Medulloblastoma Cell Viability Research Articles

Abstract 5114: Deciphering the implications of THOC7 in medulloblastoma: Unraveling its role in tumor growth and progression

Abstract Background: Medulloblastoma (MB) is the most common malignant brain cancer in children. Despite the progress made in treating MB, the 5-year survival rate for high-risk tumors remains poor and risk of recurrence within 2 years of treatment is still high. Unfortunately, patients who do survive have reduced quality of life because of the highly toxic side effects of radiation and chemotherapy. Those facts underline the importance of identifying new drivers of MB and understanding the mechanism by which those drivers may promote MB. Using CRISPR-based dependency analysis, we discovered THO Complex Subunit 7 Homolog (THOC7) as one of the highly differentially expressed genes that may uniquely provide survival advantage to MB cells. THOC7 is a component of the THO subcomplex of the TREX complex, which is known to couple mRNA transcription, processing and nuclear export associated with spliced mRNA. Methods: To understand the role of THOC7 in MB, we silenced the expression of THOC7 using siRNAs in multiple MB cell lines. The knockdown efficiency was confirmed by western blot and qPCR. The effect of THOC7 silencing on the viability of MB cells was assessed by short-term viability and long-term colony formation assays. The effect of THOC7 silencing on MB cell proliferation was measured using an IncuCyte. To assess whether THOC7 may affect MB progression, we performed Transwell migration assay. To understand the mechanism by which THOC7 may play a role in medulloblastomogenesis, we measured the effect of THOC7 silencing on the cell cycle progression and apoptosis. As presence of cancer stem cells is linked to relapse and makes MB refractory to radiation therapy, we asked whether THOC7 may support MB stem cell proliferation using neurosphere assay. Results: Our analysis revealed that MB is highly addicted to THOC7. Importantly, higher expression of THOC7 is strongly correlated with lower overall survival of MB patients. We showed that depletion of THOC7 inhibited the short- and long-term viability as well as proliferation of MB cells. In addition, silencing of THOC7 inhibited migration/invasion of MB cells. THOC7 depletion led to cell cycle arrest and increased apoptosis of MB cells. Furthermore, our neurosphere assay demonstrated that silencing of THOC7 blocked the MB stem cell proliferation. Conclusion: Our results suggest that THOC7 may be a novel driver of MB growth and progression. Inhibition of MB stem cell proliferation in THOC7 depleted cells indicates that THOC7 inhibition may sensitize refractory MB to radiation therapy. Experiments are currently underway to understand the mechanism by which THOC7 may support MB growth and progression. In conclusion, our results showing no effect of THOC7 silencing on normal cell viability suggest that approaches aimed at silencing THOC7 may be a safe and viable approach for treating MB. Citation Format: Shahad M. Abdulsahib, Santosh Timilsina, Saif Nirzhor, Daisy Medina, Prabhakar V. Pitta, Panneerdoss Subbarayalu, Manjeet K. Rao. Deciphering the implications of THOC7 in medulloblastoma: Unraveling its role in tumor growth and progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5114.

MiRNA-211 maintains metabolic homeostasis in medulloblastoma through its target gene long-chain acyl-CoA synthetase 4

The prognosis of childhood medulloblastoma (MB) is often poor, and it usually requires aggressive therapy that adversely affects quality of life. microRNA-211 (miR-211) was previously identified as an important regulator of cells that descend from neural cells. Since medulloblastomas primarily affect cells with similar ontogeny, we investigated the role and mechanism of miR-211 in MB. Here we showed that miR-211 expression was highly downregulated in cell lines, PDXs, and clinical samples of different MB subgroups (SHH, Group 3, and Group 4) compared to normal cerebellum. miR-211 gene was ectopically expressed in transgenic cells from MB subgroups, and they were subjected to molecular and phenotypic investigations. Monoclonal cells stably expressing miR-211 were injected into the mouse cerebellum. miR-211 forced expression acts as a tumor suppressor in MB both in vitro and in vivo, attenuating growth, promoting apoptosis, and inhibiting invasion. In support of emerging regulatory roles of metabolism in various forms of cancer, we identified the acyl-CoA synthetase long-chain family member (ACSL4) as a direct miR-211 target. Furthermore, lipid nanoparticle-coated, dendrimer-coated, and cerium oxide-coated miR-211 nanoparticles were applied to deliver synthetic miR-211 into MB cell lines and cellular responses were assayed. Synthesizing nanoparticle-miR-211 conjugates can suppress MB cell viability and invasion in vitro. Our findings reveal miR-211 as a tumor suppressor and a potential therapeutic agent in MB. This proof-of-concept paves the way for further pre-clinical and clinical development.Graphical

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.

