Subset Of Medulloblastomas Research Articles

REST-dependent downregulation of von Hippel-Lindau tumor suppressor promotes autophagy in SHH-medulloblastoma

The RE1 silencing transcription factor (REST) is a driver of sonic hedgehog (SHH) medulloblastoma genesis. Our previous studies showed that REST enhances cell proliferation, metastasis and vascular growth and blocks neuronal differentiation to drive progression of SHH medulloblastoma tumors. Here, we demonstrate that REST promotes autophagy, a pathway that is found to be significantly enriched in human medulloblastoma tumors relative to normal cerebella. In SHH medulloblastoma tumor xenografts, REST elevation is strongly correlated with increased expression of the hypoxia-inducible factor 1-alpha (HIF1α)—a positive regulator of autophagy, and with reduced expression of the von Hippel-Lindau (VHL) tumor suppressor protein – a component of an E3 ligase complex that ubiquitinates HIF1α. Human SHH-medulloblastoma tumors with higher REST expression exhibit nuclear localization of HIF1α, in contrast to its cytoplasmic localization in low-REST tumors. In vitro, REST knockdown promotes an increase in VHL levels and a decrease in cytoplasmic HIF1α protein levels, and autophagy flux. In contrast, REST elevation causes a decline in VHL levels, as well as its interaction with HIF1α, resulting in a reduction in HIF1α ubiquitination and an increase in autophagy flux. These data suggest that REST elevation promotes autophagy in SHH medulloblastoma cells by modulating HIF1α ubiquitination and stability in a VHL-dependent manner. Thus, our study is one of the first to connect VHL to REST-dependent control of autophagy in a subset of medulloblastomas.

The long non-coding RNA OTX2-AS1 promotes tumor growth and predicts response to BCL-2 inhibition in medulloblastoma

PurposePrimary brain tumors are a leading cause of cancer-related death in children, and medulloblastoma is the most common malignant pediatric brain tumor. The current molecular characterization of medulloblastoma is mainly based on protein-coding genes, while little is known about the involvement of long non-coding RNAs (lncRNAs). This study aimed to elucidate the role of the lncRNA OTX2-AS1 in medulloblastoma.MethodsAnalyses of DNA copy number alterations, methylation profiles, and gene expression data were used to characterize molecular alterations of OTX2-AS1 in medulloblastoma tissue samples. In vitro analyses of medulloblastoma cell models and orthotopic in vivo experiments were carried out for functional characterization of OTX2-AS1. High-throughput drug screening was employed to identify pharmacological inhibitors, while proteomics and metabolomics analyses were performed to address potential mechanisms of drug action.ResultsWe detected amplification and consecutive overexpression of OTX2 and OTX2-AS1 in a subset of medulloblastomas. In addition, OTX2-AS1 promoter methylation was linked to OTX2-AS1 expression. OTX2-AS1 knockout reduced medulloblastoma cell viability and cell migration in vitro and prolonged survival in the D283 orthotopic medulloblastoma mouse xenograft model. Pharmacological inhibition of BCL-2 suppressed the growth of OTX2-AS1 overexpressing medulloblastoma cells in vitro.ConclusionsOur study revealed a pro-tumorigenic role of OTX2-AS1 in medulloblastoma and identified BCL-2 inhibition as a potential therapeutic approach to target OTX2-AS1 overexpressing medulloblastoma cells.

Cross-Talk Between Histone Methyltransferases and Demethylases Regulate REST Transcription During Neurogenesis.

The RE1 Silencing Transcription Factor (REST) is a major regulator of neurogenesis and brain development. Medulloblastoma (MB) is a pediatric brain cancer characterized by a blockade of neuronal specification. REST gene expression is aberrantly elevated in a subset of MBs that are driven by constitutive activation of sonic hedgehog (SHH) signaling in cerebellar granular progenitor cells (CGNPs), the cells of origin of this subgroup of tumors. To understand its transcriptional deregulation in MBs, we first studied control of Rest gene expression during neuronal differentiation of normal mouse CGNPs. Higher Rest expression was observed in proliferating CGNPs compared to differentiating neurons. Interestingly, two Rest isoforms were expressed in CGNPs, of which only one showed a significant reduction in expression during neurogenesis. In proliferating CGNPs, higher MLL4 and KDM7A activities opposed by the repressive polycomb repressive complex 2 (PRC2) and the G9A/G9A-like protein (GLP) complex function allowed Rest homeostasis. During differentiation, reduction in MLL4 enrichment on chromatin, in conjunction with an increase in PRC2/G9A/GLP/KDM7A activities promoted a decline in Rest expression. These findings suggest a lineage-context specific paradoxical role for KDM7A in the regulation of Rest expression in CGNPs. In human SHH-MBs (SHH-α and SHH-β) where elevated REST gene expression is associated with poor prognosis, up- or downregulation of KDM7A caused a significant worsening in patient survival. Our studies are the first to implicate KDM7A in REST regulation and in MB biology.

