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Supplementary Figure S1 from Inhibition of Phosphatidylinositol 3-Kinase Destabilizes Mycn Protein and Blocks Malignant Progression in Neuroblastoma

Medulloblastoma (MB) is the most common pediatric brain malignancy. MB comprises 5 major subgroups known as WNT, SHH p53wt, SHH p53mut, Group 3 and Group 4. Among the four MB subgroups SHH group is the most dominant molecular subgroup in infants and adults. These tumors are proposed to arise from cerebellar granule neuron precursors (CGNPs), whose developmental expansion requires SHH signaling from the neighboring Purkinje neurons. Previous reports suggest that SHH group features a unique tumor microenvironment compared with other MB groups. To better understand how SHH MB cells interact with Tumor Microenvironment, we performed cytokine array analysis of culture media from SHH group Patient Tumor cells, spontaneous SHH MB mouse tumor cells and SHH MB cell lines. Further, confirmed these results using ELISA, Western blot, and immunofluorescence from human SHH MB cell lines, Smo/A1 mouse tumor primary cells and PZp53Med cell lines. In continuation to the observation of IGFBP2 expression in various cell types in single cell analysis, we analyzed the presence of IGFBP2 in astrocytes using Smo/A1 mouse tumor Immunohistochemistry. Our data showed increased levels of IGFBP2 produced by SHH MB cell lines compared to others. We analyzed the role of IGFBP2 in SHH MB tumor growth and metastasis. IGFBP2 knock-down stable cell lines showed phenotypic changes including reduced cell proliferation, cell migration and EMT. Further western blot analysis of IGFBP2 KD cells showed reduced expression of EMT markers also reduced the activation of STAT3. Our preliminary in vitro data suggest IGFBP2 exerts it metastasis-promoting role in SHH MB by regulating the expression of EMT marker proteins and matrix remodeling proteins. Further functional studies suggest that in SHH MB, IGFBP2 may regulate a STAT3-mediated EMT program to metastasize. These findings provide a strong rationale for further pursuing how IGFBP2 promotes medulloblastoma tumor cell growth and migration in vivo.

BACKGROUND Medulloblastoma (MB) is the most common pediatric brain tumor. MB can be dividing into 4 genetically distinct subgroups (Sonic Hedgehog (SHH), Wingless (WNT), Group 3, and Group 4). Most patients receive radiation and cisplatin backbone therapy. SHH and Group3 patients demonstrate poor prognosis, especially in cases of MYC amplification or p53 mutation. Given the continued reliance on DNA damage therapies, and our groups previous findings that YB1 regulates IGF2 transcription in SHH MB, we chose to investigate the role of YB1 in the DNA damage response and we performed RIPseq to understand mRNAs regulated by YB1 that may be targeted as novel therapeutics. METHODS Cell lines include primary MBCs derived from NeuroD2-SmoA1 SHH mice, ONS-76 human SHH, Daoy human SHH, D341 and D425 Human Group 3. YB1 overexpression or knockdown followed by radiation and assessment of yH2AX (a marker of DNA de-condensation following damage), Comet assay (a marker of physical damage), and proliferation time courses were used to assess effects of YB1 modulation on radiation response. RIPseq and YB1 knockdown were used to assess the effects of YB1 on PLXND1 levels. Scratch assay and western blotting of EMT markers were used to assess effects of PLXND1 silencing on migration. RESULTS (Radiation Studies) Overexpression of YB1 in primary MBCs followed by allograft into BL6 mice results in decreased overall survival. YB1 silencing followed by radiation results in faster resolution of yH2AX, faster resolution of damage, and lack of pRPA32 accumulation. YB1 silencing sensitizes cells to radiation resulting in substantial decreases in proliferation. (RNA Binding Protein Studies) YB1 binds and positively regulates PLXND1 translation. Silencing PLXND1 results in decreased migration and EMT mark expression. CONCLUSIONS YB1 drives a more error prone non-homologous End-Joining based mechanism of repair and binds and regulates PLXND1 post-transcriptionally. PLXND1 drives a migratory phenotype in SHH MB.

Children with Group 3 medulloblastoma (MB) have a very poor long-term outcome and many do not survive beyond 5 years. Several drivers for Group 3 MB have been identified but none have resulted in targeted therapy to date. LIN28B is a stem cell factor upregulated in Group 3 MB and is associated with worse survival. Here we investigate the role of the LIN28B pathway in Group 3 MB development. Pharmacologic inhibition of the LIN28B pathway is feasible and may provide a unique opportunity to target this tumor. Using LIN28B knockdown and overexpression in Group 3 MB cells we test LIN28B’s effect on proliferation, self-renewal and metastasis. We investigate the effect of LIN28B knockdown on survival as well as proliferation and apoptosis markers using orthotopic xenografts in vivo. We also investigate the role of let-7 and its downstream target PBK on Group 3 MB proliferation. Finally, we use a LIN28 inhibitor 1632 and a PBK inhibitor HITOPK032 to treat Group 3 MB cell lines and then assess their impact on proliferation and apoptosis. We find that down-regulation of LIN28B or PBK using shRNA results in significant reduction in cell proliferation. In contrast overexpression of LIN28B increases Group 3 cell proliferation and tumor sphere formation. LIN28B knockdown also significantly (p< 0.01) increases survival in mice with orthotopic Group 3 tumors. The LIN28 inhibitor 1632 also leads to significant reduction in MB growth through decreased cell cycle entry and increased apoptosis. In addition, HITOPK032 also demonstrates significant reduction in Group 3 MB cell proliferation at low micromolar concentration. We also demonstrate that HITOPK032 in combination with CDK 4/6 inhibition can additively inhibit proliferation of Group 3 MB cells. Our study establishes a critical role for the LIN28B-let-7-PBK pathway in Group3 MB and provides encouraging preliminary preclinical results for drugs that target this pathway.

Medulloblastoma is a malignant pediatric tumor that arises from neural progenitors in the cerebellum. Despite a five-year survival rate of ~70%, nearly all patients incur adverse side effects from current treatment strategies that drastically impact quality of life. Roughly one-third of medulloblastoma are driven by aberrant activation of the Sonic Hedgehog (SHH) signaling pathway. However, the scarcity of genetic mutations in medulloblastoma has led to investigation of other mechanisms contributing to cancer pathogenicity including epigenetic regulation of gene expression. Here, we show that Helicase, Lymphoid Specific (HELLS), a chromatin remodeler with epigenetic functions including DNA methylation and histone modification, is induced by Sonic Hedgehog (SHH) in SHH-dependent cerebellar progenitor cells and the developing murine cerebella. HELLS is also up-regulated in mouse and human SHH medulloblastoma. Others have shown that HELLS activity generally results in a repressive chromatin state. Our results demonstrate that increased expression of HELLS in our experimental systems is regulated by the oncogenic transcriptional regulator YAP1 downstream of Smoothened, the positive transducer of SHH signaling. Elucidation of HELLS as one of the downstream effectors of the SHH pathway may lead to novel targets for precision therapeutics with the promise of better outcomes for SHH medulloblastoma patients.

Medulloblastomas, the most common malignant pediatric brain tumors, have been genetically defined into four subclasses, namely WNT-activated, Sonic Hedgehog (SHH)-activated, Group 3, and Group 4. Approximately 30% of medulloblastomas have aberrant SHH signaling and thus are referred to as SHH-activated medulloblastoma. The tumor suppressor gene TP53 has been recently recognized as a prognostic marker for patients with SHH-activated medulloblastoma; patients with mutant TP53 have a significantly worse outcome than those with wild-type TP53. It remains unknown whether p53 activity is impaired in SHH-activated, wild-type TP53 medulloblastoma, which is about 80% of the SHH-activated medulloblastomas. Utilizing the homozygous NeuroD2:SmoA1 mouse model with wild-type Trp53, which recapitulates human SHH-activated medulloblastoma, it was discovered that the endogenous Inhibitor 2 of Protein Phosphatase 2A (SET/I2PP2A) suppresses p53 function by promoting accumulation of phospho-MDM2 (S166), an active form of MDM2 that negatively regulates p53. Knockdown of I2PP2A in SmoA1 primary medulloblastoma cells reduced viability and proliferation in a p53-dependent manner, indicating the oncogenic role of I2PP2A. Importantly, this mechanism is conserved in the human medulloblastoma cell line ONS76 with wild-type TP53. Taken together, these findings indicate that p53 activity is inhibited by I2PP2A upstream of PP2A in SHH-activated and TP53-wildtype medulloblastomas. Implications: This study suggests that I2PP2A represents a novel therapeutic option and its targeting could improve the effectiveness of current therapeutic regimens for SHH-activated or other subclasses of medulloblastoma with wild-type TP53.