Targeting B7‑H3 through EZH2 inhibition in MYC‑positive Group 3 medulloblastoma.

The most aggressive subtype of medulloblastoma (MB), Group 3, is characterized by MYC amplifications. However, targeting MYC has proven unsuccessful, and there remains a lack of therapeutic targets for treating MB. Studies have shown that the B7 homolog 3 (B7‑H3) promotes cell proliferation and tumor cell invasion in a variety of cancers. Similarly, it was recently revealed that B7‑H3 promotes angiogenesis in Group 3 MB and likely facilitates MB metastasis through exosome biogenesis. While therapies targeting B7‑H3 remain in the early stages of development, targeting upstream regulators of B7‑H3 expression may be more effective for halting MB progression. Notably, MYC and the enhancer of zeste homolog 2 (EZH2) are known to regulate B7‑H3 expression, and a previous study by the authors suggested that B7‑H3 amplifications present in MB are likely the result of EZH2‑MYC mediated activities. In the present study, it was reported that overexpression of EZH2 is associated with lower overall survival in Group 3 MB patients. It was also revealed that inhibition of EZH2 significantly reduces B7‑H3 and MYC transcript levels and upregulates miR‑29a, indicating that EZH2 post‑transcriptionally regulates B7‑H3 expression in Group 3 MB cells. Pharmacological inhibition of EZH2 using EPZ005687 attenuated MB cell viability and reduced the expression of B7‑H3. Similarly, pharmacological inhibition and knockdown of EZH2 led to the downregulation of MYC, B7‑H3, and H3K27me3. Further, EZH2 silencing induced apoptosis and reduced colony‑forming ability in MB cells, whereas EZH2 inhibition in MYC‑amplified C17.2 neural stem cells induced G2/M phase arrest while downregulating B7‑H3 expression. Collectively, the current study positions EZH2 as a viable target for the future development of MB treatments and that targeting EZH2 in combination with B7‑H3 immunotherapy may be an effective treatment for halting MB progression.

EXTH-86. BH3 PROFILING IDENTIFIES ONC201/TIC10 AS A PROMISING PARTNER OF ABT-263 IN MEDULLOBLASTOMA IN VITRO

Abstract OBJECTIVE Medulloblastoma represents one of the most common brain tumors in children. In this study, we identified by BH3 profiling that ONC201/TIC10 sensitizes for Bcl-xL/Bcl-2 inhibition in medulloblastoma and performed a preclinical testing of a combined treatment with ONC201/TIC10 and the Bcl-xL/Bcl-2 inhibitor ABT-263. METHODS BH3 profiling was performed to examine ONC201/TIC10-mediated dependencies on anti-apoptotic Bcl-2 family proteins. The combination treatment with ONC201/TIC10 and ABT-263 was tested in different medulloblastoma cells using MTT assays. Isobolograms were calculated to characterize the drug-drug interaction. Tumor spheroid and chorioallantoic membrane assays were used to examine the effects of the combination therapy in a 3D setting. Annexin V/PI staining and flowcytometry were used to detect pro-apoptotic effects. Western blot analyses and knockdown experiments with siRNA were performed for molecular analysis. Extracellular flux analyses served at examining effects on the tumor cell metabolism. RESULTS BH3 profiling showed that ONC201/TIC10 sensitizes medulloblastoma cells to Bcl-xL/Bcl-2 inhibition. In line with this finding, combined treatment with ONC201/TIC10 and ABT-263 led to a predominantly synergistic inhibitory effect on the cell viability of established (D425, D458, DAOY, HD-MB03), primary cultured (PC322) and stem-like (SC322) medulloblastoma cells in 2D and 3D models. The response towards the combination therapy was independent of baseline c-myc expression. On the molecular level, treatment with ONC201/TIC10 led to a dose-dependent decrease of Mcl-1. Moreover, the combination caused enhanced cleavage of caspases 9 and 3. On the metabolic level, the combination therapy led to a reduction in both, oxidative phosphorylation and the glycolytic rate and a reduced expression of respiratory chain proteins. CONCLUSION Combined treatment with ONC201/TIC10 and ABT-263 had a predominantly synergistic inhibitory effect on the cell viability of medulloblastoma cells. This effect was associated with downregulation of Mcl-1. Moreover, the combination treatment resulted in a metabolic reprogramming which likely creates a state of energy deprivation.