502 Spy1 as a Novel Cell Cycle Target Against Medulloblastoma

INTRODUCTION: The current treatment strategies for medulloblastoma (MB) are highly aggressive, cytotoxic, and ultimately unsuccessful. One recent therapeutic approach is the use of synthetic cyclin-dependent kinase inhibitors (CKIs). Speedy (Spy1) is a cyclin-like protein that primes its complexes with CDKs insensitive to natural CKIs and promotes uncontrolled cell proliferation by overriding the safety cell cycle checkpoints. Spy1 has been implicated in the expansion of stem-like populations of therapy-resistant tumour initiating cells (TICs) in neural systems. METHODS: We established an in vitro neurosphere platform as well as in vivo high-throughput Zebrafish (ZF) xenograft model using ONS-76 and DAOY MB cell lines derived from three patients. Fluorescent Activated Cell Sorting (FACS) was used to enrich for specific CD133+/- or CD15 +/- populations of MB. Spy1 levels were manipulated using lentivirus to obtain overexpression or knockdown of Spy1 protein levels in the derived TICs as well as heterogenous cell populations. RESULTS: We found that downregulation of Spy1 levels in the employed cell lines resulted in a decrease in neurospheres formation efficiency as well as significantly lower tumour burden in Zebrafish when compared to control (p<0.001). Spy1 overexpression in CD133 or CD15 negative MB populations resulted in the upregulation of Msi1 mRNA levels. Finally, in vivo treatment of ZF xenografts with R-roscovitine, a synthetic CKI, demonstrated declined migration of Spy1 KD MB cells in comparison to control cells (p<0.05). CONCLUSION: This study shows that Spy1 plays a role in the regulation of stemness and aggressive characteristics of TICs in medulloblastoma. The assessed effect on CKI treatment efficacy on the tumours in vivo demonstrated that downregulation of Spy1 may sensitize, at least a subset of MB, to therapy. This work may contribute toward optimizing the design of CKIs and their use in combination therapy for patients with MB.

Melanotic Neuroectodermal Tumor of Infancy (MNTI) and Pineal Anlage Tumor (PAT) Harbor A Medulloblastoma Signature by DNA Methylation Profiling.

Simple SummaryMelanotic neuroectodermal tumor of infancy (MNTI) is a rare tumor of uncertain origin, morphologically overlapping other rare neoplasms such as pineal anlage tumor (PAT) and a subset of medulloblastomas (i.e., melanotic medulloblastoma). Despite the similarities with MNTI, their possible histogenetic relationship has been traditionally disregarded based on their aggressive behavior and dismal prognosis. The aim of this study was to further characterize the molecular features of MNTI and PAT based on DNA-methylation and copy number variation profiling analysis. We found that MNTI shares a methylation profile with group 3 high-risk medulloblastoma, and potentially with PAT, suggesting a common histogenesis. Most MNTIs in our series lacked copy number variation alterations, whereas their presence in the one PAT deserves further study in larger cohorts to better determine their impact in prognosis and biologic behavior.MNTI is a rare tumor of indeterminate histogenesis and molecular signature. We performed methylation and copy number variation (CNV) profiles in patients with MNTI (n = 7) and PAT (n = 1) compared to the methylation brain tumor classifier v11b4 (BT-C) and the medulloblastoma (MB) classifier group 3/4 v1.0 (MB3/4-C). The patients’ mean age was 8 months (range: 4–48). The BT-C classified five MNTIs and one PAT (relapse) as class family MB-G3/G4, subclass group 3 (score: >0.9). The remaining two MNTIs and PAT (primary) were classified as class family plexus tumor, subclass pediatric (scores: >0.45). The MB3/4-C classified all MNTIs as high-risk MB-G3, Subtype II (score: >0.45). The primary PAT was classified as subtype III (score: 0.99) and its relapse as subtype II/III. MNTI and PAT clustered close to MB-G3. CNV analysis showed multiple rearrangements in one PAT and two MNTIs. The median follow-up was 54 months (four MNTIs in remission, one PAT died). In conclusion, we demonstrated that MNTI shares a homogenous methylation profile with MB-G3, and possibly with PAT. The role of a multipotent progenitor cell (i.e., early cranial neural crest cell) in their histogenesis and the influence of the anatomical site, tumor microenvironment, and other cytogenetic events in their divergent biologic behavior deserve further investigation.