BACKGROUND: Children with Group 3 medulloblastoma (MB) have a very poor long-term outcome and many do not survive beyond 5 years. Several drivers for Group 3 MB have been identified but none have resulted in targeted therapy to date. LIN28B is a stem cell factor that is upregulated in Group 3 medulloblastoma and is associated with worse survival. Here we investigate the role of the LIN28B pathway in Group 3 MB development. Pharmacologic inhibition of the LIN28B pathway is feasible and may provide a unique opportunity to target this tumor. METHODS: Using LIN28B knockdown and overexpression in G3 MB cells we test LIN28B’s effect on proliferation, self-renewal and metastasis. Similarly, we used shRNAs targeting PBK and demonstrate a similar effect on G3 MB growth. We also investigate the role of let-7 as a target of LIN28B by introducing let-7 mimetics and overexpression vectors into MB cells. Finally, we use a LIN28 inhibitor 1632 and a PBK inhibitor HITOPK032 to treat G3 MB cell lines and then assess their impact on proliferation and apoptosis. RESULTS: We find that down-regulation of LIN28B or PBK using shRNA results in significant reduction in cell proliferation. In contrast overexpression of LIN28B increases Group 3 cell proliferation and tumor sphere formation. LIN28B knockdown also significantly (p< 0.01) increases survival in mice with orthotopic Group 3 tumors. The LIN28 inhibitor 1632 also leads to significant reduction in G3 MB growth through decreased cell cycle entry and increased apoptosis. In addition, HITOPK032 also demonstrates significant reduction in Group 3 MB cell proliferation at low (nanomolar to low micromolar) concentration. CONCLUSIONS: Our study establishes a critical role for the LIN28B-let-7-PBK pathway in Group3 MB and provides encouraging preliminary preclinical results for drugs that target this pathway.

BackgroundMedulloblastoma (MB) is the most common malignant pediatric brain tumor that originates in the cerebellum and brainstem. Frequent somatic mutations and deregulated expression of epigenetic regulators in MB highlight the substantial role of epigenetic alterations. 5-hydroxymethylcytosine (5hmC) is a highly abundant cytosine modification in the developing cerebellum and is regulated by ten-eleven translocation (TET) enzymes.ResultsWe investigate the alterations of 5hmC and TET enzymes in MB and their significance to cerebellar cancer formation. We show total abundance of 5hmC is reduced in MB, but identify significant enrichment of MB-specific 5hmC marks at regulatory regions of genes implicated in stem-like properties and Nanog-binding motifs. While TET1 and TET2 levels are high in MBs, only knockout ofTet1in the smoothened (SmoA1)mouse model attenuates uncontrolled proliferation, leading to a favorable prognosis. The pharmacologicalTet1inhibition reduces cell viability andplatelet-derived growth factorsignaling pathway-associated genes.ConclusionsThese results together suggest a potential key role of 5hmC and indicate an oncogenic nature for TET1 in MB tumorigenesis, suggesting it as a potential therapeutic target for MBs.

The sonic hedgehog subtype of medulloblastoma (SHH MB) is associated with treatment failure and poor outcome. Current strategies utilizing whole brain radiation therapy result in deleterious off-target effects on the normal developing childhood brain. Most conventional chemotherapies remain limited by ineffective blood-brain barrier (BBB) penetrance. These challenges signify an unmet need for drug carriers that can cross the BBB and deliver drugs to targeted sites with high drug-loading efficiency and long-term stability. We herein leverage the enhanced stability and targeting ability of engineered high-density lipoprotein-mimetic nanoparticles (eHNPs) to cross the BBB and deliver a SHH inhibitor effectively to the cancer stem-like cell population in SHH MB. Our microfluidic technology enabled highly reproducible production of multicomponent eHNPs incorporated with apolipoprotein A1, anti-CD15, and a SHH inhibitor (LDE225). We demonstrate the dual-targeted delivery and enhanced therapeutic effect of eHNP-A1-CD15-LDE225 via scavenger receptor class B type 1 (SR-B1) and CD15 on brain SHH MB cells in vitro, ex vivo, and in vivo. Moreover, we show that eHNP-A1 not only serves as a stable drug carrier, but also has a therapeutic effect itself through SR-B1-mediated intracellular cholesterol depletion in SHH MB cells. Through the facilitated and targeted cellular uptake of drugs and direct therapeutic role of this engineered biomimetic nanocarrier in SHH MB, our multifunctional nanoparticle provides intriguing therapeutic promise as an effective and potent nanomedicine for the treatment of SHH MB.

Medulloblastoma (MB) is the most common pediatric malignant brain tumor and is currently divided into WNT, SHH, Group 3 and Group 4 subtypes. Even with multimodal chemotherapy, radiotherapy and surgery, many children with Group 3 MBs do not survive. While the molecular aberrations underlying WNT- and SHH-driven MBs are relatively well understood, the oncogenic drivers that lead to Group 3/4 MBs are poorly defined, limiting therapeutic progress. In addition to genetic mutations and alterations, cancers display dysregulated transcription and translation. RNA-binding proteins (RBPs) play key roles in both transcription and translation, and a subset of RBPs are differentially expressed in many different cancers. Indeed, we have previously demonstrated an oncogenic role for the RBP LIN28B in neuroblastoma and it is known to be upregulated in Wilms tumor, hepatoblastoma, germ cell tumors, leukemia among others. LIN28B is a key regulator of let-7 family miRNAs, which in turn inhibit LIN28B and other oncogenes. We hypothesize that LIN28B plays an important role in Group 3 MB and that a better understanding of LIN28B and LIN28B-driven networks will reveal novel therapeutic vulnerabilities. In support of our hypothesis we find that among the four subtypes, LIN28B levels are highest in Group 3 MB, and that overexpression is associated with significantly worse survival. Down-regulation of LIN28B results in significant reduction in cell proliferation by CellTiter-Glo and increased apoptosis by Caspase-Glo (as well as induction of cleaved PARP on immunoblots). In contrast overexpression of LIN28B increases Group 3 cell proliferation and tumor sphere formation. In addition we find that PDZ-binding kinase (PBK) a downstream target of LIN28B is downregulated when LIN28B is depleted. PBK knock down also leads to decreased proliferation of Group 3 MB cells. Finally, in order to robustly define the signaling networks downstream from LIN28B that are involved in Group 3 MB metastasis, we have performed who transcriptome RNA-seq profiling of two group 3 cell lines following LIN28B depletion and plan to interrogate a subset of these based on expression change and functional relevance to LIN28B-mediated Group 3 MB metastasis. This work will help define the role for LIN28B in Group 3 MB aggressiveness and pave the way for similar studies in other cancers. Citation Format: Shubin W. Shahab, Jo Lynne Rokita, Kyle Juraschka, Sachin Kumar, Michael Taylor, Robert W. Schnepp, Tobey J. MacDonald, Anna M. Kenney. Targeting the RNA binding protein LIN28B in Group 3 medulloblastoma decreases proliferation and promotes apoptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3024.

Medulloblastoma (MB) is the most common pediatric malignant brain tumor and is currently divided into WNT, SHH, Group 3 and Group 4 subtypes. Even with multimodal chemotherapy, radiotherapy and surgery, many children with Group 3 MBs do not survive. We have previously demonstrated an oncogenic role for the RNA-binding protein (RBP) LIN28B in neuroblastoma. LIN28B is a key regulator of let-7 family miRNAs, which in turn inhibit LIN28A/B and other oncogenes. LIN28B has also been found to be upregulated in Wilms tumor, hepatoblastoma, germ cell tumors, leukemia among others. We hypothesize that LIN28B plays an important role in Group 3 MB and that a better understanding of LIN28B and LIN28B-driven networks will reveal novel therapeutic vulnerabilities. LIN28B levels are highest in Group 3 MB patients, and its overexpression is associated with significantly worse survival. Here we demonstrate that down-regulation of LIN28B using shRNA results in significant reduction in cell proliferation by CellTiter-Glo and increased apoptosis by Caspase-Glo (as well as induction of cleaved PARP on immunoblots). In contrast overexpression of LIN28B increases Group 3 cell proliferation and tumor sphere formation The LIN28 inhibitor 1632 also leads to significant reduction in G3 MB cell proliferation. In addition, we find that PDZ-binding kinase (PBK) a downstream target of LIN28B is downregulated when LIN28B is depleted. PBK knock down also leads to decreased proliferation of Group 3 MB cells. Finally RNA-seq profiling following LIN28B depletion reveals additional components of the LIN28B pathway which may be amenable to therapeutic targeting. This work will help define the role for LIN28B in Group 3 MB aggressiveness and establish LIN28B and LIN28B-driven networks as novel therapeutic targets in these patients.