PRMT1 is an important factor for medulloblastoma cell proliferation and survival

Aberrant expression of protein arginine methyltransferases (PRMTs) has been implicated in a number of brain tumors, but the role of PRMT1 in medulloblastoma, the most common malignant pediatric brain tumor, remains unexplored. By examining the publicly available databases of pediatric brain tumor collection, we found that PRMT1 was predominantly expressed in medulloblastomas across all the pediatric brain tumors and that the high-level expression of PRMT1 correlated with poor survival of medulloblastoma patients. To determine the role of PRMT1 in medulloblastoma cells, we established an inducible knockdown system and demonstrated that PRMT1 depletion decreased medulloblastoma cell proliferation and induced cell apoptosis. Furthermore, the diamidine compounds, previously shown to exhibit selective PRMT1 inhibition, suppressed medulloblastoma cell viability in a dose-dependent manner. Finally, we observed induction of medulloblastoma cell apoptosis by the potent diamidine compounds at low micromolar concentrations. Together, our results suggest that PRMT1 could be an actionable therapeutic target in medulloblastoma.

G9a/EHMT2 is a Potential Prognostic Biomarker and Molecular Target in SHH Medulloblastoma.

Changes in epigenetic programming are associated with cancer development during childhood. Components of the epigenetic machinery involved in normal embryonic development and hijacked by pediatric cancers include enzymes mediating post-translational modifications of DNA and histones that regulate chromatin structure, such as histone methyltransferases (HMTs). Overexpression of the HMT G9a (euchromatic histone lysine methyltransferase 2, EHMT2) has been described in several cancer types. Medulloblastoma (MB), the main type of malignant brain tumor afflicting children, is currently classified into four molecular subgroups. Here, we show that expression level of the G9a/Ehmt2 gene is higher in MB tumors belonging to the SHH, Group 3, and Group 4 subgroups, compared to Wnt tumors. Remarkably, high G9a expression was significantly associated with shorter overall survival in MB patients. We also present evidence that G9a inhibition dose-dependently reduces MB cell viability. Our findings suggest that higher transcription of G9a may be a predictor of poor prognosis in patients with SHH MB, and that inhibiting G9a activity can display antitumor effects in MB.

ONC201/TIC10 Is Empowered by 2-Deoxyglucose and Causes Metabolic Reprogramming in Medulloblastoma Cells in Vitro Independent of C-Myc Expression.

The purpose of this study was to examine whether the imipridone ONC201/TIC10 affects the metabolic and proliferative activity of medulloblastoma cells in vitro. Preclinical drug testing including extracellular flux analyses (agilent seahorse), MTT assays and Western blot analyses were performed in high and low c-myc-expressing medulloblastoma cells. Our data show that treatment with the imipridone ONC201/TIC10 leads to a significant inihibitory effect on the cellular viability of different medulloblastoma cells independent of c-myc expression. This effect is enhanced by glucose starvation. While ONC201/TIC10 decreases the oxidative consumption rates in D458 (c-myc high) and DAOY (c-myc low) cells extracellular acidification rates experienced an increase in D458 and a decrease in DAOY cells. Combined treatment with ONC201/TIC10 and the glycolysis inhibitor 2-Deoxyglucose led to a synergistic inhibitory effect on the cellular viability of medulloblastoma cells including spheroid models. In conclusion, our data suggest that ONC201/TIC10 has a profound anti-proliferative activity against medulloblastoma cells independent of c-myc expression. Metabolic targeting of medulloblastoma cells by ONC201/TIC10 can be significantly enhanced by an additional treatment with the glycolysis inhibitor 2-Deoxyglucose. Further investigations are warranted.

