MYCN-driven Medulloblastoma Research Articles

Abstract 4599: A novel small molecule inhibitor targeting MYC oncogenic signaling as an enhancer of HDAC inhibitors for the treatment of high-risk medulloblastoma

Abstract Background: Medulloblastoma (MB) is the most prevalent malignant childhood brain tumour. Although prognosis has improved over time, 95% of children with high-risk or relapsed MB eventually succumb to the disease. A key driver gene in high-risk MB is the c-MYC oncogene, for which there are currently no approved inhibitors. Histone deacetylases (HDACs) are transcriptional repressors that are dysregulated in many cancers, making them an attractive therapeutic target. Although several HDAC inhibitors have been FDA-approved for cancer treatment, they have been associated with high toxicity and limited efficacy as single agents. Therefore, simultaneously targeting c-MYC and HDACs may result in synergistic effects that could overcome drug resistance in high-risk MB. Methods and Results: We previously reported a novel pyrido-benzimidazole analogue, SE486-11, which enhanced the therapeutic effect of HDAC inhibitors in MYCN-driven cancers. We recently developed analogues (UNSW-SC compounds) with more potent activity (IC50: 0.017 to 3.70 µM). Here, we showed that these compounds significantly reduced cell viability and induced apoptosis in MB cells. MYC status was a key determinant of sensitivity of UNSW-SC compounds, with more than 3-fold higher cytotoxicity in high MYC compared to low MYC MB cells. The lead compound, UNSW-SC-22, was shown to reduce MYC protein expression and was found to be highly synergistic in enhancing the efficacy of HDAC inhibitors in MB cells. Most importantly, we demonstrated that UNSW-SC-22 was able to freely cross the blood brain barrier, reaching a concentration of 10.8 μM, with a half-life of 1.22 hours. UNSW-SC-22 directly bound to c-MYC and MYCN proteins at low micromolar equilibrium dissociation constant (KD) values which was demonstrated by surface plasmon resonance assay. In a MYCN-driven MB mouse model, we found that UNSW-SC-22 as a single agent at 90 mg/kg decreased tumor growth and prolonged survival. Through RNA-sequencing, we identified 512 differentially expressed genes after in vitro treatment with UNSW-SC-22. Importantly, GSEA analysis revealed MYC targets and cell cycle pathways as highly downregulated pathways, and upregulation in the p53 pathway. Several genes, such as MELK and USP1 were validated as down-stream targets of UNSW-SC-22. As UNSW-SC-22 is a first-in-class anticancer drug, which we are currently testing extensively in other MYC-driven MB mice models. Conclusion: Collectively, our data strongly suggest that c-Myc and MYCN are the molecular targets of UNSW-SC compounds, and these compounds have strong potential to serve as specific targeted therapy to treat subgroup 3 and 4 of MB patients with c-Myc and MYCN overexpression and amplification. Citation Format: Sin Wi Ng, Satyanarayana Gadde, Qian Wang, Belamy B. Cheung, Glenn M. Marshall. A novel small molecule inhibitor targeting MYC oncogenic signaling as an enhancer of HDAC inhibitors for the treatment of high-risk medulloblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4599.

OMIC-03. TRANSLATIONAL CONTROL IN MYC AND MYCN MEDULLOBLASTOMA

Medulloblastoma has been extensively characterized at the genomic and transcriptional levels, but little is known about how alterations in translational control underlie tumor development. Myc and Mycn are often deregulated in medulloblastoma and play important roles in tumor initiation, maintenance and progression. Although both proteins have similar structures and are functionally redundant in hindbrain development, their amplification in cerebellar granule neural precursor cells leads to different medulloblastoma subtypes. In this project we are employing ribosome profiling on mouse medulloblastoma tumors generated from granule neural precursor cells with enforced expression of Myc or Mycn. Ribosome-protected mRNA sequencing allows us to quantitatively assess the specific transcripts regulated at the level of translation, identify translation regulatory sequences within the mammalian transcriptome, and understand genotype-to-phenotype processes. We discovered that Myc- and Mycn-driven tumors exhibit many more changes at the translational rather than at the transcriptional level. In particular, we found that Mycn-driven medulloblastoma upregulates the translation of Myc target genes, while mRNA levels of those genes show no difference between Myc- and Mycn-driven tumors. Furthermore, we find that the most significant translationally upregulated Myc target genes in the Mycn tumors are transcripts that encode ribosome biogenesis factors. We will further study the role of Myc and Mycn on translational regulation of the medulloblastoma transcriptome using our xenograft model of human iPSC-derived neuroepithelial stem cells overexpressing Myc or Mycn. Our goal is to understand the regulatory function of the translational landscape in Myc- and Mycn-driven medulloblastoma and to decipher the oncogenic signaling cascades leading to different medulloblastoma subtypes.