Medulloblastoma (MB) is the most common pediatric brain malignancy. MB comprises 5 major subgroups known as WNT, SHH p53wt, SHH p53mut, Group 3 and Group 4. Among the four MB subgroups SHH group is the most dominant molecular subgroup in infants and adults. These tumors are proposed to arise from cerebellar granule neuron precursors (CGNPs), whose developmental expansion requires SHH signaling from the neighboring Purkinje neurons. Previous reports suggest that SHH group features a unique tumor microenvironment compared with other MB groups. Recently, we performed cytokine array analysis of culture media from different MB cell lines. Interestingly, our data showed increased levels of IGFBP2 produced by SHH MB cell lines compared to others. We confirmed these results using ELISA and Western blotting from 3 human SHH MB cell lines, and Smo/A1 mouse tumor cells. IGFBP2 is a member of IGFBP super family of proteins; it plays important roles in tumor cell proliferation, metastasis and drug resistance. We analyzed the role of IGFBP2 in SHH group medulloblastoma tumor growth and metastasis. IGFBP2 knock-down stable cell lines showed phenotypic changes including reduced cell proliferation, cell migration and colony size. Our preliminary in vitro data suggest IGFBP2 exerts it metastasis-promoting role in SHH MB by regulating the expression of EMT marker proteins such as N cadherin, slug etc. and matrix remodeling proteins like MMPs and TIMPs. We are currently performing functional studies in organotypic tumor slice cultures to validate these findings and establish IGFBP2 as a novel regulator of aggressive tumor growth and spread in SHH MB.

Medulloblastoma (MB) is the most common pediatric central nervous system malignancy. Although the current standard of care leads to ~70% patient survival, the therapies are highly toxic, leading to life-long side effects, and recurrence due to therapeutic resistance is fatal. We sought to investigate mediators of radiation response in mouse models for the Sonic hedgehog (SHH) subgroup MB as well as human cell lines. We previously identified Y-box binding protein 1 (YB1) as a downstream effector of YAP-mediated MB radiation resistance. YB1 is a crucial, yet understudied, protein highly expressed across all 4 subgroups of MB. Through its DNA- and RNA-binding cold shock domain, YB1 mediates both transcriptional and translational changes important for tumor maintenance and therapeutic response. We show that following ionizing radiation, YB1 mediates DNA repair through PARP and that PARP inhibition abrogates YB1-mediated DNA repair in cells overexpressing YB1. Additionally, through its inhibitory effects on p53, YB1 is capable of mediating anti-apoptotic effects in response to genotoxic insult. By targeting YB1 with short hairpin RNA, we show that cells are more amenable to ionizing radiation induced double strand breaks. Additionally, we utilize RNA binding protein immunoprecipitation sequencing to investigate post transcriptional regulation of RNAs bound by YB1. We show that YB1 binds numerous transcripts critical for the identity of early cerebellar progenitor cells, the putative cell of origin for SHH subgroup tumors, in addition to transcripts important for cell cycling and migration.

microRNAs (miRNAs) have wide-ranging effects on large-scale gene regulation. As such, they play a vital role in dictating normal development, and their aberrant expression has been implicated in cancer. There has been a large body of research on the role of miRNAs in medulloblastoma, the most common malignant brain tumor of childhood. The identification of the 4 molecular subgroups with distinct biological, genetic, and transcriptional features has revolutionized the field of medulloblastoma research over the past 5 years. Despite this, the growing body of research on miRNAs in medulloblastoma has largely focused on the clinical entity of a single disease rather than the molecular subgroups. This review begins by highlighting the role of miRNAs in development and progresses to explore their myriad of implications in cancer. Medulloblastoma is characterized by increased proliferation, inhibition of apoptosis, and maintenance of stemness programs—features that are inadvertently regulated by altered expression patterns in miRNAs. This review aims to contextualize the large body of work on miRNAs within the framework of medulloblastoma subgroups. The goal of this review is to stimulate new areas of research, including potential therapeutics, within a rapidly growing field.

Medulloblastoma, which is the most common malignant paediatric brain tumour, has a 70% survival rate, but standard treatments often lead to devastating life-long side effects and recurrence is fatal. One of the emerging strategies in the search for treatments is to determine the roles of tumour microenvironment cells in the growth and maintenance of tumours. The most attractive target is tumour-associated macrophages (TAMs), which are abundantly present in the Sonic Hedgehog (SHH) subgroup of medulloblastoma. Here, we report an unexpected beneficial role of TAMs in SHH medulloblastoma. In human patients, decreased macrophage number is correlated with significantly poorer outcome. We confirm macrophage anti-tumoural behaviour in both ex vivo and in vivo murine models of SHH medulloblastoma. Taken together, our findings suggest that macrophages play a positive role by impairing tumour growth in medulloblastoma, in contrast to the pro-tumoural role played by TAMs in glioblastoma, another common brain tumour., The Sonic Hedgehog subgroup of medulloblastoma are characterised by the high infiltration of tumour-associated macrophages (TAMs). Here, the authors show that TAM numbers in patients are associated with better prognosis and that, consistently, in a murine model of medulloblastoma, these TAMs have anti-tumoural properties.

Cerebellar development is a highly regulated process involving numerous factors acting with high specificity, both temporally and by location. Part of this process involves extensive proliferation of cerebellar granule neuron precursors (CGNPs) induced by Sonic Hedgehog (SHH) signaling, but downstream effectors of mitogenic signaling are still being elucidated. Using primary CGNP cultures, a well-established model for SHH-driven proliferation, we show that SHH-treated CGNPs feature high levels of hypoxia-inducible factor 1α (HIF1α), which is known to promote glycolysis, stemness, and angiogenesis. In CGNPs cultured under normoxic conditions, HIF1α is posttranslationally stabilized in a manner dependent upon reactive oxygen species (ROS) and NADPH oxidase (NOX), both of which are also upregulated in these cells. Inhibition of NOX activity resulted in HIF1α destabilization and reduced levels of cyclin D2, a marker of CGNP proliferation. As CGNPs are the putative cells of origin for the SHH subtype of medulloblastoma and aberrant SHH signaling is implicated in other neoplasms, these studies may also have future relevance in the context of cancer. Taken together, our findings suggest that a better understanding of nonhypoxic HIF1α stabilization through NOX-induced ROS generation can provide insights into normal cell proliferation in cerebellar development and SHH-driven cell proliferation in cancers with aberrant SHH signaling.

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.

Post-natal proliferation of cerebellar granule neuron precursors (CGNPs), proposed cells-of-origin for the SHH-associated subgroup of medulloblastoma (MB), is driven by Sonic Hedgehog (Shh) and Insulin-like Growth Factor (IGF) in the developing cerebellum. Shh induces the oncogene Yes-associated protein (YAP), which drives IGF2 expression in CGNPs and mouse Shh-associated medulloblastomas. To determine how IGF2 expression is regulated downstream of YAP, we carried out an unbiased screen for transcriptional regulators bound to IGF2 promoters. We report that Y-box binding protein-1 (YB-1), an onco-protein regulating transcription and translation, binds to IGF2 promoter P3. We observed that YB-1 is up-regulated across human medulloblastoma subclasses as well as in other varieties of pediatric brain tumors. Utilizing the cerebellar progenitor model for the Shh-subgroup of MB in mice, we show for the first time that YB-1 is induced by Shh in CGNPs. Its expression is YAP-dependent and it is required for IGF2 expression in CGNPs. Finally, both gain-of function and loss-of-function experiments reveal that YB-1 activity is required for sustaining CGNP and medulloblastoma cell (MBC) proliferation. Collectively, our findings describe a novel role for YB-1 in driving proliferation in the developing cerebellum and medulloblastoma cells and they identify the SHH:YAP:YB1:IGF2 axis as a powerful target for therapeutic intervention in medulloblastomas.