Abstract A21: Enhancing chemotherapeutic responses in CNS malignancy through suppression of hyperactive DNA damage repair pathways

Abstract Introduction: We are targeting DNA repair pathways to enhance existing chemoradiotherapeutic strategies against medulloblastoma (MB) and malignant glioma (MG), two highly invasive tumors of the central nervous system (CNS). Current methods to treat childhood medulloblastoma are highly intrusive and lead to poor quality of life while the three-year survival rate of patients afflicted with malignant glioma remains abysmal (<5%). Recurrence of these exceedingly malignant tumors is pervasive as they can adopt several mechanisms to resist anti-cancer therapeutics including activation and up-regulation of DNA repair pathways that act to resolve DNA damage elicited by radiation and chemotherapeutic agents (chemo-radiotherapy). DNA repair inhibitors like Poly (ADP-Ribose) Polymerase (PARPi), DNA-dependent protein kinase (DNA-PKi) and Ataxia Telengiectasia Mutated (ATMi) have shown promise to sensitize tumors to DNA damaging chemo-radiotherapeutics as these inhibitors specifically target single yet highly critical DNA repair response pathway enzymes. In combination with anti-tumor agents, these sensitizers can significantly augment anti-cancer therapeutic success. However, differing tumors have variable expression/activity of these enzymes and their corresponding repair pathway(s), therefore; their identification, characterization and the development of strategies to modulate their expression can enhance current anti-tumor treatment efficacy. Methods: I will identify specific differentially-regulated DNA repair enzymes/pathways by gene expression methodology. From these findings, I will inhibit these DNA repair enzymes via shRNA or enzyme-specific inhibitors (if available), to chemosensitize MB and MG cells to DNA damaging therapeutics in an effort to reduce DNA repair in these cells thereby boosting tumor genotoxicity and cell death. Extent of DNA damage will be measured using novel high-throughput DNA repair assays combined with unique cellular DNA damage reporters/sensors to facilitate these studies. Results: In comparing subsets of MB and MG cell lines with existing DNA repair inhibitors, I have found differing DNA repair pathways as potential targets to sensitize these CNS tumors to chemo-radiotherapeutics. MB cells rely on DNA-PK/SSBR (single strand break repair) dependent pathways to resolve induced genotoxicity, while MG utilizes the ATM/SSBR pathways. My focus is now to expand this dataset in additional pertinent tumors and to perform a detailed comparative analysis to identify specific highly active tumor-specific DNA repair enzymes with which to target in order to enhance tumor cell death. These include the use of DNA damage repair response PCR arrays to compare the expression levels of individual DNA repair enzyme/pathway members amongst these CNS tumors. Conclusion: I have identified unique DNA repair enzymes, which may mediate specific differential chemo-radioresistant phenotypes in MB and MG. An expanded analysis is currently underway to further differentiate DNA repair mechanisms and therapeutic responses between these two brain tumor types. I hope to translate these findings into pre-clinical models whereby my data may lead to identifying next-generation brain cancer treatments with improved patient survival and quality-of-life. Citation Format: Marina Mostafizar, Sachin Katyal. Enhancing chemotherapeutic responses in CNS malignancy through suppression of hyperactive DNA damage repair pathways [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr A21.

