MYCN Overexpression Research Articles

N-Myc overexpression increases cisplatin resistance in neuroblastoma via deregulation of mitochondrial dynamics.

N-Myc is a global transcription factor that regulates the expression of genes involved in a number of essential cellular processes including: ribosome biogenesis, cell cycle and apoptosis. Upon deregulation, N-Myc can drive pathologic expression of many of these genes, which ultimately defines its oncogenic potential. Overexpression of N-Myc has been demonstrated to contribute to tumorigenesis, most notably for the pediatric tumor, neuroblastoma. Herein, we provide evidence that deregulated N-Myc alters the expression of proteins involved in mitochondrial dynamics. We found that N-Myc overexpression leads to increased fusion of the mitochondrial reticulum secondary to changes in protein expression due to aberrant transcriptional and post-translational regulation. We believe the structural changes in the mitochondrial network in response to N-Myc amplification in neuroblastoma contributes to two important aspects of tumor development and maintenance—bioenergetic alterations and apoptotic resistance. Specifically, we found that N-Myc overexpressing cells are resistant to programmed cell death in response to exposure to low doses of cisplatin, and demonstrated that this was dependent on increased mitochondrial fusion. We speculate that these changes in mitochondrial structure and function may contribute significantly to the aggressive clinical ph9enotype of N-Myc amplified neuroblastoma.

Neuroblastoma.

Neuroblastoma is the most common extracranial solid tumour occurring in childhood and has a diverse clinical presentation and course depending on the tumour biology. Unique features of these neuroendocrine tumours are the early age of onset, the high frequency of metastatic disease at diagnosis and the tendency for spontaneous regression of tumours in infancy. The most malignant tumours have amplification of the MYCN oncogene (encoding a transcription factor), which is usually associated with poor survival, even in localized disease. Although transgenic mouse models have shown that MYCN overexpression can be a tumour-initiating factor, many other cooperating genes and tumour suppressor genes are still under investigation and might also have a role in tumour development. Segmental chromosome alterations are frequent in neuroblastoma and are associated with worse outcome. The rare familial neuroblastomas are usually associated with germline mutations in ALK, which is mutated in 10-15% of primary tumours, and provides a potential therapeutic target. Risk-stratified therapy has facilitated the reduction of therapy for children with low-risk and intermediate-risk disease. Advances in therapy for patients with high-risk disease include intensive induction chemotherapy and myeloablative chemotherapy, followed by the treatment of minimal residual disease using differentiation therapy and immunotherapy; these have improved 5-year overall survival to 50%. Currently, new approaches targeting the noradrenaline transporter, genetic pathways and the tumour microenvironment hold promise for further improvements in survival and long-term quality of life.

Characterization of pancreatic glucagon-producing tumors and pituitary gland tumors in transgenic mice overexpressing MYCN in hGFAP-positive cells.

Amplification or overexpression of MYCN is involved in development and maintenance of multiple malignancies. A subset of these tumors originates from neural precursors, including the most aggressive forms of the childhood tumors, neuroblastoma and medulloblastoma. In order to model the spectrum of MYCN-driven neoplasms in mice, we transgenically overexpressed MYCN under the control of the human GFAP-promoter that, among other targets, drives expression in neural progenitor cells. However, LSL-MYCN;hGFAP-Cre double transgenic mice did neither develop neural crest tumors nor tumors of the central nervous system, but presented with neuroendocrine tumors of the pancreas and, less frequently, the pituitary gland. Pituitary tumors expressed chromogranin A and closely resembled human pituitary adenomas. Pancreatic tumors strongly produced and secreted glucagon, suggesting that they derived from glucagon- and GFAP-positive islet cells. Interestingly, 3 out of 9 human pancreatic neuroendocrine tumors expressed MYCN, supporting the similarity of the mouse tumors to the human system. Serial transplantations of mouse tumor cells into immunocompromised mice confirmed their fully transformed phenotype. MYCN-directed treatment by AuroraA- or Brd4-inhibitors resulted in significantly decreased cell proliferation in vitro and reduced tumor growth in vivo. In summary, we provide a novel mouse model for neuroendocrine tumors of the pancreas and pituitary gland that is dependent on MYCN expression and that may help to evaluate MYCN-directed therapies.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Proliferation and Survival of Embryonic Sympathetic Neuroblasts by MYCN and Activated ALK Signaling.