Abstract 2473: Combined BET-bromodomain and CDK2 inhibition in MYC-driven medulloblastoma

Abstract Misexpression of MYC genes (MYC and MYCN) occurs commonly in medulloblastoma (MB), the most frequent malignant childhood brain tumor. We previously showed that tumors are addicted to MYCN and that MYCN stabilization is required for MB development in mice (Swartling et al, Genes & Dev, 2010; Cancer Cell, 2012). Targeted MYCN suppression completely depleted MYCN-driven MB cells in vivo. Immediate transcriptional changes from such MYCN blockade were found by RNA-Seq and showed similarities to changes that occurred after CDK2 suppression or when inhibiting BET bromodomains. CDK2 and BET inhibitors both inhibited MYC protein expression and effectively induced cell cycle arrest or apoptosis. Compared with either agent alone a sustained combination treatment over 7-10 days displayed synergy and effectively abolished tumor cell proliferation in vitro. The combined treatment further reduced tumor growth in orthotopical MB transplants and significantly prolonged survival as compared to single agent therapy. Our data suggest that dual inhibition of CDK2 and BET Bromodomains could be a novel treatment approach in suppressing medulloblastoma by targeting MYC proteins. Citation Format: Sara Bolin, Anna Borgenvik, Camilla Persson, Gabriela Rosén, Anders Sundström, Jun Qi, James E. Bradner, William A. Weiss, Yoon-Jae Cho, Holger Weishaupt, Fredrik J. Swartling. Combined BET-bromodomain and CDK2 inhibition in MYC-driven medulloblastoma. [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 2473.

Downregulation of MYCN through PI3K Inhibition in Mouse Models of Pediatric Neural Cancer.

The MYCN proto-oncogene is associated with poor outcome across a broad range of pediatric tumors. While amplification of MYCN drives subsets of high-risk neuroblastoma and medulloblastoma, dysregulation of MYCN in medulloblastoma (in the absence of amplification) also contributes to pathogenesis. Since PI3K stabilizes MYCN, we have used inhibitors of PI3K to drive degradation. In this study, we show PI3K inhibitors by themselves induce cell cycle arrest, with modest induction of apoptosis. In screening inhibitors of PI3K against MYCN, we identified PIK-75 and its derivative, PW-12, inhibitors of both PI3K and of protein kinases, to be highly effective in destabilizing MYCN. To determine the effects of PW-12 treatment in vivo, we analyzed a genetically engineered mouse model for MYCN-driven neuroblastoma and a model of MYCN-driven medulloblastoma. PW-12 showed significant activity in both models, inducing vascular collapse and regression of medulloblastoma with prominent apoptosis in both models. These results demonstrate that inhibitors of lipid and protein kinases can drive apoptosis in MYCN-driven cancers and support the importance of MYCN as a therapeutic target.

Combined MYC and P53 Defects Emerge at Medulloblastoma Relapse and Define Rapidly Progressive, Therapeutically Targetable Disease

SummaryWe undertook a comprehensive clinical and biological investigation of serial medulloblastoma biopsies obtained at diagnosis and relapse. Combined MYC family amplifications and P53 pathway defects commonly emerged at relapse, and all patients in this group died of rapidly progressive disease postrelapse. To study this interaction, we investigated a transgenic model of MYCN-driven medulloblastoma and found spontaneous development of Trp53 inactivating mutations. Abrogation of p53 function in this model produced aggressive tumors that mimicked characteristics of relapsed human tumors with combined P53-MYC dysfunction. Restoration of p53 activity and genetic and therapeutic suppression of MYCN all reduced tumor growth and prolonged survival. Our findings identify P53-MYC interactions at medulloblastoma relapse as biomarkers of clinically aggressive disease that may be targeted therapeutically.