TB-08MECHANISMS OF IMMUNE RESISTANCE IN PEDIATRIC POSTERIOR FOSSA TUMORS

TB-08. MECHANISMS OF IMMUNE RESISTANCE IN PEDIATRIC POSTERIOR FOSSA TUMORS William Brugmann1, Alvaro Laureano2, Jonathan Torchia5, Rashieda Hatcher1, Bridget Kennis1, Soumen Khatua1, Wafik Zaky1, Gianluca Sbardella3, Judy Moyes1, Stewart Goldman4, Rishi Lulla4, Jason Fangusaro4, David Sandberg7, Lucia Silla2, Veena Rajaram6, Cynthia Hawkins5, Annie Huang5, Laurence Cooper1, Dean Lee1, and Vidya Gopalakrishnan1; University of Texas MD Anderson Cancer Center, Houston, TX, USA; Hospital de Clinicas de Porto Alegre, Porto Allegre, Brazil; Universita di Salerno, Fisciano, Salerno, Italy; Lurie Children’s Hospital, Northwestern University, Chicago, IL, USA; Hospital for Sick Children, Toronto, ON, Canada; University of Texas Southwestern Medical Center, Dallas, TX, USA; Universityof TexasatHouston, Houston, TX, USA Posterior fossa tumors (PFT) such as medulloblastoma (MB) and atypical teratoid rhabdoid tumor (ATRT) are aggressive and malignant pediatric brain tumors. Tumor recurrence is common with no available cure. The current standard-of-care can cause significant morbidities including a sharply elevated risk for secondary malignancies, ototoxicity and neurophysiological sequelae.Here,weofferanewtherapeutic approach forchildrenwith recurrent/refractory PFT,which harnesses the killing capacity of naturalkiller (NK) cells. This is predicated on our ability to deliver ex vivo expanded and activated clinical grade NK cells to the tumor microenvironment through an ommaya reservoir. In pre-clinical studies we observed that many MBs and ATRT were susceptible to NK cells in vitro and in vivo. These observations provided support for a first in humans Phase I trial (NCT02271711) to examine the safety and feasibility of infusing NK cells into the fourth ventricle of patients with recurrent/refractory PFTs. Our in vitro studies also identified resistance to NK-mediated lysis in a subset of MBs and ATRTs. This resistance was caused by the epigenetic upregulation of the growth and differentiation factor-1 (GDF-1), a member of the TGF-beta family, in ATRTs and the elevated expression of the programmed death-ligand 1 (PD-L1) in MBs. Their genetic and pharmacological manipulation countered tumor cell resistance to cytolysis by NK cells. Mis-regulation of these molecules was also observed in patient samples, setting the stage for their development as biomarkers to predict tumor response, and providing justification for subsequent clinical trials combining NK cells with epigenetic modifiers. Neuro-Oncology 18:iii169–iii173, 2016. doi:10.1093/neuonc/now084.5 #The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Bromodomain Inhibitor Review: Bromodomain and Extra-terminal Family Protein Inhibitors as a Potential New Therapy in Central Nervous System Tumors.

The bromodomain and extraterminal (BET) family proteins associate with transcriptional activation through interaction with acetylated chromatin, therefore playing a key role as epigenetic regulators. BET proteins serve to regulate the expression of importance oncogenes, including those involved in apoptosis as well as cell cycle progression. Due to this potential as an epigenetic target, small molecule inhibition of BET proteins have been investigated and demonstrate promising activity in both solid and hematologic malignancies, including brain tumors. Glioblastoma multiforme (GBM), subsets of medulloblastoma, and diffuse intrinsic pontine glioma (DIPG) are types of brain tumors with dismal prognoses, and as such have been the subjects of preclinical studies using BET inhibitors both in vivo and in vitro. While results from these preclinical investigations have shown promise, clinical trials are in early phases at this time. In this review, we will summarize the current literature on BET family proteins, their potential as therapeutic targets in brain tumors as well as other malignancies, and the preclinical and clinical investigations that have been undertaken to date.

WIP1 modulates responsiveness to Sonic Hedgehog signaling in neuronal precursor cells and medulloblastoma.

High-level amplification of the protein phosphatase PPM1D (WIP1) is present in a subset of medulloblastomas (MBs) that have an expression profile consistent with active Sonic Hedgehog (SHH) signaling. We found that WIP1 overexpression increased expression of Shh target genes and cell proliferation in response to Shh stimulation in NIH3T3 and cerebellar granule neuron precursor (cGNP) cells in a p53-independent manner. Thus, we developed a mouse in which WIP1 is expressed in the developing brain under control of the Neurod2 promoter (ND2:WIP1). The external granule layer in early post-natal ND2:WIP1 mice exhibited increased proliferation and expression of Shh downstream targets. MB incidence increased and survival decreased when ND2:WIP1 mice were crossed with a Shh-activated MB mouse model. Conversely, Wip1 knock out significantly suppressed MB formation in two independent mouse models of Shh-activated MB. Furthermore, Wip1 knock-down or treatment with a WIP1 inhibitor suppressed the effects of Shh stimulation and potentiated the growth inhibitory effects of SHH pathway-inhibiting drugs in Shh-activated MB cells in vitro. This suggests an important cross-talk between SHH and WIP1 pathways that accelerates tumorigenesis and supports WIP1 inhibition as a potential treatment strategy for MB.