MYCN overexpression combined with activated anaplastic lymphoma kinase (ALK) is sufficient to induce neuroblastoma (NB) in mouse sympathoadrenal cells. To address cellular and molecular effects elicited by MYCN/ALK cooperation, we used cultures of chick sympathetic neuroblasts. We demonstrate that MYCN increases proliferation but not survival, whereas long-term expression of ALKF1174L elicits cell-cycle exit, differentiation, and survival of postmitotic neurons. Combined MYCN/ALKF1174L expression allows long-term proliferation and survival of neuroblasts with differentiated characteristics. In the presence of ALKF1174L signaling, MYCN induces the expression of the ubiquitin ligase SKP2 (S-phase kinase-associated protein 2), which targets p27 for degradation and is also upregulated in high-risk NB. SKP2 inhibition supports a function for SKP2 in the maintained neuroblast proliferation downstream of MYCN/ALK, which may represent an early step toward tumorigenesis.

N-Myc Induces an EZH2-Mediated Transcriptional Program Driving Neuroendocrine Prostate Cancer

The transition from castration-resistant prostate adenocarcinoma (CRPC) to neuroendocrine prostate cancer(NEPC) has emerged as an important mechanism of treatment resistance. NEPC is associated with overexpression and gene amplification of MYCN (encoding N-Myc). N-Myc is an established oncogene in several rare pediatric tumors, but its role in prostate cancer progression is not well established. Integrating a genetically engineered mouse model and human prostate cancer transcriptome data, we show that N-Myc overexpression leads to the development of poorly differentiated, invasive prostate cancer that is molecularly similar to human NEPC. This includes an abrogation of androgen receptor signaling and induction of Polycomb Repressive Complex 2 signaling. Altogether, our data establishes N-Myc as an oncogenic driver of NEPC.

Protein arginine methyltransferase 1 is a novel regulator of MYCN in neuroblastoma.

Amplification or overexpression of MYCN is associated with poor prognosis of human neuroblastoma. We have recently defined a MYCN-dependent transcriptional signature, including protein arginine methyltransferase 1 (PRMT1), which identifies a subgroup of patients with high-risk disease. Here we provide several lines of evidence demonstrating PRMT1 as a novel regulator of MYCN and implicating PRMT1 as a potential therapeutic target in neuroblastoma pathogenesis. First, we observed a strong correlation between MYCN and PRMT1 protein levels in primary neuroblastoma tumors. Second, MYCN physically associates with PRMT1 by direct protein-protein interaction. Third, depletion of PRMT1 through siRNA knockdown reduced neuroblastoma cell viability and MYCN expression. Fourth, we showed that PRMT1 regulates MYCN stability and identified MYCN as a novel substrate of PRMT1. Finally, we demonstrated that mutation of putatively methylated arginine R65 to alanine decreased MYCN stability by altering phosphorylation at residues serine 62 and threonine 58. These results provide mechanistic insights into the modulation of MYCN oncoprotein by PRMT1, and suggest that targeting PRMT1 may have a therapeutic impact on MYCN-driven oncogenesis.

Identification of Neuroblastoma Subgroups Based on Three-Dimensional Telomere Organization

Using 3D telomere quantitative fluorescence in situ hybridization, we determined the 3D telomere organization of 74 neuroblastoma tissue samples. Hierarchical cluster analysis of the measured telomere parameters identified three subgroups from our patient cohort. These subgroups have unique telomere profiles based on telomere length and nuclear architecture. Subgroups with higher levels of telomere dysfunction were comprised of tumors with greater numbers of telomeres, telomeric aggregates, and short telomeres (P<.0001). Tumors with greater telomere dysfunction were associated with unfavorable tumor characteristics (greater age at diagnosis, unfavorable histology, higher stage of disease, MYCN amplification, and higher MYCN expression) and poor prognostic risk (P<.001). Subgroups with greater telomere dysfunction also had higher intratumor heterogeneity. MYCN overexpression in two neuroblastoma cell lines with constitutively low MYCN expression induced changes in their telomere profile that were consistent with increased telomere dysfunction; this illustrates a functional relationship between MYCN and 3D telomere organization. This study demonstrates the ability to classify neuroblastomas based on the level of telomere dysfunction, which is a novel approach for this cancer.