EXTH-68. DUAL METABOLIC REPROGRAMMING BY ONC201/TIC10 AND 2-DEOXYGLUCOSE HAS A STRONG ANTIPROLIFERATIVE EFFECT ON MEDULLOBLASTOMA CELLS

Abstract BACKGROUND Medulloblastoma represents one of the most common brain tumors in children. While the understanding of the molecular characteristics of this disease has very much advanced, more efficient and less toxic therapeutics are still in high demand. In this study we examined whether the imipridone ONC201/TIC10 affects the metabolic and proliferative activity of medulloblastoma cells alone and in combination with 2-Deoxyglucose in vitro. METHODS Extracellular flux (agilent seahorse) and Western blot analyses were performed to assess effects on tumor cell metabolism and the expression of proteins of the respiratory chain in established medulloblastoma cells. MTT assays and spheroid assays were performed to examine anti-proliferative effects in a 2-D and 3-D setting. RESULTS Treatment with ONC201/TIC10 has a strong inihibitory effect on the cellular viability of different medulloblastoma cells independent of c-Myc expression. While ONC201/TIC10 decreases the oxidative consumption rates in D458 (c-Myc high) and DAOY (c-Myc low) cells, extracellular acidification rates experienced an increase in D458 and a decrease in DAOY cells. Treatment with ONC201/TIC10 in combination with the glycolysis inhibitor 2-Deoxyglucose synergistically inhibited the cellular viability of medulloblastoma cells and impaired the growth of spheroids. CONCLUSION Overall, ONC201/TIC10 profoundly inhibits the proliferative activity of medulloblastoma cells in a c-Myc-independent manner. Additional treatment with the glycolysis inhibitor 2-Deoxyglucose synergistically enhances the anti-medulloblastoma activity of ONC201/TIC10. This promising approach warrants further investigations.

STAT3 inhibitor in combination with irradiation significantly inhibits cell viability, cell migration, invasion and tumorsphere growth of human medulloblastoma cells

ABSTRACT Persistent activation of signal transducer and activator of transcription 3 (STAT3) is frequently reported in cancers and plays important roles in tumor progression. Therefore, directly targeting persistent STAT3 signaling is an attractive cancer therapeutic strategy. The aim of this study is to test the inhibitory efficacy of novel STAT3 small molecule inhibitors, LLY17 and LLL12B, in combination with irradiation in human medulloblastoma cells. Both LLY17 and LLL12B inhibit the IL-6-induced and persistent STAT3 phosphorylation in human medulloblastoma cells. Irradiation using 4 Gy alone exhibits some inhibitory effects on medulloblastoma cell viability, and these effects are further enhanced by combining with either STAT3 inhibitor. Irradiation alone also shows certain inhibitory effects on medulloblastoma cell migration and invasion and the combination of LLY17 or LLL12B with irradiation further demonstrates greater inhibitory effects than monotherapy. STAT3 inhibitor alone or irradiation alone exhibits some suppression of medulloblastoma tumorsphere growth, and the combination of LLY17 or LLL12B and irradiation exhibits greater suppression of tumorsphere growth than monotherapy. Combining either STAT3 inhibitor with irradiation reduces the expression of STAT3 downstream targets, Cyclin D1 and Survivin, and induces apoptosis in medulloblastoma cells. These results support that combination of a potent STAT3 inhibitor such as LLY17 or LLL12B with irradiation is an effective and novel therapeutic approach for medulloblastoma.

Geniposide exhibits anticancer activity to medulloblastoma cells bydownregulating microRNA-373.