The Ketogenic Diet Does Not Affect Growth of Hedgehog Pathway Medulloblastoma in Mice.

The altered metabolism of cancer cells has long been viewed as a potential target for therapeutic intervention. In particular, brain tumors often display heightened glycolysis, even in the presence of oxygen. A subset of medulloblastoma, the most prevalent malignant brain tumor in children, arises as a consequence of activating mutations in the Hedgehog (HH) pathway, which has been shown to promote aerobic glycolysis. Therefore, we hypothesized that a low carbohydrate, high fat ketogenic diet would suppress tumor growth in a genetically engineered mouse model of medulloblastoma. However, we found that the ketogenic diet did not slow the growth of spontaneous tumors or allograft flank tumors, and it did not exhibit synergy with a small molecule inhibitor of Smoothened. Serum insulin was significantly reduced in mice fed the ketogenic diet, but no alteration in PI3 kinase activity was observed. These findings indicate that while the ketogenic diet may be effective in inhibiting growth of other tumor types, it does not slow the growth of HH-medulloblastoma in mice.

Molecular Subgrouping of Medulloblastoma: Impact Upon Research and Clinical Practice.

Medulloblastoma, the most common primary pediatric malignant brain tumor is a molecularly heterogeneous disease with different developmental origins, distinct phenotypes, diverse biological behaviour, and contrasting clinical outcomes. The current clinico-radiological risk-classification fails to take account of this heterogeneity and existent prognostic variability. It is widely accepted that dysregulation of normal developmental processes constitute a key mechanism of tumorigenesis in at least a subset of medulloblastomas. Several attempts at biological classification have successfully identified distinct subgroups with subgroup-specific gene signatures, demographics, histologic subtypes, and rates of metastases. Several research groups have classified medulloblastoma into molecular subgroups using a variety of different genomic approaches and platforms such as gene expression profiling, microRNA profiling and methylation arrays. Recently, a consensus has emerged that classifies medulloblastoma into four distinct molecular subgroups named as wingless (WNT), sonic hedgehog (SHH), Group 3 and Group 4 respectively. However, such integrative approaches have limited applicability in the clinic due to the need of fresh-frozen tissues and elaborate molecular biology tools. In parallel, some groups have proposed and validated traditional antibody-based approaches using immunohistochemistry on archival specimen for rapid and reliable molecular subgrouping to be applied in any basic neuropathology laboratory. Heterogeneity within each of these four consensus subgroups has also been demonstrated that needs to be considered in the design of future clinical trials. There is a compelling need to integrate molecular biomarkers with clinico-pathologic outcome indicators to refine risk-stratification as well as develop novel molecularly targeted agents for optimizing therapeutic index and personalizing therapy.

Abstract IA17: Targeting placental growth factor/ neuropilin-1 pathway inhibits growth and spread of medulloblastoma