Abstract B78: Targeting the translational apparatus in MYCN-driven medulloblastoma

Abstract Amplification of MYCN is associated with high-risk SHH-driven and group 4 medulloblastoma, while mis-expression and amplification of MYC is associated with aggressive group 3 tumors. The biology of these tumors, particularly the group 3/4 tumors, is poorly understood, with no effective targeted therapies. We have recently developed a model of group 4 medulloblastoma in which high levels of mutationally-stabilized MYCN protein drive oncogenesis. We have shown in other tumor types that modulation of the translational apparatus downstream of the mammalian target of rapamycin (mTOR) is required for MYC-driven oncogenesis and that there is a synergistic relationship between MYC and the mTOR axis to promote tumor formation. mTOR signals through two primary outputs, rpS6 kinase (S6K) and the translation initiation factor eIF4E. A new class of clinical mTOR active site inhibitors disrupts signaling through both effectors, whereas the allosteric binder rapamycin disrupts only S6K. We describe genetic and therapeutic studies aimed at distinguishing the effects of S6K and eIF4E in both tumor formation and in therapy. These mechanistically distinct activities have enormous therapeutic implications, as in our medulloblastoma model, active site inhibitors of mTOR, but not rapamycin, demonstrate efficacy. Taken together, this supports the idea that signaling through S6K is dispensable, whereas eIF4E is required for MYC-driven oncogenesis. Citation Format: Justin G. Meyerowitz, W. Clay Gustafson, Erin Nekritz, Fredrik Swartling, Kevan M. Shokat, Davide Ruggero, William A. Weiss. Targeting the translational apparatus in MYCN-driven medulloblastoma. [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 B78.

Abstract 996: Metastasis and tumor recurrence from rare SOX9-positive cells in Group 4 medulloblastoma

Abstract Tumor recurrence is the main cause of death in children with medulloblastoma (MB). MYCN is a marker for poor prognosis which is amplified in SHH and Group 4 but rarely in WNT and Group 3 subgroups of MB. The exact mechanisms for MB relapse are not known but recent findings (Ramaswamy et al. Lancet Oncol., 2013) suggest temporal differences within the four MB subgroups. For instance, SHH tumors recur locally while SHH-independent Group 4 tumors develop distant metastases. In order to study these processes further, we used a previously described transgenic mouse model of MYCN-driven SHH-independent MB (GTML mouse) to recreate the metastatic recurrence of such brain tumors in vivo. We previously showed that the expression of SOX9 transcription factor correlates well with SHH tumors and that only a few scattered SOX9-positive cells are found in SHH-independent GTML tumors similarly to Group 4 human MB. By using a combination of Tet-ON and Tet-OFF inducible systems we managed to target this rare population of SOX9-positive GTML tumor cells in vivo. These cells were able to form a distant recurrence after the tumor bulk in the mice was removed by using dox-inducible oncogene depletion. These relapses showed similar morphology and immunoreactivity of defined MB markers but presented generally higher levels of SOX9 compared to the primary GTML tumors. Additionally, we overexpressed SOX9 in cerebellar stem cells transfected with a mutationally stabilized MYCNT58A and injected them back into the cerebellum of adult mice. Surprisingly, the MB-like tumors that formed migrated and developed in the forebrain in contrast to the cerebellar tumors induced by the same cells transfected with MYCNT58A only. These findings suggest that increased levels of SOX9 drives migration of MYCN-driven MB cells. A similar correlation was found in Group 4 MB patients where isolated metastases had consistently higher SOX9 levels as compared to the corresponding primary tumor. Significance: We have developed a new mouse model for MB recurrence. We show how a rare population of SOX9-positive cells in SHH-independent MB is capable of initiating recurrence after primary tumor removal. The relapsed MB has similar characteristics to the initial tumor but develops at a distant site in the brain, in line with recent data from human tumors. Further characterization of cells with such properties will help to improve our understanding of the mechanisms behind metastatic MB recurrence and to develop treatments against those migrating cell populations. Citation Format: Vasil Savov, Grammatiki Fotaki, Marc Remke, Adrian M. Dubuc, Vijay Ramaswamy, Matko Cancer, Holger Weishaupt, Michael D. Taylor, Fredrik J. Swartling. Metastasis and tumor recurrence from rare SOX9-positive cells in Group 4 medulloblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 996. doi:10.1158/1538-7445.AM2014-996

Abstract LB-201: MYC and TP53 defects interact at medulloblastoma relapse to define rapidly progressive disease and can be targeted therapeutically