Medulloblastoma is a common tumor originates from central nervous system in children with metastatic potential. Geniposide is the major active ingredient separated from the fruit of Gardenia jasminoides Ellis. Herein, we tested the possible anticancer activity of geniposide on human medulloblastoma cells, as well as the potential underlying molecular mechanisms. Firstly, followed by geniposide incubation, cell viability, proliferation, apoptosis, migration, and invasion of medulloblastoma Daoy cells, along with microRNA-373 (miR-373) expression were tested, respectively. Then, the influences of miR-373 overexpression in the reduction of medulloblastoma cell proliferation, migration, and invasion and the elevation of apoptosis, triggered by geniposide treatment, were re-investigated. Finally, the Ras/Raf/MEK/ERK pathway activity was analyzed. Geniposide treatment inhibited medulloblastoma cell viability, proliferation, migration, and invasion, but promoted cell apoptosis. Surprisingly, miR-373 expression in medulloblastoma cells was obviously downregulated by geniposide treatment. miR-373 overexpression reversed the effects of geniposide on Daoy cells. Furthermore, geniposide hindered the Ras/Raf/MEK/ERK pathway by downregulating miR-373 expression. Geniposide exhibited anticancer activity on human medulloblastoma cells and blocked Ras/Raf/MEK/ERK pathway by downregulating miR-373 expression.

Abstract 255: Antagonizing Na-K-Cl cotransporter NKCC1 impairs medulloblastoma cell viability

Abstract Medulloblastoma, a common pediatric brain cancer, is comprised of four molecular subgroups: Wnt-activated; Shh-activated; group 3; and group 4. Group 3 subgroup patients have the lowest survival rate and the highest rates of relapse and metastasis [1]. The standard-of-care for medulloblastoma consists of surgical removal of the tumor, radiation, and chemotherapy [1]. However, the standard-of-care poses many risk factors including hearing loss, permanent damage to endocrine and neurocognitive function, and secondary tumors [1]. There is a high demand for new treatment options for medulloblastoma. Interestingly, group 3 medulloblastoma tumors have high expression of GABRA5 and SLC12A2, which code for the α5-subunit of the ligand-gated ionotropic γ-aminobutyric acid type A receptor (GABAAR) and the sodium-potassium-chloride cotransporter isoform-1 (NKCC-1), respectively [2]. Several recent studies have provided evidence that ion channel activity contributes to brain cancer progression [3]. If so, then modulating ion channel(s) function may impair cancer cell viability. We have already shown that use of a positive allosteric modulator of the GABAAR can impair viability of group 3 medulloblastoma cells in vitro and in vivo and with greater specificity and potency than standard-of-care chemotherapeutic [4, 5]. Others have shown that inhibition or down-regulation of NKCC-1 can reduce cell viability, increase apoptosis, and decrease migration and invasion of cells derived from various cancer types [3]. We have examined how modulating NKCC-1 and GABAAR alone and simultaneously impacts the viability of group 3 medulloblastoma cells. We find that there is a synergistic effect in the inhibition of NKCC-1 and positive modulation of GABAAR. We will report on these observations and our model for the mechanism of action.