Sonic hedgehog (Shh) signaling is closely coupled with bioenergetics of medulloblastoma, the most common malignant pediatric brain tumor. Shh-associated medulloblastoma arises from cerebellar granule neuron precursors (CGNP), a neural progenitor whose developmental expansion requires signaling by Shh, a ligand secreted by the neighboring Purkinje neurons. Previous observations show that Shh signaling inhibits fatty acid oxidation although driving increased fatty acid synthesis. Proliferating CGNPs and mouse Shh medulloblastomas feature high levels of glycolytic enzymes in vivo and in vitro . Because both of these metabolic processes are closely linked to mitochondrial bioenergetics, the role of Shh signaling in mitochondrial biogenesis was investigated. This report uncovers a surprising decrease in mitochondrial membrane potential (MMP) and overall ATP production in CGNPs exposed to Shh, consistent with increased glycolysis resulting in high intracellular acidity, leading to mitochondrial fragmentation. Ultrastructural examination of mitochondria revealed a spherical shape in Shh-treated cells, in contrast to the elongated appearance in vehicle-treated postmitotic cells. Expression of mitofusin 1 and 2 was reduced in these cells, although their ectopic expression restored the MMP to the nonproliferating state and the morphology to a fused, interconnected state. Mouse Shh medulloblastoma cells featured drastically impaired mitochondrial morphology, restoration of which by ectopic mitofusin expression was also associated with a decrease in the expression of Cyclin D2 protein, a marker for proliferation. Implications: This report exposes a novel role for Shh in regulating mitochondrial dynamics and rescue of the metabolic profile of tumor cells to that of nontransformed, nonproliferating cells and represents a potential avenue for development of medulloblastoma therapeutics. Mol Cancer Res; 14(1); 114–24. ©2015 AACR . This article is featured in Highlights of This Issue, [p. 1][1] [1]: /lookup/volpage/14/1?iss=1

BACKGROUND: Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Current treatment involves multi-agent chemotherapy and/or radiation, but this one-size-fits-all treatment often results in long term side effects. The development of novel therapeutics has been slowed, limited by the lack of representative samples of individual patient MB; therefore, we adapted a novel drug testing assay to address this issue: organotypic ex vivo brain tumor slice culture (OSC). We hypothesize that OSC can be a robust real-time screening tool for evaluating drug efficacy for individual MB patients. We tested novel inhibitors for STAT3 (WP1066) and YB1 (fisetin) in an OSC assay, with cisplatin and DMSO vehicle as positive and negative controls respectively. OBJECTIVES: To determine the feasibility of performing our OSC on human MB and to evaluate WP1066 and fisetin on patient-derived samples. METHODS: MB was taken directly from the OR at time of initial resection. Tumors were sectioned into 300-μm-thick slices with a vibratome. The slices were cultured, treated with WP1066, fisetin, cisplatin, or vehicle and after 48hrs, the slices were fixed in 4% paraformaldehyde. The slices were immunostained with antibodies against cleaved caspase 3, and confocal images were obtained. Analysis and quantification of staining was done with FIJI and IMARIS imaging software. RESULTS: We were able to perform our assay on three patient samples: two non-WNT/non-SHH and one SHH tumor. WP1066 and fisetin treatment induced cell death in all three samples, similar to cisplatin, and significantly more than vehicle. We also found increased cell death in the nestin positive tumor stem-like cells. We correlated phenotypic response with gene expression, as determined by single-cell RNA-sequencing performed on one MB and whole exome sequencing of another MB. CONCLUSIONS: We have successfully performed a novel OSC assay for real-time drug testing, thus illustrating a possible new personalized medicine tool in MB.

Mitochondrial utilization of the carbohydrate fuel for energy production is required for postnatal cerebellar development., While deregulation of mitochondrial metabolism and cytosolic glycolysis has been well recognized in tumor cells, the role of coordinated mitochondrial oxidation and cytosolic fermentation of pyruvate, a key metabolite derived from glucose, in physiological processes is not well understood. Here, we report that knockout of PTPMT1, a mitochondrial phosphoinositide phosphatase, completely blocked postnatal cerebellar development. Proliferation of granule cell progenitors, the most actively replicating cells in the developing cerebellum, was only moderately decreased, and proliferation of Purkinje cell progenitors did not seem to be affected in knockout mice. In contrast, generation of functional Bergmann glia from multipotent precursor cells (radial glia), which is essential for cerebellar corticogenesis, was totally disrupted. Moreover, despite a low turnover rate, neural stem cells were impaired in self-renewal in knockout mice. Mechanistically, loss of PTPMT1 decreased mitochondrial aerobic metabolism by limiting utilization of pyruvate, which resulted in bioenergetic stress in neural precursor/stem cells but not in progenitor or mature cells, leading to cell cycle arrest through activation of the AMPK-p19/p21 pathway. This study suggests that mitochondrial oxidation of the carbohydrate fuel is required for postnatal cerebellar development, and identifies a bioenergetic stress–induced cell cycle checkpoint in neural precursor/stem cells.

In the developing cerebellum, Sonic hedgehog (SHH) signaling is required for expansion of cerebellar granule neural progenitors, proposed to be cells-of-origin for the SHH-driven pediatric brain tumor medulloblastoma. In this issue of Developmental Cell, Chang et al. (2019) show that the transcription factor Atoh1/MATH1 regulates primary cilium formation, enabling SHH signaling.

Sonic hedgehog (Shh) signaling is closely coupled with the bioenergetics of medulloblastoma, the most common malignant pediatric solid tumor. We have reported earlier that Shh causes deregulation of mitochondrial biogenesis by suppressing mitofusins, leading to fragmented mitochondria in vitro as well as in SmoA1 mouse medulloblastomas (MB) in vivo. Ectopic expression of mitofusins restored mitochondrial fusion accompanied by a rescue in proliferation to the non-proliferative phenotype. In the present study, we report that a rescue in proliferation is also observed in organotypic slice cultures implanted with mitofusin overexpressing SmoA1 neurospheres. When mitofusin overexpressing tumor cells were injected into the cerebella of postnatal day 2 mice, tumor development was significantly delayed. We have significant evidence indicating that Shh induces high increases levels of Reactive Oxygen Species (ROS) in Cerebellar Granule Neuron Precursor cells (CGNPs). When total ROS in a cell was scavenged by treating with N-Acetyl Cytosine (NAC), the fragmented mitochondrial morphology was rescued to their fused morphology. NADPH Oxidase 4 (NOX4) is a known producer of ROS in cells. When we treated CGNPs with apocynin, an NADPH oxidase inhibitor, a significant reduction in proliferation was observed. NOX4 was also found to localise in the peri-vascular niche in SmoA1 tumors. This could have possible implications for a role of ROS in promoting the proliferation of tumor re-populating cells post irradiation. Our goal is to determine if manipulating ROS-mediated mitochondrial dynamics can restore the metabolic profile of tumor cells to that of non-transformed, non-proliferating cells. This would suggest a potential novel treatment paradigm for medulloblastoma that may reduce the requirement for high dose radiation.

Development of the cerebellum occurs postnatally and is marked by a rapid proliferation of cerebellar granule neuron precursors (CGNPs). CGNPs are the cells-of-origin for SHH-driven medulloblastoma, the most common malignant brain tumor in children. Here, we investigated the role of ERK, JNK, and p38 mitogen-activated protein kinases (MAPKs) in CGNP proliferation. We found high levels of p38α in proliferating CGNPs. Concomitantly, members of the p38 pathway, such as ASK1, MKK3 and ATF-2, were also elevated. Inhibition of the Shh pathway or CGNP proliferation blunts p38α levels, irrespective of Shh treatment. Strikingly, p38α levels were high in vivo in the external granule layer (EGL) of the postnatal cerebellum, Shh-dependent mouse medulloblastomas and human medulloblastomas of the SHH subtype. Finally, knocking down p38α by short hairpin RNA-carrying lentiviruses as well as the pharmacologically inhibiting of its kinase activity caused a marked decrease in CGNP proliferation, underscoring its requirement for Shh-dependent proliferation in CGNPs. The inhibition of p38α also caused a decrease in Gli1 and N-myc transcript levels, consistent with reduced proliferation. These findings suggest p38 inhibition as a potential way to increase the efficacy of treatments available for malignancies associated with deregulated SHH signaling, such as basal cell carcinoma and medulloblastoma.

Medulloblastoma (MB), the most common pediatric brain tumor, has a 70% survival rate but standard treatments often lead to devastating life-long side effects, and recurrence is fatal. MB was classified based on molecular and genetic profiles and resulted in four distinct subgroups. The most common subclass of MB is SHH, which accounts for approximately 30% of cases. This class has been successfully modeled in vivo in murine models, which closely recapitulate human disease, providing a convenient and relevant model system for analyzing the SHH MB subclass in vivo. To better understand the mechanisms of tumor growth and recurrence, recent attention has been focused on determining the composition and role of non-tumor cells comprising the tumor microenvironment (TME). Tumor-associated macrophages (TAM) are a key component of the TME that could have two opposite effects on tumor. TAMs can help tumors to evade the immune system by suppressing other immune cell functions, and contribute to tumor growth by promoting angiogenesis. On the other hand, they could suppress tumor growth and delay tumor development. Recently, it was reported that of the subgroups, human SHH MB has the greatest number of TAMs, as well as increased expression of macrophage-associated genes. However, to date, there are no studies addressing the functional role of TAMs in SHH MB. In this work, we demonstrate a functional role of TAMs in both in vitro and in vivo models. We show that reduction of macrophage numbers leads to increased animal mortality in a murine model of SHH MB. Further investigation of the TME and TAMs in MB has the potential to elucidate immune system involvement in the disease and lead to development of novel treatment options.