Abstract 2456: Synergy between loss of NF1 and overexpression of MYCN in neuroblastoma is mediated by the GAP-related domain

Abstract Earlier reports indicated that the role of Nf1 tumor suppressor gene in limiting sympathoadrenal cell growth during embryologic development is independent of its ability to down-modulate RAS-MAPK signaling. This finding raised the question of whether neuroblastoma pathogenesis was also accelerated by loss of a similar non-canonical function of NF1. To elucidate how loss of the NF1 tumor suppressor gene contributes to the development of high-risk neuroblastoma, we relied on a transgenic zebrafish model that overexpresses MYCN and harbors loss-of-function nf1 mutations. We show here that loss of nf1 leads to aberrant activation of RAS signaling in MYCN-induced neuroblastoma, promoting both increased tumor cell survival and rapid tumor cell proliferation. We demonstrate further that the GTPase-activating protein (GAP) activity of the (GAP)-related domain (GRD) is sufficient to suppress accelerated initiation of neuroblastoma in nf1-deficient zebrafish, even though this transgene is unable to restrict abnormal sympathoadrenal cell growth during embryologic development. Hence NF1 exhibits different activities in vivo in the normal development and tumorigenesis of the peripheral sympathetic nervous system. Our findings establish nf1-deficient zebrafish that overexpress MYCN as an ideal animal model system for investigating new strategies to improve treatment of very high risk neuroblastomas with aberrant RAS-MAPK activation. We are currently performing high-throughput in vivo drug analysis using these zebrafish with primary tumors. Citation Format: Shuning He, Marc R. Mansour, Mark W. Zimmerman, Dong Hyuk Ki, Hillary M. Layden, Koshi Akahane, Eric D. de Groh, Antonio R. Perez-Atayde, Shizhen Zhu, Jonathan A. Epstein, A Thomas Look. Synergy between loss of NF1 and overexpression of MYCN in neuroblastoma is mediated by the GAP-related domain. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2456.

Abstract 887: N-Myc drives neuroendocrine prostate cancer

Abstract Emerging observations from clinical trials suggest that a subset of castration resistant prostate adenocarcinomas (CRPC) eventually evolve or progress to a predominantly neuroendocrine phenotype (NEPC). This transition is emerging as an important mechanism of treatment resistance. This cell plasticity is characterized by loss of androgen receptor (AR) and prostate specific antigen (PSA), and significant over-expression and gene amplification of MYCN (encoding N-Myc). While N-Myc is a bona fide driver oncogene in several rare tumor types, the molecular mechanisms that underlie N-Myc driven NEPC have yet to be characterized. Integrating a novel genetically engineered mouse (GEM) model of prostate specific N-Myc overexpression, human prostate cancer cell line modeling, and human prostate cancer transcriptome data, we found that N-Myc over-expression leads to the development of poorly differentiated, invasive prostate cancer that is molecularly similar to human NEPC tumors. To determine if N-Myc plays a causal role in driving the NEPC phenotype, we generated GEM lines that carry a CAG-driven lox-stop-lox human MYCN gene integrated into the ROSA26 (LSL-MYCN) locus and either a Tmprss2 driven tamoxifen-activated Cre recombinase (T2-Cre) or probasin (Pb)-Cre. Since PTEN deletion is a frequent alteration in CRPC and PI3K/AKT signaling can enhance N-Myc protein stability we also engineered the mice with a floxed Pten locus. N-Myc over-expression in the context of Ptenf/+ at 3 months post-induction leads to focal mouse high-grade prostatic intraepithelial neoplasia (mHGPIN). T2-Cre; Ptenf/f; LSL-MYCN+/+ mice develop highly proliferative, diffuse mHGPIN which consists of proliferations of cells with nuclear atypia that expand the glands, imparting irregular borders and inducing a mild stromal response, mitotic figures, and incipient necrosis. RNAseq data from N-Myc these mHGPIN lesions show they are molecularly similar to NEPC based on RNAseq data from 203 human CRPC and NEPC samples. At 6 months, Pb-Cre; Ptenf/f; LSL-MYCN+/+ mice develop poorly differentiated, highly proliferative, invasive prostate cancer. Based on the RNAseq data from the N-Myc GEM line, GEM-derived mouse prostate cancer organoid cultures and isogenic cell lines, we found that N-Myc regulates a specific NEPC-associated molecular program that includes a repression of AR signaling, enhanced AKT signaling and repression of Polycomb Repressive Complex 2 target genes. We further showed that N-Myc interacts with AR and this interaction depends on Enhancer of Zeste Homolog 2 (EZH2). Finally, N-Myc expressing cell lines and organoids displayed an enhanced sensitivity to inhibitors targeting the AKT pathway, EZH2 and Aurora-A. Altogether, our data shows that N-Myc drives the neuroendocrine phenotype in prostate cancer and provides rationale for the development of new therapeutic strategies for treating this aggressive subtype of prostate cancer. Citation Format: Etienne Dardenne, Himisha Beltran, Kaitlyn Gayvert, Matteo Benelli, Adeline Berger, Loredana Puca, Joanna Cyrta, Andrea Sboner, Zohal Noorzad, Theresa MacDonald, Cynthia Cheung, Dong Gao, Yu Chen, Martin Eilers, Juan Miguel Mosquera, Brian D. Robinson, Mark A. Rubin, Olivier Elemento, Francesca Demichelis, David S. Rickman. N-Myc drives neuroendocrine prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 887.