Targeting Glucose Metabolism in Medulloblastoma

Medulloblastoma is the most common malignant brain tumor of childhood. Although current treatment approaches for this cancer have improved overall survival, they leave devastating and lifelong sequelae and the mortality remains high. Novel targeted therapies are therefore crucial to improve survival in these children and decrease treatment‐related toxicity.Medulloblastoma development is largely driven by deregulation of normal cerebellar proliferation wherein the sonic hedgehog (Shh) pathway plays a critical role. Activating mutations of Shh genes are frequent in medulloblastoma and promote their rapid growth. In addition, highly aggressive Shh‐active medulloblastomas also exhibit mutations of the Ras/MAPK pathway and increased HIF‐1α, which are observed to stimulate glycolysis by activating the 6‐phosphofructo‐2‐kinase/fructose‐2,6 bisphosphatase family of enzymes (PFKFB1‐4). The PFKFBs produce fructose‐2,6‐bisphosphate (F26BP), a potent activator of 6‐phosphofructo‐1‐kinase, a rate‐limiting step in glycolysis. Recent data indicate that the PFKFB4 family member is increased in multiple human tumors and cancer lines, required for cell survival in aggressive brain tumors and strongly induced by hypoxia. We have found that the PFKFB4 enzyme was highly expressed in a series of patient‐derived medulloblastomas and correlated significantly with expression of the Shh pathway effector Smoothened (SMO) and GLI1. Through HIF‐1α upregulation, hypoxia was observed to strongly induce PFKFB4 expression in Shh‐active medulloblastoma cells and silencing PFKFB4 in these cells markedly suppressed F26BP and glycolytic flux to lactate and significantly decreased cell survival and proliferation, more prominently in hypoxia, indicating that PFKFB4 may be preferentially required for survival and growth under hypoxia. In addition, we found that co‐silencing PFKFB4 with SMO or GLI1 markedly decreased cell survival. We have recently developed a novel selective small molecule inhibitor of PFKFB4 and examined the effects of inhibiting PFKFB4 and the Shh pathway simultaneously. We found that simultaneous administration of the PFKFB4 inhibitor and inhibitors of the Shh pathway synergistically decreased Shh‐active medulloblastoma cell viability. Taken together, our data indicate that targeting PFKFB4 may be a valid therapeutic option against medulloblastoma and strongly support the further examination of simultaneous inhibition of PFKFB4 and the Shh pathway as a treatment strategy in medulloblastoma.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Blocking interleukin-6 signaling inhibits cell viability/proliferation, glycolysis, and colony forming activity of human medulloblastoma cells.

Elevated levels of the pro-inflammatory cytokine interleukin-6 (IL-6) have tumor-promoting activity and are associated with poor survival outcomes in many cancers. Additionally, the IL-6/GP130/STAT3 axis has been widely studied due to its pivotal role in tumor development and maintenance in a number of tissue types, including the cerebellum. However, the connection between IL-6 signaling and medulloblastoma progression is largely unexplored. In the present study, we observed that IL-6 induced medulloblastoma cell viability, cell proliferation and glycolysis. Furthermore, it also upregulated the expression of phosphorylated STAT3, indicating that the IL-6/GP130/STAT3 pathway plays a central role in medulloblastoma. The FDA-approved drug bazedoxifene, a blocker of the formation of the hexameric IL-6/IL-6R/GP130 complex, was re-purposed in this study to inhibit the IL-6/GP130/STAT3 signaling pathway. Bazedoxifene not only inhibited IL-6 mediated cell viability and cell proliferation, and increased phosphorylated STAT3 expression, but it also decreased cell glycolysis, demonstrating a certain level of therapeutic efficacy in vitro. Collectively, our findings offer new insight into the molecular mechanism underlying the biological aggressiveness of medulloblastoma, the roles of IL-6 in these processes and a possible efficacious adjuvant therapy for medulloblastoma.

MicroRNA-206 inhibits the viability and migration of medulloblastoma cells by targeting LIM and SH3 protein 1.

MicroRNA (miR)-206 has been found to be deregulated in various types of human cancer, including medulloblastoma. However, the regulatory mechanism of miR-206 in medulloblastoma growth and metastasis remains largely unclear. In the present study, reverse transcription-quantitative polymerase chain reaction data indicated that miR-206 was significantly downregulated in medulloblastoma tissues compared with adjacent non-tumor tissues (P<0.01). Furthermore, low expression of miR-206 was significantly associated with seeding at presentation and anaplastic histology (P<0.01), but not with sex, age, or residual tumors. Overexpression of miR-206 significantly reduced the viability and migration of medulloblastoma D341 cells (P<0.01). LIM and SH3 protein 1 (LASP1) was further identified as a novel target of miR-206 in D341 cells. mRNA levels of LASP1 were significantly higher in medulloblastoma tissues compared to adjacent non-tumor tissues (P<0.01), with an inverse correlation to the miR-206 levels in medulloblastoma tissues. In addition, protein expression levels of LASP1 ere negatively regulated by miR-206 in D341 cells. Further investigation showed that overexpression of LASP1 significantly eliminated the inhibitory effects of miR-206 on the migration and invasion of D341 cells (P<0.01). In conclusion, our study demonstrates that miR-206 has a suppressive role in medulloblastoma cell viability and invasion, partly at least, via the targeting of LASP1. Our study highlights the importance of the miR-206/LASP1 in medulloblastoma.