Malignant high-grade gliomas (HGGs), neoplasms of glial cells and their precursors, such as glioblastomas (GBMs) and diffuse intrinsic pontine gliomas (DIPGs), are the most deadly primary brain tumors in children, and are incurable with current therapies. The most frequent genetic lesions in pediatric HGGs (pHGGs) include amplification/mutation/overexpression of receptor tyrosine kinases (RTKs), such as PDGFRA, and neomorphic K27M mutations in histone H3 proteins that dominantly block H3-K27 methylation and prevent normal epigenetic gene silencing. Current data indicates that RTK signaling and H3-K27M mutations cooperate to drive tumor pathology and confer therapeutic resistance. To understand how these mutations drive tumorigenesis, my research program uses a multidisciplinary approach that includes functional screening in Drosophila glioma models and drug testing in patient-derived HGG tumor stem cell cultures. Our studies yielded strong preliminary evidence that the YAP transcriptional activator is overexpressed and activated in RTK-mutant pHGG cells, and that YAP transcriptional activity is required for proliferation and survival of pHGG cells, particularly in pHGGs harboring H3-K27M mutations. YAP family transcription factors can normally stimulate expansion of stem/progenitor cell populations in response to physiological RTK signaling. Established YAP transcriptional target genes, which are normally controlled by methylation and epigenetic silencing, include genes that can drive HGG tumor cell self-renewal, proliferation, and therapeutic resistance when overexpressed. Therefore, we hypothesize that, as a consequence of oncogenic RTK alterations, YAP becomes constitutively activated to drive a gene expression program that promotes uncontrolled expansion of neural stem/progenitor cells to create malignant tumors, and that this is exacerbated by H3-K27M epigenetic dysregulation. In testing this hypothesis, we generated compelling preliminary data showing that inhibition of YAP specifically provokes growth arrest and cell death of RTK-overexpressing H3-K27M-mutant pHGG cells. Our research indicates that YAP inhibition may be a promising therapeutic strategy for pHGGs.

Radiation therapy remains the standard of care for many cancers, including the malignant pediatric brain tumor medulloblastoma. Radiation leads to long-term side effects, whereas radioresistance contributes to tumor recurrence. Radio-resistant medulloblastoma cells occupy the perivascular niche. They express Yes-associated protein (YAP), a Sonic hedgehog (Shh) target markedly elevated in Shh-driven medulloblastomas. Here we report that YAP accelerates tumor growth and confers radioresistance, promoting ongoing proliferation after radiation. YAP activity enables cells to enter mitosis with un-repaired DNA through driving insulin-like growth factor 2 (IGF2) expression and Akt activation, resulting in ATM/Chk2 inactivation and abrogation of cell cycle checkpoints. Our results establish a central role for YAP in counteracting radiation-based therapies and driving genomic instability, and indicate the YAP/IGF2/Akt axis as a therapeutic target in medulloblastoma.

Aberrant hedgehog signaling has been implicated in many human cancers, including medulloblastoma (MB), the most common malignant pediatric brain tumor. In fact, 30% of MB tumors are driven by aberrant activity of the Sonic hedgehog (SHH) signaling pathway. Our efforts focus on unraveling the downstream components of this pathway to better understand medulloblastoma and better inform therapeutic treatment development and decisions. While some of the signaling alterations in SHH MB are due to gene mutations or amplifications, others may have their roots in epigenetics. Therefore, we examined expression levels of several candidate epigenetic regulators in our systems and identified lymphoid-specific helicase (HELLS) as a gene whose expression is markedly induced by SHH. HELLS is a unique member of the SNF2 family of chromatin remodelers with multiple epigenetic functions in DNA methylation, histone acetylation and methylation, and chromatin remodeling. Additional roles in transcription activation and DNA repair have also been reported. Of interest, HELLS was shown to delay senescence by inhibiting the expression of CDKN2A, the genetic locus of P16INK4A, a key tumor suppressor whose inactivation has recently been reported as critical to MB progression. Confirming our initial finding, we observed considerably higher levels of HELLS in primary cultures of cerebellar granule neuron precursors (CGNPs) treated with SHH and in SmoA1 mouse MB tumor tissue when compared to adjacent normal cerebellum. Our preliminary analysis of a large cohort of MB patients also indicates overexpression of HELLS in human SHH MB. Bioinformatic promoter analysis and experiments with SHH pathway inhibitors suggest regulation of HELLS expression through members of the SHH proliferation program. Downstream effectors of SHH include GLI1/2 and YAP1. Inhibition of GLI1 and GLI2 with Gant61 in both CGNPs and cultured mouse MB cells resulted in a reduction of HELLS, but an increase of apoptosis markers may indicate this reduction is due to cell death rather than specific downregulation of HELLS. In contrast, verteporfin, an inhibitor of the interaction between the transcriptional co-activator YAP1 and the transcription factor TEAD1, resulted in a reduction of HELLS at the mRNA and protein level without a concomitant increase of apoptosis markers. These results suggest that transcriptional upregulation of HELLS in mouse cerebellar progenitors and MB tumors is mediated by the SHH effector YAP1. Experiments to confirm direct involvement of YAP1 in HELLS gene transcription are underway. Future studies to understand the role of HELLS in SHH MB have the potential to provide a better understanding of the disease and new avenues for development of targeted treatments to improve the lives of these patients. Citation Format: M. Hope Robinson, Anna M. Kenney. HELLS is upregulated in Sonic hedgehog-associated medulloblastoma and proliferating cerebellar progenitors in a YAP-dependent manner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4134.

Medulloblastoma is the most common malignant pediatric brain tumor. Currently, the standard therapy comprising radiation, surgery and chemotherapy “cures” about 70% of medulloblastoma patients. However, these aggressive and non-differential treatment modalities cause lifelong side effects in patients. Medulloblastoma patients are classified into four different subgroups based on transcriptional and molecular profiles, namely WNT, SHH, Group 3, and Group 4. The SHH subgroup accounts for approximately 30% of all medulloblastoma cases and is further subcategorized into TP53 wild type and TP53 deficient subtypes. The importance of TP53 status in SHH medulloblastoma patients has been recently recognized by the association between TP53 mutation and poor prognosis for these patients. However, research findings illustrating TP53 signaling in preclinical models of SHH medulloblastoma are quite limited. Apart from the fact that TP53 mutation is associated with poor survival, 80% of medulloblastoma cases are TP53 wild type. TP53 protein is a tumor suppressor which acts as a checkpoint protein for all types of cells. Cancer cells either mutate TP53 gene or employ other mechanisms such as overexpressing MDM2 to compromise TP53 protein function. Therefore we hypothesize that TP53 function is compromised by highly active MDM2 in TP53 wild type Shh medulloblastoma. Using the SmoA1 mouse model, which closely recapitulates human SHH medulloblastoma, we aim to investigate whether TP53 signaling has been disrupted in Shh medulloblastoma. Surprisingly, we observed relatively higher TP53 protein levels in tumor tissue lysates as compared to that in neighboring normal cerebellum using SmoA1 mice. However, MDM2, the major suppressor of TP53, showed higher phosphorylation at Ser166, which would stabilize MDM2, resulting in degradation of newly-produced TP53 protein. We also found significantly increased protein levels of I2PP2A, an endogenous inhibitor of phosphatase 2A, which can dephosphorylate MDM2 at Ser166. Using COG112, a small molecule inhibitor of I2PP2A, we showed the reactivation of PP2A, concurrent with a decrease of p-MDM2 and an increase of TP53 in a time-dependent manner. From these experiments, we conclude that 1) TP53 pathway is functional in our SmoA1 mouse model; 2) I2PP2A upregulation could be the reason for rapid degradation of TP53 protein in SmoA1 mice. These findings have translational implications as they suggest that targeting I2PP2A in TP53 wild type SHH medulloblastoma patients could be a method of driving tumor cell death, thus resulting in better clinical outcomes for these patients. Citation Format: Yun Wei, Victor Maximov, Anna Kenney. Increased I2PP2A compromises TP53 function by stabilizing MDM2 in Shh medulloblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4133.