Abstract Disease recurrence following multi-modal therapy is the single most adverse event in medulloblastoma (MB). Currently >90% of relapsing patients die, accounting for ∼10% of childhood cancer deaths. MB is heterogeneous at diagnosis, comprising four molecular subgroups with distinct clinicopathological and molecular features and outcomes. The relevance of these features at relapse is unknown, making characterisation, modelling and targeted therapy of relapse biology essential to improve outcomes. However, relapsed MBs are not routinely biopsied in clinical practice. We undertook a first comprehensive investigation of the molecular, clinical and pathological features of 29 relapsed MBs and paired tumour samples taken at diagnosis, including the assessment of features with established significance at diagnosis (e.g. chromosome 17 and TP53 pathway status, MYC family (MYC, MYCN) gene amplification, polyploidy, CTNNB1 mutation and molecular subgroup status). Molecular subgroup was concordant at diagnosis and relapse, however evidence of alteration of all other features examined was found in relapsed tumours, with the majority of changes (30/44) representing acquired high-risk events. Most notably, MYC family gene amplifications and TP53 pathway defects commonly emerged in combination at relapse following conventional multimodal treatment (P=0.02, 7/22, 32%) and predicted rapid progression to death (P=0.016). These observations suggested aberrant activation of MYC family genes synergizes with TP53 inactivation in the genesis of biologically aggressive MB. To investigate any such relationship, we examined Trp53 status in our transgenic mouse model of spontaneously-arising MYCN-driven MB (GTML; Glt1-tTA/TRE-MYCN-Luc). Somatic Trp53 mutations were found in 83% of tumors (n=10/12). Direct modelling of this interaction in GTML/Trp53KI/KI mice dramatically enhanced MB formation with 100% penetrance (43/43, median survival 47 days) in GTML/Trp53KI/KI versus 6% (3/50) in GTML; P<0.0001), faithfully mimicked clinicopathological characteristics of TP53-MYC family gene-associated relapsed human tumors, and validated the essential role of TP53 in potentiating the growth of MYCN-driven MB. Finally, therapeutic inhibition of Aurora-A kinase using MLN8237 in these tumours, and in derived neurospheres in vitro, promoted degradation of MYCN, reduced tumor growth and prolonged survival. In summary, while subgroup status remains stable, MBs display altered molecular, pathological and clinical features at relapse, and the emergence of combined TP53-MYC family gene defects is common following conventional therapy. Their association with rapid demise, coupled with their biological validation as driving and therapeutically exploitable events in a novel mouse MB model, strongly support further investigation and routine biopsy of relapse disease to drive future individualised therapeutic strategies. Citation Format: Rebecca M. Hill, Sanne Kuijper, Janet Lindsey, Ed C. Schwalbe, Karen Barker, Jessica Boult, Daniel Williamson, Zai Ahmad, Albert Hallsworth, Sarra Ryan, Evon Poon, Simon Robinson, Ruth Ruddle, Florence Raynaud, Louise Howell, Colin Kwok, Abhijit Joshi, Sarah Nicholson, Stephen Crosier, Stephen Wharton, Tom Jacques, Keith Robson, Antony Michalski, Darren Hargrave, Barry Pizer, Simon Bailey, Fredrik J. Swartling, Kevin Petrie, William A. Weiss, Louis Chesler, Steve Clifford. MYC and TP53 defects interact at medulloblastoma relapse to define rapidly progressive disease and can be targeted therapeutically. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-201. doi:10.1158/1538-7445.AM2014-LB-201

Abstract 3100: Glial origin for MYCN-driven medulloblastoma and targeted prosenescence therapies

Abstract MYCN-amplified medulloblastoma is a poor-prognosis childhood brain tumor with unknown cellular origin. Recently, we showed how MYCN overexpression could generate tumors with high penetrance from a glutamate transporter (GLT1) promoter in a transgenic model (GTML) of medulloblastoma. By using Rosa26-lsl-LacZ reporters we also showed that GLT1-positive neural stem cells represent presumed cells of tumor origin. Transgenic GTML mice generate SHH-independent medulloblastoma 2-3 months after birth. Before tumor onset GTML mice presented significantly more proliferation in thalamic forebrain cells and in Bergmann glia in the cerebellum as compared to corresponding normal brain. Strikingly, adult GTML mice also show proliferative capacity in the Bergmann glia cell population in response to MYCN oncogene activity, further connecting this population to medulloblastoma development. Retroviral tagging using the RCAS/tv-a system marked BLBP- or GFAP-positive tumor initiating cells and expression analysis could further determine genetic profiles of glial origin. Interestingly, MYCN levels and early proliferation of brain tumors could be reduced by specific CDK2-inhibition by Milciclib or by using the bromodomain inhibitor JQ1. Treatment studies revealed a need for sustained treatment over 7-10 days to effectively abolish tumor cell proliferation. Both treatment strategies induced tumor cell senescence as seen by SA-beta-gal staining and by downregulated levels of Lamin B1. Our data suggest that tumors generated arise from radial glia or Bergmann glia in this medulloblastoma model and that dual inhibition of CDK2 and bromodomains suppresses tumor growth by targeting MYCN. Citation Format: Sara M. Bolin, Jasmine Lau, Justin Chen, Vasil Savov, Anders I. Persson, Sanna-Maria Hede, William A. Weiss, Fredrik Swartling. Glial origin for MYCN-driven medulloblastoma and targeted prosenescence therapies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3100. doi:10.1158/1538-7445.AM2014-3100