Stepwise assembly of functional C-terminal REST/NRSF transcriptional repressor complexes as a drug target.

In human cells, thousands of predominantly neuronal genes are regulated by the repressor element 1 (RE1)-silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF). REST/NRSF represses transcription of these genes in stem cells and non-neuronal cells by tethering corepressor complexes. Aberrant REST/NRSF expression and intracellular localization are associated with cancer and neurodegeneration in humans. To date, detailed molecular analyses of REST/NRSF and its C-terminal repressor complex have been hampered largely by the lack of sufficient amounts of purified REST/NRSF and its complexes. Therefore, the aim of this study was to express and purify human REST/NRSF and its C-terminal interactors in a baculovirus multiprotein expression system as individual proteins and coexpressed complexes. All proteins were enriched in the nucleus, and REST/NRSF was isolated as a slower migrating form, characteristic of nuclear REST/NRSF in mammalian cells. Both REST/NRSF alone and its C-terminal repressor complex were functionally active in histone deacetylation and histone demethylation and bound to RE1/neuron-restrictive silencer element (NRSE) sites. Additionally, the mechanisms of inhibition of the small-molecule drugs 4SC-202 and SP2509 were analyzed. These drugs interfered with the viability of medulloblastoma cells, where REST/NRSF has been implicated in cancer pathogenesis. Thus, a resource for molecular REST/NRSF studies and drug development has been established.

RITA displays anti-tumor activity in medulloblastomas independent of TP53 status.

Current therapy of medulloblastoma, the most common malignant brain tumor of childhood, achieves 40–70% survival. Secondary chemotherapy resistance contributes to treatment failure, where TP53 pathway dysfunction plays a key role. MDM2 interaction with TP53 leads to its degradation. Reactivating TP53 functionality using small-molecule inhibitors, such as RITA, to disrupt TP53-MDM2 binding may have therapeutic potential. We show here that RITA decreased viability of all 4 analyzed medulloblastoma cell lines, regardless of TP53 functional status. The decrease in cell viability was accompanied in 3 of the 4 medulloblastoma cell lines by accumulation of TP53 protein in the cells and increased CDKN1A expression. RITA treatment in mouse models inhibited medulloblastoma xenograft tumor growth. These data demonstrate that RITA treatment reduces medulloblastoma cell viability in both in vitro and in vivo models, and acts independently of cellular TP53 status, identifying RITA as a potential therapeutic agent to treat medulloblastoma.

Usp7 promotes medulloblastoma cell survival and metastasis by activating Shh pathway

The ubiquitin-specific protease Usp7 plays roles in multiple cellular processes through deubiquitinating and stabilizing numerous substrates, including P53, Pten and Gli. Aberrant Usp7 activity has been implicated in many disorders and tumorigenesis, making it as a potential target for therapeutic intervention. Although it is clear that Usp7 is involved in many types of cancer, its role in regulating medulloblastoma (MB) is still unknown. In this study, we show that knockdown of Usp7 inhibits the proliferation and migration of MB cells, while Usp7 overexpression exerts an opposite effect. Furthermore, we establish Usp7 knockout MB cell line using the CRISPR/Cas9 system and further confirm that Usp7 knockout also blocks MB cell proliferation and metastasis. In addition, we reveal that knockdown of Usp7 compromises Shh pathway activity and decrease Gli protein levels, while P53 level and P53 target gene expression have no obvious changes. Finally, we find that Usp7 inhibitors apparently inhibit MB cell viability and migration. Taken together, our findings suggest that Usp7 is important for MB cell proliferation and metastasis by activating Shh pathway, and is a putative therapeutic target for MBs.