Sonic hedgehog (Shh) signaling is closely coupled with the bioenergetics of medulloblastoma, the most common malignant pediatric solid tumor. We have previously reported that Shh causes deregulation of mitochondrial biogenesis by suppressing mitofusins. This led to fragmented mitochondria in vitro in primary cultures of mouse cerebellar progenitor cells, putative cells-of-origin of the SHH molecular subclass of medulloblastoma. This phenomenon was also observed in electron micrographs of SmoA1 mouse medulloblastomas in vivo. Ectopic expression of mitofusins in both proliferating progenitor cells and primary mouse medulloblastoma cell cultures restored mitochondrial fusion and led to a non-proliferative phenotype (1). We have observed high levels of Reactive Oxygen Species (ROS) in SmoA1 mouse medulloblastoma cells in vitro. When total ROS in primary cultures of mouse medulloblastoma cells was scavenged by N-Acetyl Cytosine (NAC) treatment, the mitochondria regained their fused morphology, leading to a reduction in cell proliferation. NAC treatment of organotypic cerebellar slice cultures implanted with SmoA1 neurospheres also resulted in a reduction in proliferation as well as impaired spread of tumor cells within normal cerebellar tissue. Medulloblastoma tumor repopulating cells, which are responsible for radiation resistance and tumor recurrence, are primarily located in the perivascular niche (PVN) of Medulloblastoma (2). We therefore focused our attention on the role played by ROS in regulating mitochondrial structure of tumor repopulating cells, to decipher if this affects the survival of these cells. Indeed we have observed that NAC treatment combined with irradiation restores mitochondrial morphology, leading to enhanced cell death within the PVN of Medulloblastoma. We further carried out gene expression analysis of ROS-regulating genes in the Shh subgroup of Medulloblastoma. NADPH Oxidase 4 (NOX4), which is a known producer of cytoplasmic ROS in cells (3), was found to be upregulated as compared to NOX1 and 3. This elevation was observed across all subgroups of medulloblastoma, but was most evident in the Shh subgroup. We have also found that NOX4 co-localizes in the PVN of medulloblastoma. Treatment with Apocynin, a NOX2/4 inhibitor, led to enhanced cell death in the PVN, similar to what we observed after NAC treatment. Taken together, our results show that anti-oxidant treatment restores the fused morphology of mitochondria in the PVN and leads the cells to apoptosis, thereby preventing tumor recurrence post-irradiation, and they implicate Nox4 as a potential therapeutic target. 1. Malhotra A, Dey A, Prasad N, Kenney AM. 2015. Mol Cancer Res 2. Hambardzumyan D, Becher OJ, Rosenblum MK, Pandolfi PP, Manova-Todorova K, Holland EC. 2008. Genes Dev 22: 436-48 3. Bedard K, Krause KH. 2007. Physiol Rev 87: 245-313 Citation Format: Anshu Malhotra, Abhinav Dey, Anna M. Kenney. Reactive Oxygen Species regulates tumor stem cell survival in medulloblastoma via mitochondrial biogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2411.

The Sonic hedgehog (Shh)-subgroup of the pediatric brain tumor medulloblastoma (MB) has the highest frequency of post-radiotherapy local recurrence, which is fatal. Recently, there have been major advances in characterizing the genomic divergence during MB tumor recurrence (1) and the role of spatial heterogeneity in MB (2). However, there is no evidence that demonstrates actionable targets in the Shh-pathway for prevention of tumor recurrence post-radiation. Previously we have shown that Shh-target Y-box binding protein 1 (YB1) drives MB cell proliferation (3) and controls the expression of IGF2, which contributes to radiation resistance in MB (4). Stem-like tumor cells occupying the peri-vascular niche (PVN) are proposed to drive MB recurrence through their radiation-resistant properties (4, 5). In the SmoA1 mouse model for Shh MB, we observe that these cells feature elevated levels of the oncogene YB1. Ectopic expression of YB1 in this model significantly reduced survival, while phosphomutant YB1 inhibited tumor formation. Using mouse organotypic MB slice cultures we found that YB1 is essential for expansion of the cancer stem-like cell population from the PVN post-radiation. We also demonstrate localization of endogenous phosphorylated YB1 to sites of DNA damage post-radiation, and we show that ectopic expression of YB1 in primary MB cell (MBC) cultures drives rapid DNA repair in a PARP-dependent manner. Mechanistically, we show that inhibiting YB-1 phosphorylation using the flavonoid Fisetin disrupts the physical interaction of YB1 and PARP, thus, derailing the DNA repair process post-radiation in MBCs. Subsequently, using a novel ex vivo assay of tumor Neurosphere-implantation on a cerebellar Brain Slice (NoBS) culture, we demonstrate the effectiveness of Fisetin in preventing the survival of cancer stem-like cells in the MB tumor microenvironment. Our findings demonstrate a novel role for YB1 in promoting Shh MB tumor growth, and they reveal a critical requirement for YB1-phosphorylation in driving radiation resistance through PARP-mediated DNA repair, enabling escape from DNA damage-induced apoptosis. Moreover, our results imply that YB1 phosphorylation inhibitors can be used to enhance radiation responsiveness, thereby reducing incidence of medulloblastoma recurrence. Reference: 1. Morrissy AS, Garzia L, Shih DJ, Zuyderduyn S, Huang X, et al. 2016. Nature 529: 351-72. 2. Morrissy AS, Cavalli FMG, Remke M, Ramaswamy V, Shih DJH, et al. 2017. Nat Genet 49: 780-83. 3. Dey A, Robitaille M, Remke M, Maier C, Malhotra A, et al. 2016. Oncogene 35: 4256-684. 4. Fernandez LA, Squatrito M, Northcott P, Awan A, Holland EC, et al. 2012. Oncogene 31: 1923-375. 5. Hambardzumyan D, Becher OJ, Rosenblum MK, Pandolfi PP, Manova-Todorova K, Holland EC. 2008. Genes Dev 22: 436-48 Citation Format: Abhinav Dey, Anshu Malhotra, Anna Kenney. YB1-phosphorylation mediates YB1-PARP interaction to regulate DNA-repair post-radiation in Shh medulloblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4125.

Medulloblastoma is the most common solid malignant pediatric brain tumor. These tumors arise in the cerebellum and can be molecularly subdivided into 4 consensus subgroups, one of which is marked by amplification and activation of Sonic hedgehog (Shh) pathway components and downstream targets. This subclass is proposed to arise from the oncogenic transformation of cerebellar granule neuron precursors (CGNPs), whose expansion during post-natal brain development requires activation of the Shh pathway and downstream targets. These tumors often demonstrate similarities with normal cerebellar development at the molecular level, thus allowing us to use primary CGNP cultures as a model system for the Sonic hedgehog (Shh) driven subclass of medulloblastoma. In addition to mitogens driving proliferation in cancer, it has been shown in the past that low levels of intracellular reactive oxygen species (ROS) can contribute to proliferation through, amongst other methods, phosphatase inhibition and subsequent deregulation of key pathways including pathways that collaborate with Shh signaling. To this end we've studied a reported ROS-generating effector of insulin and insulin-like growth factor signaling, NADPH oxidase 4 (Nox4). It is thought that Nox4 is both upregulated by insulin signaling and synergizes with it downstream via ROS-induced sustained phosphorylation of Akt. Our work in CGNPs revealed a marked induction of Nox4 in response to Shh at the mRNA and protein levels as well as an increase in total reactive oxygen species content. Subsequent studies suggest that Nox4 activity is critical to sustaining proliferation of Shh driven CGNPs. Western blots of shRNA knockdowns of Nox4 showed reduction of proliferative marker CyclinD2. The knockdowns also precipitated a drop in phosphorylated Akt perhaps leaving the Shh pathway without the full effects of one of its major signaling partners, the PI3K/Akt pathway.

Amplification of MYCN occurs commonly in neuroblastoma. We report that phosphatidylinositol 3-kinase (PI3K) inhibition in murine neuroblastoma (driven by a tyrosine hydroxylase-MYCN transgene) led to decreased tumor mass and decreased levels of Mycn protein without affecting levels of MYCN mRNA. Consistent with these observations, PI3K inhibition in MYCN-amplified human neuroblastoma cell lines resulted in decreased levels of Mycn protein without affecting levels of MYCN mRNA and caused decreased proliferation and increased apoptosis. To clarify the importance of Mycn as a target of broad-spectrum PI3K inhibitors, we transduced wild-type N-myc and N-myc mutants lacking glycogen synthase kinase 3β phosphorylation sites into human neuroblastoma cells with no endogenous expression of myc. In contrast to wild-type N-myc, the phosphorylation-defective mutant proteins were stabilized and were resistant to the antiproliferative effects of PI3K inhibition. Our results show the importance of Mycn as a therapeutic target in established tumors in vivo, offer a mechanistic rationale to test PI3K inhibitors in MYCN-amplified neuroblastoma, and represent a therapeutic approach applicable to a broad range of cancers in which transcription factors are stabilized through a PI3K-dependent mechanism. (Cancer Res 2006; 66(16): 8139-46)

High-grade gliomas (HGGs), including glioblastoma (GBM) and diffuse intrinsic pontine glioma (DIPG), are the most aggressive malignant high-grade gliomas in children, and are incurable with current therapies. The most frequent genetic lesions that drive formation of HGGs include amplification/overexpression, mutation, and/or activation of receptor tyrosine kinases (RTK), such as PDGFRA, and K27M mutations in histone H3 proteins. We have discovered that the Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) transcriptional activators, which encode paralogous transcriptional activators, are frequently overexpressed in RTK-mutant/overexpressing pediatric HGGs. We have also discovered that YAP/TAZ activity is required for to promote stemness and self-renewal in tumor cells. We hypothesize that, because of oncogenic RTK alterations, YAP/TAZ become constitutively activated to drive a gene expression program that promotes uncontrolled expansion of neural stem/progenitor cells to create malignant tumors, and that this can be exacerbated by H3-K27M epigenetic dysregulation. In testing this hypothesis, we have generated compelling preliminary data showing that pharmacologic inhibition of YAP/TAZ activity specifically provokes growth arrest and cell death of RTK-overexpressing HGG cells, particularly in the context of H3-K27M mutations. Our ongoing research is focused on identifying the YAP/TAZ transcriptional program that drives tumorigenesis. Our research indicates that YAP/TAZ inhibition may be a promising therapeutic strategy for pediatric HGGs.