Abstract 2433: Loss of chd5-mediated gene repression synergizes with MYCN to accelerate neuroblastoma tumorigenesis in zebrafish

Abstract Neuroblastoma is a malignancy of the peripheral sympathetic nervous system (PSNS) and accounts for 10-15% of cancer deaths among children. For the 40% of patients presenting with high-risk disease, current therapeutic approaches are insufficient and long-term survival is less than 50%. Along with genomic amplification of the MYCN oncogene, hemizygous loss of the 1p36 chromosomal region is a major risk factor in neuroblastoma. The human CHD5 gene is a neuronal specific chromatin remodeling helicase that maps to 1p36, and is thus frequently lost in high-risk neuroblastoma. Our laboratory has previously generated a faithful model of pediatric neuroblastoma in the zebrafish driven by overexpression of the MYCN oncogene in the PSNS (dbh:MYCN). Additionally, zebrafish chd5 mutant alleles were created using the newly developed gene editing technologies TALEN and CRISPR-Cas9. The resulting chd5 mutant fish exhibit abnormal development of the PSNS in the form of expansion of the superior cervical ganglia and enlargement of the interrenal gland (adrenal medulla). Haploinsufficiency for Chd5 combined with dbh:MYCN expression accelerates the onset and increases the penetrance of neuroblastoma tumorigenesis in zebrafish, indicating a tumor suppressive function. Elevated p-ERK and PCNA+ cells in tumor tissue indicates that loss of Chd5, cooperates with MYCN overexpression to accelerate neuroblast proliferation in vivo. Chd5 (in addition to Chd3 and Chd4) is a core member of the epigenetic regulatory NuRD complex, which also contains HDAC1-2, MTA1-3, MBD2-3, GATAD2A/B and RBBP4/7. The conserved biological function of Chd5 is to silence gene expression through the maintenance of a repressed chromatin state. Tumors deficient for Chd5 expression exhibit reduced levels of the H3K27me3 histone modification, a marker of facultatively repressed genes. Future studies will further explore the mechanism and function of Chd5 so that the pathways mediating tumor suppression can be elucidated and that essential proteins in these pathways can be targeted in ways that exploit the synthetic lethal relationships that are established. Citation Format: Mark W. Zimmerman, Shuning He, Jimann Shin, Shizhen Zhu, Feng Guo, Marc Mansour, Deepak Reyon, J Keith Joung, Jinhua Quan, Timur Yusufzai, A Thomas Look. Loss of chd5-mediated gene repression synergizes with MYCN to accelerate neuroblastoma tumorigenesis in zebrafish. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2433.

Synergy between loss of NF1 and overexpression of MYCN in neuroblastoma is mediated by the GAP-related domain.

Earlier reports showed that hyperplasia of sympathoadrenal cell precursors during embryogenesis in Nf1-deficient mice is independent of Nf1's role in down-modulating RAS-MAPK signaling. We demonstrate in zebrafish that nf1 loss leads to aberrant activation of RAS signaling in MYCN-induced neuroblastomas that arise in these precursors, and that the GTPase-activating protein (GAP)-related domain (GRD) is sufficient to suppress the acceleration of neuroblastoma in nf1-deficient fish, but not the hypertrophy of sympathoadrenal cells in nf1 mutant embryos. Thus, even though neuroblastoma is a classical "developmental tumor", NF1 relies on a very different mechanism to suppress malignant transformation than it does to modulate normal neural crest cell growth. We also show marked synergy in tumor cell killing between MEK inhibitors (trametinib) and retinoids (isotretinoin) in primary nf1a-/- zebrafish neuroblastomas. Thus, our model system has considerable translational potential for investigating new strategies to improve the treatment of very high-risk neuroblastomas with aberrant RAS-MAPK activation.