Abstract Background: Medulloblastoma (MB) is the most common pediatric brain tumor and a heterogeneous disease. Several studies have characterized tumor genetic heterogeneity with the hope of identifying targets for personalized therapy. In fact, inhibitors of Sonic Hedgehog (Shh) - a developmental pathway aberrantly activated in a subset of MBs - are currently being evaluated. Unfortunately, mutations and potential developmental adverse effects in the pediatric population can limit these therapies. We propose targeting the tumor stroma as an efficacious alternative treatment option. In particular, we aim to identify and target crucial cancer cell-stromal cell crosstalk signaling that sustains tumor growth and spread. In this context, we have evaluated the effects of blocking signaling of Placental Growth Factor (PIGF) - a member of the vascular endothelial growth factor (VEGF) family - that is dispensable in development but relevant during disease, in the growth and progression of MB. Here, we show the expression of PIGF and its receptor Neuropilin-1 (NRP-1) across primary tumors; dissect the mechanisms that regulate PIGF secretion and downstream signaling; and demonstrate the dramatic efficacy of specific anti-PIGF and anti-NRP-1 blocking antibodies in three MB models in vivo. Methods: Expression of PIGF and NRP-1 was analyzed in a cohort of 32 surgical samples of pediatric MBs by immunohistochemistry, array-comparative genomic hybridization, and deep-gene sequencing. The correlation between NRP-1 expression and 5-year overall survival was evaluated retrospectively in an independent, clinically annotated cohort of 42 MBs. Human orthotopic xenograft (D283-Med; D341-Med) and spontaneous (Smo/Smo) established tumors were treated with anti-PIGF (Thrombogenics or Genentech) or anti-NRP-1 (Genentech) antibodies. Tumor growth, progression and response to therapy was followed by 1) clinical evaluation of posterior fossa symptoms and body weight; 2) whole-body luminescence and blood GLuc levels; 3) small animal MRI; or 4) Optical Frequency Domain Imaging (OFDI). Results: We report that: 1) PIGF is expressed by more than 90% of primary pediatric MBs, regardless of their genetic subtype or histological classification; and high expression of NRP-1 correlates with poor overall survival of patients; 2) MB cells release Shh ligands that stimulate paracrine production of stromal PIGF by cerebellar granule neurons; 3) PIGF signals through NRP-1 - and independent of vascular endothelial growth factor receptor 1 (VEGFR1) - to activate the Erk/MEK pathway and sustain tumor survival; 4) Blockade of PIGF or NRP-1 with specific antibodies causes dramatic regression of MB, decreases spinal metastatic burden, and prolongs survival in in vivo orthotopic and spontaneous mouse models. Conclusion: This work identifies PIGF as the first common target across MB subgroups and provides insight into the critical role of tumor-stroma interactions. The study demonstrates that targeting PIGF/NRP-1 may provide a safe and efficient treatment option for pediatric MBs and lays the ground for evaluation of anti-PIGF therapy in clinical trials. Reference: M. Snuderl et al, Cell, 152(2):1065-76 (2013). Citation Format: Matija Snurdel, Ana Batista, Nathaniel Kirkpatrick, Carmen Almodovar, Lars Riedemann, Teresa Peterson, Napoleone Ferrara, Michael Klagsbrun, Dan G. Duda, Dai Fukumura, Lei Xu, Peter Carmeliet, Rakesh Jain. Targeting placental growth factor/ neuropilin-1 pathway inhibits growth and spread of 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 IA17.

Smoothened (SMO) receptor mutations dictate resistance to vismodegib in basal cell carcinoma

Basal cell carcinomas (BCCs) and a subset of medulloblastomas are characterized by loss-of-function mutations in the tumor suppressor gene, PTCH1. PTCH1 normally functions by repressing the activity of the Smoothened (SMO) receptor. Inactivating PTCH1 mutations result in constitutive Hedgehog pathway activity through uncontrolled SMO signaling. Targeting this pathway with vismodegib, a novel SMO inhibitor, results in impressive tumor regression in patients harboring genetic defects in this pathway. However, a secondary mutation in SMO has been reported in medulloblastoma patients following relapse on vismodegib to date. This mutation preserves pathway activity, but appears to confer resistance by interfering with drug binding.Here we report for the first time on the molecular mechanisms of resistance to vismodegib in two BCC cases. The first case, showing progression after 2 months of continuous vismodegib (primary resistance), exhibited the new SMO G497W mutation. The second case, showing a complete clinical response after 5 months of treatment and a subsequent progression after 11 months on vismodegib (secondary resistance), exhibited a PTCH1 nonsense mutation in both the pre- and the post-treatment specimens, and the SMO D473Y mutation in the post-treatment specimens only. In silico analysis demonstrated that SMOG497W undergoes a conformational rearrangement resulting in a partial obstruction of the protein drug entry site, whereas the SMO D473Y mutation induces a direct effect on the binding site geometry leading to a total disruption of a stabilizing hydrogen bond network. Thus, the G497W and D473Y SMO mutations may represent two different mechanisms leading to primary and secondary resistance to vismodegib, respectively.

Arhgap36-dependent activation of Gli transcription factors

Hedgehog (Hh) pathway activation and Gli-dependent transcription play critical roles in embryonic patterning, tissue homeostasis, and tumorigenesis. By conducting a genome-scale cDNA overexpression screen, we have identified the Rho GAP family member Arhgap36 as a positive regulator of the Hh pathway in vitro and in vivo. Arhgap36 acts in a Smoothened (Smo)-independent manner to inhibit Gli repressor formation and to promote the activation of full-length Gli proteins. Arhgap36 concurrently induces the accumulation of Gli proteins in the primary cilium, and its ability to induce Gli-dependent transcription requires kinesin family member 3a and intraflagellar transport protein 88, proteins that are essential for ciliogenesis. Arhgap36 also functionally and biochemically interacts with Suppressor of Fused. Transcriptional profiling further reveals that Arhgap36 is overexpressed in murine medulloblastomas that acquire resistance to chemical Smo inhibitors and that ARHGAP36 isoforms capable of Gli activation are up-regulated in a subset of human medulloblastomas. Our findings reveal a new mechanism of Gli transcription factor activation and implicate ARHGAP36 dysregulation in the onset and/or progression of GLI-dependent cancers.