METASTASIS AND TUMOR RECURRENCE FROM RARE SOX9-POSITIVE CELLS IN MYCN-DRIVEN MEDULLOBLASTOMA

Metastasis and tumor recurrence from rare SOX9-positive cells in MYCN-driven medulloblastoma

Abstract 357: A glial origin for medulloblastoma and inhibition of MYCN stabilization.

Abstract The origin of MYCN-driven medulloblastoma, a poor-prognosis childhood brain tumor is not known. We recently showed that MYCN could generate tumors with high penetrance from a glutamate transporter (GLT1) promoter in a transgenic model (GTML) of medulloblastoma. By using Rosa26-lsl-LacZ reporters we also showed that GLT1-positive neural stem cells (NSCs) from normal brain represent putative cells of tumor origin. GTML mice generate aggressive MYCN-driven medulloblastoma 2-3 months after birth. BrdU-labeling of 3-week-old GTML mice detected significantly more proliferation in thalamic forebrain cells and of BrdU/BLBP-double positive Bergmann glia in the cerebellum as compared to corresponding normal brain. Retroviral tagging with the RCAS/tv-a system marked BLBP- or GFAP-positive tumor initiating cells and expression analysis could further determine genetic profiles of tumor origin. Interestingly, MYCN levels and early proliferation of brain tumors could be reduced by specific inhibition of CDK2 or by using the bromodomain inhibitor JQ1. Both treatment strategies induced tumor cell senescence as seen by SA-beta-gal activity and by downregulated Lamin B1. Our results propose a radial glial or a Bergmann glial origin for MYCN-driven medulloblastoma and further suggest ways to inhibit tumor growth by repressing MYCN stabilization. Citation Format: Sara Bolin, Jasmine Lau, Vasil Savov, Justin Chen, Anders I. Persson, Sanna-Maria Hede, William A. Weiss, Fredrik J. Swartling. A glial origin for medulloblastoma and inhibition of MYCN stabilization. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 357. doi:10.1158/1538-7445.AM2013-357

Abstract 1426: MiRNA expression profiling of the murine GTML medulloblastoma model reveals similarities with human tumors and identifies novel candidate miRNAs

Abstract MicroRNAs (miRNAs) are small molecules which regulate gene expression post-transcriptionally and aberrant expression of many miRNAs is associated with different forms of cancer, including the childhood brain tumor medulloblastoma. Amplification and over-expression of either MYC or MYCN transcription factors are particularly frequent in some of the most clinically aggressive subtypes of this disease. The GTML transgenic mouse is a recently established model for highly aggressive subtypes of this tumor, in which over-expression of MYCN is targeted to the central nervous system utilizing a glutamate transporter (GLT1). Tumors exhibit two distinguishable histopathological phenotypes characteristic of specific human medulloblastoma subtypes: classic and large cell/anaplastic (LCA). Here, we evaluate the deregulation of miRNAs in tumors derived from GTML mice in order to assess the contribution of miRNAs to medulloblastoma pathogenesis. The expression of 591 miRNAs was analyzed using TaqMan low density arrays (Applied Biosystems) in 8 cerebella (controls) and 15 tumors exhibiting either LCA (n=12), and classic (n=3) phenotype. Cluster analyses performed on the overall expression of miRNAs showed a clear segregation between tumors and normal cerebella, but no definitive difference between tumor LCA and classic histopathology. We identified 37 miRNAs that were differentially expressed between tumors and controls (>2-fold; p-value< 0.05 after Bonferroni correction for multiple comparisons). Interestingly 25 of these miRNAs were conserved between mouse and human genomes, and 8 were previously associated with human medulloblastoma. Among 17 up-regulated miRNAs, four were members of the miR-17∼92a polycistronic cluster (a cluster of oncomiRs up-regulated by MYCN). We also identified miR-182, miR-96, miR-183 and miR-183*, all members of the same cluster, as up-regulated. Interestingly over-expression of miR-182 and miR-183 has been previously reported within the Wnt subtype of human medulloblastoma. Among 20 miRNAs under-expressed in the mouse tumors, miR-135a and miR-135b are conserved in human and predicted to target MYCC. Moreover miR-135a is deleted in human classic medulloblastoma with high frequency (33%). The association of miR-135b with human medulloblastoma has been previously suggested, although the role of this miRNA in medulloblastoma is controversial, since either deletion or amplification of the relevant genetic locus occurs in human tumors. Our study represents the first miRNA profiling of the recently established GTML mouse model of aggressive MYCN-driven medulloblastoma. The degree of similarity in miRNA expression observed between murine and human tumors indicates that this model is a suitable tool for the assessment of the biologic role and suitability of miRNAs as therapeutic targets in human medulloblastoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1426. doi:1538-7445.AM2012-1426