Abstract A19: Transformation of primary neural crest cells to model pediatric cancers

Abstract Neural crest cells (NCCs) are a multipotent, highly migratory cell population specified from the neural tube during embryonic development. NCCs undergo EMT (epithelial to mesenchymal transition) then migrate throughout the body forming diverse lineages including neurons, Schwann cells, melanocytes, and osteoblasts. Precursor cells from NCC lineages are thought to be the cell of origin for several pediatric and adult cancers including neuroblastoma, peripheral primitive neuroectodermal tumors (pPNET), malignant peripheral nerve sheath tumors (MPNST), cranial-facial osteosarcoma, and melanoma. The most common pediatric cancer in infants is neuroblastoma, which arises from the sypathoadrenal lineage of trunk NCCs. To study early events in neuroblastoma oncogenesis we established a system based on transformation of primary mouse NCCs. Trunk NCCs were isolated from day E9.5 embryos, and resulted in a population that was &amp;gt;95% positive for the NCC markers Sox10, p75, and Ascl1 (MASH1) by immunofluorescence. Culturing primary NCCs in neurogenic differentiation media resulted in neurons positive for Tuj1, Map2, and tyrosine hydroxylase (TH). Since a prominent category of high-risk neuroblastoma involves amplification of MYCN, we next determined whether MYCN overexpression was sufficient to transform wild-type NCCs in our assay. NCCs were isolated from C57Black6 mice and infected with MYCN retrovirus within 48hr of isolation, and then 10,000 cells/mouse were injected subcutaneously. MYCN overexpression alone generated a single tumor in 1 out of 12 mice, and the resulting tumor expressed the neuronal markers synaptophysin and tyrosine hydroxylase (TH) by immunohistochemistry (IHC). To determine whether loss of p53 could increase the transformation efficiency, NCCs from p53-compromised mice were infected with MYCN. These tumors had increased penetrance with 19 out of 19 mice developing tumors whether cells were injected into nude mice or into syngeneic C57Black6 mice with an intact immune system. Based on pathological analysis, these tumors were classified as primitive neuroectodermal tumors with divergent differentiation. Tumor regions which were diffusely positive for neuronal markers appeared neuroblastoma-like or PNET-like. Osteosarcoma was also a prominent feature in tumors with loss of p53. We also observed an additional tumor type which was negative by IHC for neuronal, Schwannian, and melanoma markers, and is not yet fully characterized. In general, mice with the fastest onset of tumor growth displayed micrometastases to the lung and liver, while mice with slower tumor growth had macrometastases to these organs. Injection of cell lines derived from the primary tumors also lead to aggressive metastasis to the lung and liver. Although metastasis from subcutaneous tumors is generally rare, it was a recurrent feature in our model which recapitulates the highly metastatic nature of human cancers derived from NCCs like neuroblastoma and melanoma. Lastly, we determined whether NCCs were sensitive to the BET bromodomain inhibitor JQ1. JQ1 treatment significantly inhibited growth of both primary NCCs and cell lines derived from NCC tumors, while having minimal effect on NIH-3T3 fibroblasts. Furthermore, our results imply that JQ1 sensitivity in these cells is independent of MYCN expression levels. Using primary mouse NCCs, we found that MYCN overexpression is capable of initiating cell transformation and formed a single tumor with characteristics of neuroblastoma. Additional loss of p53 resulted in a more diverse panel of tumor types, which emphasizes the ability of NCCs to contribute to multiple lineages. Our results suggest that studies utilizing primary NCCs may be beneficial to identify initiating oncogenic events in neuroblastoma and perhaps other pediatric cancers. Citation Format: Rachelle R. Olsen, Joel H. Otero, Jesus Garcia-Lopez, Kirby A. Wallace, Zhirong Yin, Kevin W. Freeman. Transformation of primary neural crest cells to model pediatric cancers. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr A19.

N-Myc Drives Neuroendocrine Prostate Cancer Initiated from Human Prostate Epithelial Cells

MYCN amplification and overexpression are common in neuroendocrine prostate cancer (NEPC). However, the impact of aberrant N-Myc expression in prostate tumorigenesis and the cellular origin of NEPC have not been established. We define N-Myc and activated AKT1 as oncogenic components sufficient to transform human prostate epithelial cells to prostate adenocarcinoma and NEPC with phenotypic and molecular features of aggressive, late-stage human disease. We directly show that prostate adenocarcinoma and NEPC can arise from a common epithelial clone. Further, N-Myc is required for tumor maintenance, and destabilization of N-Myc through Aurora A kinase inhibition reduces tumor burden. Our findings establish N-Myc as a driver of NEPC and a target for therapeutic intervention.