Therapeutic Targeting of Developmental Signaling Pathways in Medulloblastoma: Hedgehog, Notch, Wnt and Myc

During development of the cerebellum, a large number of molecular factors interact to produce an intricate brain structure. Many of these developmentally significant genes are members of signaling cascades implicated in the formation and growth of the embryonal brain tumor medulloblastoma. Genes controlling critical developmental pathways such as Hedgehog, Notch, Wnt, and Myc are known to be overexpressed and/or genetically altered in subsets of medulloblastoma. These pathways are also linked by their ability to induce or maintain stem-cell phenotypes in normal development. Their over-activation in tumors can lead to proliferation, invasion, altered metabolism, and evasion of treatment-induced cell death. The importance of these signaling cascades in medulloblastoma cells makes them attractive targets for therapeutic intervention. The development of therapeutic agents targeting these pathways may lead to improvement in patient survival and a reduction in the intensity of highly morbid radiation and chemotherapy that patients currently receive. In this review, we discuss a number of approaches to targeting these pathways in medulloblastoma. Keywords: Non-genotoxic therapy, primitive neuroectodermal tumor, gamma secretase, tankyrase, glutamine, cyclopamine, itraconazole

Novel Agents Targeting the IGF-1R/PI3K Pathway Impair Cell Proliferation and Survival in Subsets of Medulloblastoma and Neuroblastoma

The receptor tyrosine kinase (RTK)/phosphoinositide 3-kinase (PI3K) pathway is fundamental for cancer cell proliferation and is known to be frequently altered and activated in neoplasia, including embryonal tumors. Based on the high frequency of alterations, targeting components of the PI3K signaling pathway is considered to be a promising therapeutic approach for cancer treatment. Here, we have investigated the potential of targeting the axis of the insulin-like growth factor-1 receptor (IGF-1R) and PI3K signaling in two common cancers of childhood: neuroblastoma, the most common extracranial tumor in children and medulloblastoma, the most frequent malignant childhood brain tumor. By treating neuroblastoma and medulloblastoma cells with R1507, a specific humanized monoclonal antibody against the IGF-1R, we could observe cell line-specific responses and in some cases a strong decrease in cell proliferation. In contrast, targeting the PI3K p110α with the specific inhibitor PIK75 resulted in broad anti-proliferative effects in a panel of neuro- and medulloblastoma cell lines. Additionally, sensitization to commonly used chemotherapeutic agents occurred in neuroblastoma cells upon treatment with R1507 or PIK75. Furthermore, by studying the expression and phosphorylation state of IGF-1R/PI3K downstream signaling targets we found down-regulated signaling pathway activation. In addition, apoptosis occurred in embryonal tumor cells after treatment with PIK75 or R1507. Together, our studies demonstrate the potential of targeting the IGF-1R/PI3K signaling axis in embryonal tumors. Hopefully, this knowledge will contribute to the development of urgently required new targeted therapies for embryonal tumors.

Mismatch repair deficiency: a temozolomide resistance factor in medulloblastoma cell lines that is uncommon in primary medulloblastoma tumours

Background:Tumours are responsive to temozolomide (TMZ) if they are deficient in O6-methylguanine-DNA methyltransferase (MGMT), and mismatch repair (MMR) proficient.Methods:The effect of TMZ on medulloblastoma (MB) cell killing was analysed with clonogenic survival assays. Expression of DNA repair genes and enzymes was investigated using microarrays, western blot, and immunohistochemistry. DNA sequencing and promoter methylation analysis were employed to investigate the cause of loss of the expression of MMR gene MLH1.Results:Temozolomide exhibited potent cytotoxic activity in D425Med (MGMT deficient, MLH1 proficient; IC50=1.7 μℳ), moderate activity against D341Med (MGMT proficient, MLH1 deficient), and DAOY MB cells (MGMT proficient, MLH1 proficient). MGMT inhibitor O6-benzylguanine sensitised DAOY, but not D341Med cells to TMZ. Of 12 MB cell lines, D341Med, D283Med, and 1580WÜ cells exhibited MMR deficiency due to MLH1 promoter hypermethylation. DNA sequencing of these cells provided no evidence for somatic genetic alterations in MLH1. Expression analyses of MMR and MGMT in MB revealed that all patient specimens (n=74; expression array, n=61; immunostaining, n=13) are most likely MMR proficient, whereas some tumours had low MGMT expression levels (according to expression array) or were totally MGMT deficient (3 out of 13 according to immunohistochemistry).Conclusion:A subset of MB may respond to TMZ as some patient specimens are MGMT deficient, and tumours appear to be MMR proficient.