Sonic hedgehog (Shh)-mediated medulloblastoma growth requires IGF2 (Insulin-like Growth Factor 2) and we recently showed that Yes Associated Protein (YAP1) induces IGF2 expression by Y-box protein 1(YB1) in Shh-stimulated cerebellar granule neural precursors (CGNPs), proposed cells-of-origin for the Shh molecular subclass of medulloblastoma, and mouse Shh-medulloblastoma cells. We found elevated levels of YAP1, YB1 and IGF2 in tumor cells occupying the peri-vascular niche, a microenvironmental niche proposed to house so-called tumor re-populating cells that survive radiation and contribute to medulloblastoma recurrence, which is fatal. We have developed an ex vivo approach using organotypic brain tumor slice cultures to better understand how YAP1, YB1, and IGF2 regulate peri-vascular niche cell survival post-radiation. We observed that the perivascular niche cell population expressing stem cell markers increases markedly following exposure to radiation. Additionally, on targeting any component of the YAP1-YB1-IGF2 axis we observed increased level of cell death within the niche and compromised cancer stem cell niche expansion post-radiation. These findings strongly indicate that therapeutic approaches intended to impair the function of this pathway could be used to reduce the use of cranio-spinal radiation of medulloblastoma patients, which causes life-long side effects that drastically impair quality of life. Citation Format: Abhinav Dey, Caroline Maier, Anshu Malhotra, Anna Kenney. Cancer stem cell survival postradiation in medulloblastomas requires YAP, YB1, and IGF2. [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 2451.

Downstream of mitogenic Sonic hedgehog (Shh) signaling, Yes-Associated Protein (YAP) can drive proliferation in Cerebellar Granule Neural Progenitor (CGNP) cells. CGNPs are the proposed cells of origin of SHH medulloblastomas. They are a neural progenitor type whose developmental expansion requires signaling by Shh, a secreted ligand produced by the neighboring Purkinje neurons. Approximately 30% of human medulloblastomas bear an activated Sonic hedgehog pathway gene expression signature. Ectopic expression of YAP promotes highly aggressive Shh-driven medulloblastoma growth and radio-resistance (Fernandez et al., 2009). Medulloblastoma being the most common solid malignancy of childhood and a leading cause of pediatric mortality, the current standard of care results in about 60% “cure” rate. But the survivors are beset with long-term side effects, including cognitive impairment, seizures, premature aging, and susceptibility to cancer. Moreover, recurrence and metastasis are lethal. Therefore, identification of novel modes of molecular targeted therapies is critical for the improved quality of life for survivors and reduced incidence of recurrence and metastasis. Recently, there has been renewed interest in how altered metabolic patterns in tumors could be exploited for therapeutic purposes. Deregulating the metabolic machinery for aberrant energy utilization is one of the hallmarks of a proliferating cancer cell. Previously, our lab made the novel observation that Shh mitogenic/oncogenic signaling is tightly coupled to the reprogramming of mitochondrial bioenergetics: Shh inhibits fatty acid oxidation (FAO, or β-oxidation) while driving increased fatty acid synthesis (FAS), an early step of lipogenesis. The production of citrate, an essential component for fatty acid synthesis, occurs inside the mitochondrion via the Tri-carboxylic acid cycle (TCA). We analyzed the effect of Shh treatment and ectopic YAP expression on CGNPs and found that YAP increases levels of fatty acid synthase (FASN) and ATP citrate lyase (ACLY), while YAP knock-down in Shh-treated CGNPs resulted in reduced levels of these enzymes. Moreover, we also observed a surprising decrease in mitochondrial membrane potential. This prompted us to further analyze the ultrastructure of mitochondria using Transmission Electron Microscopy. Shh-treated or ectopic YAP-expressing mitochondria presented a swollen morphology, along with an expanded matrix space and deformed cristae structure, typical of morphologically aberrant mitochondria. These differences in mitochondrial structure were also visible in ultrastructures of SmoA1 tumor tissue as well as in vitro cultures of SmoA1 tumor cells (MBCs). Being dynamic structures, mitochondria undergo constant fusion and fission events, which contribute to their biogenesis. Expression of fusion genes Mitofusin 1 and 2 was reduced while DRP1, a fission promoting gene was highly induced in all samples under study. Ectopic expression of Mitofusin 1 and 2, and knock down of DRP1 in CGNPs and MBCs not only restores the membrane potential to the non-proliferating state, but also indicates a reduction in proliferation. Our study thus implicates YAP-regulated metabolic pathways and enzymes as potential targets for novel medulloblastoma therapies. Our goal is to determine whether hampering YAP-mediated mitochondrial fragmentation can restore the metabolic profile of tumor cells to that of non-transformed, non-proliferating cells, thus suggesting a potential novel treatment paradigm that may reduce or eliminate the requirement for high dose radiation. Citation Format: Anshu Malhotra, Abhinav Dey, Anna Marie Kenney. Yes-Associated Protein: A master metabolic regulator In medulloblastoma. [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 B12.

Medulloblastoma, the most common malignant paediatric brain tumour, is currently treated with nonspecific cytotoxic therapies including surgery, whole-brain radiation, and aggressive chemotherapy. As medulloblastoma exhibits marked intertumoural heterogeneity, with at least four distinct molecular variants, previous attempts to identify targets for therapy have been underpowered because of small samples sizes. Here we report somatic copy number aberrations (SCNAs) in 1,087 unique medulloblastomas. SCNAs are common in medulloblastoma, and are predominantly subgroup-enriched. The most common region of focal copy number gain is a tandem duplication of SNCAIP, a gene associated with Parkinson's disease, which is exquisitely restricted to Group 4 alpha. Recurrent translocations of PVT1, including PVT1-MYC and PVT1-NDRG1, that arise through chromothripsis are restricted to Group 3. Numerous targetable SCNAs, including recurrent events targeting TGF-beta signalling in Group 3, and NF-kappa B signalling in Group 4, suggest future avenues for rational, targeted therapy.

During development and in cancer, tissue and cell growth control requires coordinated regulation of cell proliferation and apoptosis. The tumor suppressive Hippo pathway plays a key role in size regulation and cell-contact inhibition. During the past decade, this pathway has been delineated in Drosophila and now is starting to be better understood in mammals, where an increasing level of complexity and cell context specificity is becoming evident. As we discuss, dys-regulation of this pathway at any step can lead to uncontrolled growth and tumor formation. Indeed, a majority of the pathway components have been implicated in human cancers.

Certain types of medulloblastoma, the most common solid pediatric cancer, are proposed to arise from neural precursors known as cerebellar granule neuron precursors (CGNPs), which require signaling by Sonic hedgehog (Shh) and insulin-like growth factor (IGF) for their proliferation and survival. Aberrant activity of these pathways is implicated in medulloblastoma. IGF activates the mammalian Target of Rapamycin (mTOR), a growth-promoting kinase normally kept in check by the tumor suppressive Tuberous Sclerosis Complex (TSC), comprised of TSC1 and TSC2. TSC also counteracts proliferation by stabilizing the cyclin-dependent kinase inhibitor p27(Kip1), preventing progression through G(1)- to S-phase of the cell cycle. We reported that mice with impaired TSC activity show increased susceptibility to Shh-mediated medulloblastoma. CGNPs and tumors from these mice display increased proliferation, mTOR pathway activation, glycogen synthase kinase-3 (GSK-3) alpha/beta inactivation, and atypical p27(Kip1) cytoplasmic localization. GSK-3alpha/beta inactivation was mTOR-dependent, whereas p27(Kip1) localization was uncoupled from mTOR, and was instead regulated by TSC2. These results provide insight into the molecular 'hardwiring' of the mitogenic network downstream of Shh signaling and emphasize the separate yet synergistic effects regulated by the TSC complex in (1) fueling proliferation through mTOR activation/GSK-3alpha/beta inactivation and (2) compromising checkpoint mechanisms via TSC2-dependent p27(Kip1) nuclear exclusion. Future medulloblastoma therapies targeting Shh signaling can be developed to selectively modulate these activities, to restore checkpoint control and attenuate uncontrolled hyperproliferation.