Abstract A04: Modeling pediatric malignancies by transforming primary neural crest cells

Abstract Neural crest cells (NCCs) are a highly migratory multipotent cell population first specified during neural tube closure at the neural plate border. Soon after neural tube closure NCCs undergo epithelial to mesenchymal transitioning (EMT) to allow migration throughout the body plan where they generate diverse cell types including neurons, Schwann cells, melanocytes, and osteoblasts. NCCs have been proposed to be the embryonic precursor cell population to multiple pediatric malignancies including neuroblastoma, peripheral primitive neuroectodermal tumors (pPNET), malignant peripheral nerve sheath tumors (MPNST), cranial-facial osteosarcoma as well as adult malignancies like melanoma. Despite this very little work has been done with primary NCCs in pediatric cancer research. The most common of these pediatric cancers is neuroblastoma, which arises in the sympathoadrenal lineage of trunk NCCs. To establish a system for studying the earliest events in neuroblastoma oncogenesis we developed an assay based on the oncogenic transformation of primary NCCs. Isolation of primary trunk NCCs from day 9.5 embryos resulted in a population that was &amp;gt;95% positive for the NCC markers Sox10, p75 and Ascl1 (MASH1) as assessed by immunofluorescence. These isolated NCCs were also capable of generating Tuj1, Map2, and tyrosine hydroxylase (TH) positive neurons when placed in neurogenic media. We next wanted to test the transformation of primary NCCs. Since a prominent category of high-risk neuroblastoma is N-Myc amplification, we first determined if N-Myc overexpression was sufficient to transform wild-type NCCs in our assay. For this and all other tumor experiments, NCCs were retrovirally infected within 48 hours of isolation and 10,000 cells were injected the next day subcutaneously into the flanks of mice. N-Myc alone generated a single tumor in 1 out of 16 mice with the resulting tumor being uniformly positive for the neuronal markers synaptophsyon, MAP2 and tyrosine hydroxylase (TH). This tumor also contained neuropil and ganglion cells and was characterized as neuroblastoma, differentiating subtype, stroma poor. To determine if loss of p53 could increase the efficiency of transformation, NCCs from p53-compromised mice were infected with N-Myc. This led to 100% penetrance with 19 out of 19 mice generating tumors and with equivalent frequency in nude or syngeneic C57BL/6 mice showing this approach can be used in immune competent mice. The resulting tumors from this genetic combination were characterized as primitive neuroectodermal tumors with divergent differentiation. Regions of these tumors that were diffusely positive for neuronal markers appeared neuroblastoma-like or PNET-like. Osteosarcoma was also a prominent feature in every tumor with loss of p53. Additionally we observed yet to be defined tumor types that were negative by IHC for neuronal markers, Schwannian markers and melanoma markers. Generally, in mice with the fastest tumor onset micrometastases to the lung and liver were observed, while slower onset correlated with macroscopic metastases to the same organs. Injection of cell lines derived from primary tumors lead to aggressive metastatic spread to lung and liver. Though subcutaneous tumors rarely metastasize this was a salient feature of our neural crest derived cancers, which recapitulates the highly metastatic nature of human NCC derived cancers like neuroblastoma and melanoma. N-Myc in a wild type background caused a single phenotypically accurate neuroblastoma while loss of p53 caused an increase in cancer divergence underscoring the capacity of NCCs to generate a diverse array of cancer types including osteosarcoma. From this study we believe primary NCCs can be used as a platform for identifying initiating cancer events in neuroblastoma and possibly other pediatric malignancies. Citation Format: Rachelle R. Olsen, Otero Joel, Kirby Wallace, Jerold Rehg, Kevin W. Freeman. Modeling pediatric malignancies by transforming primary neural crest cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr A04.

ALK(R1275Q) perturbs extracellular matrix, enhances cell invasion and leads to the development of neuroblastoma in cooperation with MYCN.

Overexpression of MYCN is a hallmark of neuroblastoma (NB). ALK(R1275Q), an activating mutation of ALK (anaplastic lymphoma kinase), has been found in sporadic and familial NB patients. In this report, we demonstrated that ALK(R1275Q) knock-in, MYCN transgenic compound mice developed NB with complete penetrance. Transcriptome analysis revealed that ALK(R1275Q) globally downregulated the expression of extracellular matrix (ECM)- and basement membrane (BM)-associated genes in both primary neuronal cells and NB tumors. Accordingly, ALK(R1275Q)/MYCN tumors exhibited reduced expression of ECM/BM-related proteins as compared with MYCN tumors. In addition, on MYCN transduction, ALK(R1275Q)-expressing neuronal cells exhibited increased migratory and invasive activities. Consistently, enhanced invasion and metastasis were demonstrated in ALK(R1275Q)/MYCN mice. These results collectively indicate that ALK(R1275Q) confers a malignant potential on neuronal cells that overexpress MYCN by impairing normal ECM/BM integrity and enhancing tumor growth and dissemination. Moreover, we found that crizotinib, an ALK inhibitor, almost completely inhibited the growth of ALK(R1275Q)/MYCN tumors in an allograft model. Our findings provided insights into the cooperative mechanism of the mutated ALK and overexpressed MYCN in the pathogenesis of NB and demonstrated the effectiveness of crizotinib on ALK(R1275Q)-positive tumors.