Targeting the enhancer of zeste homologue 2 in medulloblastoma

Enhancer of zeste homologue 2 (EZH2) is the catalytic subunit of Polycomb repressive complex 2 that catalyzes the trimethylation of histone H3 on Lys 27, and represses gene transcription. EZH2 enhances cancer-cell proliferation and regulates stem cell maintenance and differentiation. Here, we demonstrate that EZH2 is highly expressed in medulloblastoma, a highly malignant brain tumor of childhood, and this altered expression is correlated with genomic gain of chromosome 7 in a subset of medulloblastoma. Inhibition of EZH2 by RNAi suppresses medulloblastoma tumor cell growth. We show that 3-deazaneplanocin A, a chemical inhibitor of EZH2, can suppress medulloblastoma cell growth partially by inducing apoptosis. Suppression of EZH2 expression diminishes the ability of tumor cells to form spheres in culture and strongly represses the ability of known oncogenes to transform neural stem cells. These findings establish a role of EZH2 in medulloblastoma and identify EZH2 as a potential therapeutic target especially in high-risk tumors.

Role of MXD3 in Proliferation of DAOY Human Medulloblastoma Cells

A subset of medulloblastomas, the most common brain tumor in children, is hypothesized to originate from granule neuron precursors (GNPs) in which the sonic hedgehog (SHH) pathway is over-activated. MXD3, a basic helix-look-helix zipper transcription factor of the MAD family, has been reported to be upregulated during postnatal cerebellar development and to promote GNP proliferation and MYCN expression. Mxd3 is upregulated in mouse models of medulloblastoma as well as in human medulloblastomas. Therefore, we hypothesize that MXD3 plays a role in the cellular events that lead to medulloblastoma biogenesis. In agreement with its proliferative role in GNPs, MXD3 knock-down in DAOY cells resulted in decreased proliferation. Sustained overexpression of MXD3 resulted in decreased cell numbers due to increased apoptosis and cell cycle arrest. Structure-function analysis revealed that the Sin3 interacting domain, the basic domain, and binding to E-boxes are essential for this activity. Microarray-based expression analysis indicated up-regulation of 84 genes and down-regulation of 47 genes. Potential direct MXD3 target genes were identified by ChIP-chip. Our results suggest that MXD3 is necessary for DAOY medulloblastoma cell proliferation. However, increased level and/or duration of MXD3 expression ultimately reduces cell numbers via increased cell death and cell cycle arrest.

Abstract 1431: A novel tumor-suppressor role of MyoD, a muscle differentiation factor, in mouse models of medulloblastoma

Abstract Medulloblastoma, an embryonal tumor of the cerebellum, is the most common pediatric brain cancer. For decades, it has been known that a small subset of medulloblastomas present with myogenic differentiation. However, the mechanistic basis and significance of this biological anomaly remain to be determined. We have identified hitherto unknown expression of muscle differentiation factor, MyoD in cerebellar tumors from mouse models of medulloblastoma as well as in a subset of human medulloblastomas. At a cellular level, MyoD is expressed in mitotically active cells and intriguingly, in mouse tumors does not activate its canonical targets involved in the muscle differentiation program. To understand the functional consequence of this finding in medulloblastoma genesis, we have conducted genetic studies to assess tumor formation in the SmoA1 and SmoA2 medulloblastoma models, on a MyoD-deficient background. We demonstrate that MyoD acts as a haploinsufficient tumor suppressor as the MyoD wt/-; SmoA1 and MyoD wt/-; SmoA2 mice have an accelerated onset of tumors compared to MyoD wt/wt; SmoA1 or MyoD wt/wt; SmoA2 mice respectively. Our findings suggest that the expression of MyoD might be an oncogene-induced compensatory response in the cerebellum to suppress tumor growth by restoring balance between proliferation and differentiation, similar to the role of some other bHLH transcription factors. Harnessing this latent tumor suppressor network to induce differentiation or cell cycle arrest holds promise for future therapeutic interventions. 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 1431. doi:1538-7445.AM2012-1431