Enhanced radiation efficiency by gold nanoparticles after proton beam irradiation in medulloblastoma cell lines

Introduction: In pediatric oncology, one quarter of patients is diagnosed with a tumor of the CNS. Although outcome did improve significantly during the last decade, prognosis of malignant CNS-tumors in childhood is still lagging behind and needs further improvement. Especially for relapse patients, prognosis is worse and curative therapy approaches are missed. Besides radical resection and chemotherapy, radiotherapy is standard of care. However, secondary malignancy after treatment of CNS-tumors in childhood occurs in 5.5% of cases, thus accounting for 19% of all secondary cancers in childhood tumors. Although proton beam irradiation allows to cover target volumes comparable to those achieved by photons, the main advantage of proton beams is sparing of healthy tissues. Recent in vitro and in vivo studies demonstrated an enhanced biological effect of proton therapy in the presence of gold nanoparticles. This held true both for irradiated cancer cells in vitro and in vivo. In this study we wanted to extend these findings to preclinical medulloblastoma models using proton beams and well-defined gold nanoparticles. Materials and methods: Three different medulloblastoma cell lines (DAOY, ONS76 and UW228) were cultured and treated with proton beam irradiation after incubation with gold nanoparticles or without. Three different doses of irradiation (6, 10 and 20 Gy) were evaluated. Stock solutions of gold nanoparticles were used in serial dilutions. Viability of medulloblastoma cells was evaluated by using MTT assays 72 hours after irradiation. Results: As expected, proton beam irradiation had the main effect on medulloblastoma cell viability. However, in our study gold nanoparticles had a cytotoxic effect at high concentrations (> 400 µg/ml). Proton beam irradiation in the presence of gold nanoparticles decreased the IC50 that was observed when cells were irradiated only. Conclusion: Confirming published data we could show enhanced biological effectiveness of proton beam irradiation in medulloblastoma cell lines in the presence of gold nanoparticles. Sensitization was directly correlated with radiation dose and the amount of gold nanoparticles per cell. Further investigation on the impact of particle size on toxicity as well as on enhanced radiation efficiency is needed.

MB-20WDR5 ORCHESTRATES CANCER CELL SELF RENEWAL IN Myc DRIVEN MEDULLOBLASTOMA

MB-20. WDR5 ORCHESTRATES CANCER CELL SELF RENEWAL IN Myc DRIVEN MEDULLOBLASTOMA Swati Ghosh1, Sujatha Venkataraman1, Diane Birks1, Nicholas Foreman1,2, and Rajeev Vibhakar1,2; University of Colorado, Aurora, CO, USA; Children Hospital Colorado, Aurora, CO, USA MYC oncogene driven medulloblastoma (MB) has a poor overall outcome and there is a need for more effective therapies. Wehypothesized that epigenetic mechanisms may mediate MYC-driven medulloblastoma. To address this, we performed a functional epigenome wide RNAi screen targeting 406 epigenetic molecules associated with chromatin remodeling and histone modification in Myc MB cells. Unbiased analysis identified several key regulators of long-term viability in these Myc amplified MB cells. Remarkably we identified multiple proteins that are part of the COMPASS like macromolecular complex as critical mediators of MB cell viability. These include ASH2L, MLL2 (KMT2D), WDR5, DPY30 and HFC1. This complex is critical for methylationof histone3 lysine4 (H3K4me3) andmediating transcriptional activation. We established that WDR5 is important for MYC driven medulloblastoma cell survival. Knock down of WDR5 attenuates clonogenic growth of tumor cells, suppresses stem cell associated gene expression programs and prevents formation of new neurospheres suggesting an inhibition of self-renewal of the medulloblastoma cells. Co-immunoprecipitation studies revealed an interaction between cMYC and WDR5. Confocal microscopy using high cMYC medulloblastoma cells also demonstrated that WDR5 co-localizes with cMYC in nucleus. C-MYC overexpressing neural stem cells differentiate down a neuronal lineage upon WDR5 knockdown. We are now performing more detailed studies on the activity of WDR5 in vitro and in vivo in MYC driven medulloblastoma. Further characterization of WDR5 will provide deeper insight into understanding of group3 medulloblastoma and its therapeutic intervention. Neuro-Oncology 18:iii97–iii122, 2016. doi:10.1093/neuonc/now076.18 #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.