Effect of low doses of actinomycin D on neuroblastoma cell lines.

BackgroundNeuroblastoma is a malignant embryonal tumor occurring in young children, consisting of undifferentiated neuroectodermal cells derived from the neural crest. Current therapies for high-risk neuroblastoma are insufficient, resulting in high mortality rates and high incidence of relapse. With the intent to find new therapies for neuroblastomas, we investigated the efficacy of low-doses of actinomycin D, which at low concentrations preferentially inhibit RNA polymerase I-dependent rRNA trasncription and therefore, ribosome biogenesis.MethodsNeuroblastoma cell lines with different p53 genetic background were employed to determine the response on cell viability and apoptosis of low-dose of actinomycin D. Subcutaneously-implanted SK-N-JD derived neuroblastoma tumors were used to assess the effect of low-doses of actinomycin D on tumor formation.ResultsLow-dose actinomycin D treatment causes a reduction of cell viability in neuroblastoma cell lines and that this effect is stronger in cells that are wild-type for p53. MYCN overexpression contributes to enhance this effect, confirming the importance of this oncogene in ribosome biogenesis. In the wild-type SK-N-JD cell line, apoptosis was the major mechanism responsible for the reduction in viability and we demonstrate that treatment with the MDM2 inhibitor Nutlin-3, had a similar effect to that of actinomycin D. Apoptosis was also detected in p53−/−deficient LA1-55n cells treated with actinomycin D, however, only a small recovery of cell viability was found when apoptosis was inhibited by a pan-caspase inhibitor, suggesting that the treatment could activate an apoptosis-independent cell death pathway in these cells. We also determined whether actinomycin D could increase the efficacy of the histone deacetylase inhibitor, SAHA, which is in being used in neuroblastoma clinical trials. We show that actinomycin D synergizes with SAHA in neuroblastoma cell lines. Moreover, on subcutaneously-implanted neuroblastoma tumors derived from SK-N-JD cells, actinomycin D led to tumor regression, an effect enhanced in combination with SAHA.ConclusionsThe results presented in this work demonstrate that actinomycin D, at low concentrations, inhibits proliferation and induces cell death in vitro, as well as tumor regression in vivo. From this study, we propose that use of ribosome biogenesis inhibitors should be clinically considered as a potential therapy to treat neuroblastomas.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0489-8) contains supplementary material, which is available to authorized users.

Abstract PR12: Transcriptional regulation of metabolism by MLX and its binding partners is essential for tumor cell survival and spermatogenesis