Abstract 3451: Interactions between N-myc and Sox9 promote medulloblastoma and determine cell fate decisions in cerebellar neural stem cells

Abstract The proto-oncogene MYCN (N-myc in mice) regulates normal brain development, and is often misexpressed in childhood brain tumors, including medulloblastoma. While most medulloblastoma arise independently of Sonic hedgehog (Shh), about ∼25% of are Shh-dependent and have a more favorable prognosis. We recently induced Shh-independent classic and large cell anaplastic medulloblastoma by driving MYCN from the glial Glutamate Transporter 1 promoter in a tet-regulated transgenic mouse model (Swartling et al, 2010). To characterize cell of origin, we used BrdU labeling in premalignant transgenic mice, demonstrating increased proliferation in cerebellar Bergmann glial cells. Bergmann glia showed high levels of glial marker GFAP as well as the transcription factor Sox9. Whereas neural stem cells (NSCs) from normal perinatal cerebellum contained a high proportion of Sox9-positive cells, primary cultures of tumor spheres showed low levels of Sox9, suggesting that MYCN suppresses Sox9 during medulloblastoma progression. To clarify mechanism, we transduced wild-type (WT) or mutationally-stabilized N-myc-T58A into GFAP-positive NSCs from perinatal murine cerebellum. Transplantation of N-myc-WT NSCs was insufficient for tumor formation. N-myc-T58A cerebellar NSCs generated both Shh-dependent and Shh-independent medulloblastoma, as a function of prenatal or postnatal cerebellar age, respectively. Expression of Sox9 correlated directly with Sonic Hedgehog (Shh) dependence in both murine and human medulloblastoma samples. In addition, forced expression of Sox9 induced Shh-dependence and self-renewal in the Shh-negative mouse medulloblastoma spheres. Our data suggest 1). A glial cell of origin for MYCN-driven medulloblastoma, and 2). Sox9 as a critical target for MYCN during medulloblastoma initiation, with activation of Sox9 leading to Shh-dependence, and inhibition of Sox9 leading to Shh-independent medulloblastoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3451. doi:10.1158/1538-7445.AM2011-3451

Pleiotropic role forMYCNin medulloblastoma

Medulloblastoma (MB) is the most common malignant brain tumor of childhood. Sonic Hedgehog (SHH) signaling drives a minority of MB, correlating with desmoplastic pathology and favorable outcome. The majority, however, arises independently of SHH and displays classic or large cell anaplastic (LCA) pathology and poor prognosis. To identify common signaling abnormalities, we profiled mRNA, demonstrating misexpression of MYCN in the majority of human MB and negligible expression in normal cerebella. We clarified a role in pathogenesis by targeting MYCN (and luciferase) to cerebella of transgenic mice. MYCN-driven MB showed either classic or LCA pathologies, with Shh signaling activated in approximately 5% of tumors, demonstrating that MYCN can drive MB independently of Shh. MB arose at high penetrance, consistent with a role for MYCN in initiation. Tumor burden correlated with bioluminescence, with rare metastatic spread to the leptomeninges, suggesting roles for MYCN in both progression and metastasis. Transient pharmacological down-regulation of MYCN led to both clearance and senescence of tumor cells, and improved survival. Targeted expression of MYCN thus contributes to initiation, progression, and maintenance of MB, suggesting a central role for MYCN in pathogenesis.