MicroRNAS (miRNAs) are small endogenous non-coding RNAs that play important roles in many different biological processes including proliferation, differentiation and apoptosis through silencing of target genes. Emerging evidence indicates that miRNAs are key players in mammalian development that, when altered, contribute to tumorigenesis. However, only a few studies to date have focused on the role of miRNAs in medulloblastoma, the most common malignant pediatric brain tumor. These tumors arise in the cerebellum and may attribute their origins to deregulated proliferation of neural progenitor cells during development. Understanding the interplay between normal brain development and medulloblastoma pathogenesis is necessary in order for more efficient, less toxic targeted therapies to be developed and implemented. MiRNA expression profiling of both mouse and human medulloblastomas has led to the identification of signatures correlating with the molecular subgroups of medulloblastoma, tumor diagnosis and response to treatment, as well as novel targets of potential clinical relevance. This review summarizes the recent miRNA literature in both medulloblastoma and normal brain development.

Interactions between the developmentally essential Sonic hedgehog (Shh) and Insulin-like Growth Factor (IGF) pathways play prominent roles in medulloblastoma (MB), the most common malignant pediatric brain tumor. MB patients undergo surgery, chemotherapy, and radiation, a regimen that carries devastating side effects, emphasizing the need for targeted therapies to improve survival and quality of life. Post-natal proliferation of cerebellar granule neuron precursors (CGNPs), proposed cells-of-origin for the SHH-associated subgroup of MB, is driven by Shh and IGF in the developing cerebellum. Shh induces the oncogene Yes-associated protein (YAP), which drives IGF2 expression in CGNPs and mouse Shh-associated medulloblastomas. To determine how IGF2 expression is regulated downstream of YAP, we carried out an unbiased screen for transcriptional regulators bound to IGF2 promoters. We report that Y-box binding protein-1 (YB-1), an onco-protein regulating transcription and translation, binds to IGF2 promoter P3 and is required for IGF2 expression in CGNPs. We show that YB-1 is induced by Shh in CGNPs in a YAP-dependent manner and is found in the germinal layer of the developing cerebellum. We observed that YB-1 is up-regulated across human medulloblastoma subclasses and is elevated in a mouse model for the Shh medulloblastoma subclass. Finally, shRNA-mediated knockdown experiments reveal that YB-1 activity is required for CGNP and medulloblastoma cell (MBC) proliferation in primary cultures and organotypic slice cultures. Collectively, our findings describe a novel role for YB-1 in driving proliferation in the developing cerebellum and medulloblastoma cells and they identify the SHH:YAP:YB1:IGF2 axis as a powerful target for therapeutic intervention in medulloblastomas.

Medulloblastoma is the most common solid malignant pediatric brain tumor. These tumors arise in the cerebellum and can be molecularly subdivided into 4 consensus subgroups, one of which is marked by amplification and activation of Sonic hedgehog (Shh) pathway components and downstream targets. This subclass is proposed to arise from oncogenic transformation of cerebellar granule neuron precursors (CGNPs), whose expansion during post-natal brain development is driven by and requires activation of the Shh pathway. These tumors often demonstrate similarities with normal cerebellar development at the molecular level, thus allowing us to use primary CGNP cultures as a model system for the Sonic hedgehog (Shh) driven subclass of medulloblastoma. In addition to mitogens driving proliferation, it has been shown in the past that low levels of intracellular reactive oxygen species (ROS) are required for proliferation, through mechanisms as diverse as inhibition of receptor tyrosine phosphatases, stabilization of proliferation proteins, and modifications of metabolites, thus indicating additional roles for ROS in proliferation and perhaps tumor growth beyond their known capacity to cause DNA damage, thereby contributing to genomic instability and apoptosis. Although the Shh ligand does not bind to a receptor tyrosine kinase (RTK), it is known that Shh signaling cooperates with RTK- activated pathways such as the insulin-like growth factor pathway to drive proliferation. To determine whether intracellular ROS play a role in Shh-driven CGNP proliferation, we treated CGNPs with the ROS scavenger lipoic acid (LA) in the presence of Shh, and observed a significant decrease in proliferation. Conversely, addition of the ROS inducer tert-Butyl hydroperoxide to Shh treated CGNPs led to enhanced proliferation over Shh treatment alone. These results indicate that a certain level of ROS are required to support Shh-driven CGNP proliferation, and enhancing their levels can increase proliferation. To investigate whether Shh signaling may affect expression of ROS regulatory genes we carried out a qPCR analysis. We identified up-regulation of sod2, gstm1, and gsto1: genes known to respond to ROS and whose products neutralize ROS, over vehicle-treated CGNPs. Paradoxically, when we examined ROS regulatory enzyme expression in Shh-driven mouse medulloblastomas, we noted sharp drop in the expression of gstm1, gsto1, and sod2 compared to the adjacent cerebellum suggesting a reduced ability to inactivate ROS, which could be contributing to proliferation or DNA damage in this transgenic model. These findings suggest that normal Sonic hedgehog pathway activation contributes to the production and tight regulation of ROS via downstream effectors in addition to synergizing with ROS to drive proliferation, a highly regulated balance that may be lost in medulloblastoma. Citation Format: Chad R. Potts, Rachel D. Rotenberry, Anna M. Kenney. Reactive oxygen species in Sonic hedgehog-driven proliferation of cerebellar granule neuron precursors. [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 3978. doi:10.1158/1538-7445.AM2014-3978

A subclass of Medulloblastoma shows considerable dependence on the Sonic Hedgehog (Shh) pathway. Since cellular functions are a consequence of coordinated networks involving protein-protein interactions, it is imperative to explore different events cooperating with Shh entities in order to explore novel targets for medulloblastoma therapy. The Yes Associated Protein (YAP), a component of the Hippo pathway, has been previously established as a downstream target of the Shh pathway (Fernandez et al 2011). Several interesting gene targets featured in the networks developed by us using YAP overexpression in CGNPs. A concurrent upregulation of IGF2 and H19, both imprinted genes, was observed. Possible links between the Shh/Hippo pathways and IGF2 signaling also featured in the networks developed in the study. Important links to lipid metabolism, a process highly active in medulloblastomas and essential for the proliferation of CGNPs, were also predicted. The study includes results of signaling interactions between the Shh/Hippo pathways and entities from other relevant pathways that together play a role in medulloblastoma development and proliferation. Citation Format: Anshu Malhotra, Chad R. Potts, Africa Fernandez-Lopez, Rachel Rotenberry, Anna M. Kenney. Signaling network-based analyses of sonic hedgehog pathway components: predictions and possibilities. [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 4105. doi:10.1158/1538-7445.AM2013-4105

Medulloblastoma is a malignant brain tumor that arises in the cerebellum of young children and occasionally is observed in adults. Increasing evidence suggests that different cell populations represent distinct cells of origin for the various medulloblastoma subtypes, and that these subtypes can be associated with aberrant activation of specific signaling pathways. Current mouse models recapitulate the Shh pathway driven tumors however few models are available to study other medulloblastoma subtypes as genetic events that drive tumorigenesis remain unknown. To address this problem we used the Sleeping Beauty transposon system to uncover novel mutations that contribute to medulloblastoma. Based on earlier work in normal CGNPs, we found that the translation initiation factor eIF4E is critical for regulating CGNP proliferation and the mRNA translation machinery is uniformly upregulated across medulloblastoma subtypes. Using a mouse model wherein eIF4E is ubiquitously over-expressed under control of the -actin promoter (T-Eif4e) we investigated cerebellum development and found that the CGNPs have increased proliferation in vitro in the absence of Shh as well as an expanded proliferative zone in vivo; however these mice do not develop medulloblastoma. Because eIF4E is an oncogene and been shown to cooperate with other known oncogenes in different cell types to accelerate tumorigenesis, we investigated whether eIF4E can cooperate with other mutated genes to generate medulloblastoma. Using a CGNP-specific promoter to drive transposase expression (“Math1-SB”), we crossed these mice with two different lines that carry the Sleeping Beauty transposon (T2/Onc 68 or T2/Onc 76) on different chromosomes. The progeny were then crossed with the T-Eif4e mice and observed for medulloblastoma occurrence; no tumors arise as a result of Math1-SB-mediated transposase activity alone. However, compared to the latency of tumors generated in SmoA1 mice, which have constitutive activation of the Shh pathway, tumors in T-eIF4E/Math1-SB/T2Onc animals arose earlier and were highly proliferative based on Ki-67 staining. They also expressed Otx2, which is associated with a medulloblastoma subtype that does not contain Shh or Wnt pathway mutations. Sequencing of the tumors to identify the genes whose activity is induced (novel oncogenes) or repressed (novel tumor suppressors) by transposon insertion will reveal genes that cooperate with eIF4E to induce medulloblastoma, and may lead to development of new mouse models for medulloblastoma as well as increased insight into genetic events contributing to these tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-149.