Abstract Background: The proto-oncogene MYC encodes a bHLHLZ transcription factor that binds to its obligate DNA-binding partner, MAX, leading to heterodimer occupancy of E-Box motifs within the genome. MYC is normally mitogen-dependent and tightly regulated where it plays a role in transcriptionally coordinating metabolic sufficiency for cell cycle progression, regulating both proliferation and viability. MYC is deregulated in the majority of human cancers. Importantly, MYC exists within an extended family of related bHLHLZ transcription factors including the MYC antagonizing MAX dimerization (MXD) proteins, MYC paralogs, such as MYCN, as well as MAX-like protein X (MLX), and its nutrient-sensing heterodimerization partners MondoA and carbohydrate response element binding protein (ChREBP). We recently defined a role for the extended MAX/MLX network in the regulation of metabolism and survival of cancer cells specifically with deregulated MYC or MYCN, in which MYC-MAX or MYCN-MAX heterodimers coordinately regulate transcription of important metabolic targets with MondoA-MLX to facilitate tumor cell survival. Targeting a number of these downstream effectors is sufficient to kill cancer cells with deregulated MYC. Methods: Expanding on our initial findings we have used molecular and biochemical approaches to demonstrate non-redundant roles for both MondoA-MLX and ChREBP-MLX in the transcriptional regulation of metabolism and survival of human cancer cells with deregulated MYC, including liver and colon cancer cells. We have also characterized the metabolic and biological phenotypes associated with targeted deletion of Mlx in the mouse, combining in vivo and ex vivo analysis with both whole-body deletion and tissue-specific ablation to unmask novel functions of Mlx and its binding partners. Results: Our results demonstrate that MLX and its binding partners regulate pathways associated with mitochondrial function as well as glucose and lipid metabolism in a non-redundant, cell-type-dependent manner. The presence of MLX stabilizes MondoA and ChREBP, and deletion of Mlx in the mouse leads to decreased levels of MondoA and Chrebp proteins in vivo. Whole-body deletion of Mlx in the mouse is well tolerated but leads to male infertility and teratozoospermia. This sterility phenotype is recapitulated by specific deletion of Mlx in the Sertoli cells of the testis, a major metabolic cell type within the tissue and the central regulator of multiple steps associated with germ cell fate, including production and/or transport of critical metabolites required by the germ cells. This indicates both cell-autonomous (germ cell) and non-cell-autonomous (Sertoli cell) regulation of survival and metabolism in spermatogenesis mediated by Mlx. Importantly, human patients with teratozoospermia exhibit diminished expression of MondoA, MLX and their metabolic targets. Moreover, overexpression of MYCN, MondoA, MLX and their metabolic targets occurs in patients with germ cell-derived tumors, such as seminomas. Conclusions: Transcriptional coordination between the mitogen- and nutrient-responsive arms of the MAX/MLX network exhibits context-dependent lethality. MondoA and Chrebp exhibit non-redundant functions in both cancer cells and normal mouse tissue, and inactivation of Mlx phenocopies loss of both factors, suggesting that targeting Mlx can inhibit both MondoA and Chrebp transcriptional activity and subsequent metabolic output. Metabolic regulation by Mlx impacts normal testicular tissue homeostasis and differentiation in the mouse, and its deregulation is associated with human diseases such as loss of function associated with infertility and gain of function associated with germ cell malignancy. While deletion of Myc, Mycn or Max is embryonic lethal in the mouse, deletion of MondoA, Chrebp or Mlx is well tolerated, indicating that targeting this arm of the extended MYC network, or their critical downstream targets, could be of therapeutic value in metabolic syndrome, fertility and cancer. Citation Format: Patrick A. Carroll, Daniel Diolaiti, Pei-Feng Cheng, Haiwei Gu, Danijel Djukovic, Daniel Raftery, Donald E. Ayer, Charles H. Muller, Robert N. Eisenman. Transcriptional regulation of metabolism by MLX and its binding partners is essential for tumor cell survival and spermatogenesis. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr PR12.

The Interaction of Myc with Miz1 Defines Medulloblastoma Subgroup Identity

Four distinct subgroups of cerebellar medulloblastomas (MBs) differ in their histopathology, molecular profiles, and prognosis. c-Myc (Myc) or MycN overexpression in granule neuron progenitors (GNPs) induces Group 3 (G3) or Sonic Hedgehog (SHH) MBs, respectively. Differences in Myc and MycN transcriptional profiles depend, in part, on their interaction with Miz1, which binds strongly to Myc but not MycN, to target sites on chromatin. Myc suppresses ciliogenesis and reprograms the transcriptome of SHH-dependent GNPs through Miz1-dependent gene repression to maintain stemness. Genetic disruption of the Myc/Miz1 interaction inhibited G3 MB development. Target genes of Myc/Miz1 are repressed in human G3 MBs but not in other subgroups. Therefore, the Myc/Miz1 interaction is a defining hallmark of G3 MB development.

Neuroblastoma and Its Zebrafish Model.

Neuroblastoma, an important developmental tumor arising in the peripheral sympathetic nervous system (PSNS), accounts for approximately 10 % of all cancer-related deaths in children. Recent genomic analyses have identified a spectrum of genetic alterations in this tumor. Amplification of the MYCN oncogene is found in 20 % of cases and is often accompanied by mutational activation of the ALK (anaplastic lymphoma kinase) gene, suggesting their cooperation in tumor initiation and spread. Understanding how complex genetic changes function together in oncogenesis has been a continuing and daunting task in cancer research. This challenge was addressed in neuroblastoma by generating a transgenic zebrafish model that overexpresses human MYCN and activated ALK in the PSNS, leading to tumors that closely resemble human neuroblastoma and new opportunities to probe the mechanisms that underlie the pathogenesis of this tumor. For example, coexpression of activated ALK with MYCN in this model triples the penetrance of neuroblastoma and markedly accelerates tumor onset, demonstrating the interaction of these modified genes in tumor development. Further, MYCN overexpression induces adrenal sympathetic neuroblast hyperplasia, blocks chromaffin cell differentiation, and ultimately triggers a developmentally-timed apoptotic response in the hyperplastic sympathoadrenal cells. In the context of MYCN overexpression, activated ALK provides prosurvival signals that block this apoptotic response, allowing continued expansion and oncogenic transformation of hyperplastic neuroblasts, thus promoting progression to neuroblastoma. This application of the zebrafish model illustrates its value in rational assessment of the multigenic changes that define neuroblastoma pathogenesis and points the way to future studies to identify novel targets for therapeutic intervention.