro and inhibition of tumourigenesis in vivo. Mechanistically, we identified ATF4 as a novel regulator which coordinates with N-Myc to directly activate ASCT2 expression. Of note, ASCT2 expression, which correlates with that of N-Myc and ATF4, is markedly elevated in high-stage neuroblastoma tumour samples compared with low-stage ones. More importantly, high ASCT2 expression is significantly associated with poor prognosis and survival of neuroblastoma patients. In aggregate, these findings elucidate a novel mechanism depicting how cell autonomous insults (MYCN amplification) and microenvironmental stresses (ATF4 induction) in concert coordinate ASCT2 activation to promote aggressive neuroblastoma progression, and establish ASCT2 as a novel biomarker in patient prognosis and stratification.