Aggressive Medulloblastoma Research Articles

MDB-06. ALTERNATIVE SPLICING MEDIATED BY OTX2 DRIVES THE DEVELOPMENT OF GROUP 3 MEDULLOBLASTOMA

Abstract BACKGROUND OTX2 is a transcription factor and known molecular hallmark of Group 3 medulloblastoma (MB). While previous studies have focused on its association with active enhancer elements to modulate the chromatin landscape, here, we demonstrate an unexpected role for OTX2 in regulating Group 3 MB alternative splicing (AS). METHODS We used TurboID to broadly interrogate the Group 3 MB OTX2 protein interactome and identify associations with splicing regulators. RNA-sequencing was performed to identify OTX2-driven AS events, a subset of which were validated in Group 3 and Group 4 MB patient tumors and the developing human cerebellar rhombic lip (RL). Splice blocking morpholinos were used to mimic OTX2-regulated splicing events and to determine their role in MB cell fate decisions both in vitro and in vivo. RESULTS OTX2 associates with the Large Assembly of Splicing Regulators (LASR) complex through protein-protein interactions and modulates the localization of a LASR protein to help stabilize the complex and sustain a Group 3 MB splicing program. Motif enrichment analysis demonstrates that members of the LASR complex are also significantly enriched near OTX2-regulated alternatively spliced exons. OTX2 is found on nascent mRNA, and its role in AS is independent of DNA-regulatory functions. OTX2 controls a pro-tumorigenic splicing program that is mirrored in the human cerebellar RL origins. Among the OTX2-regulated spliced genes, PPHLN1 is expressed in the most primitive rhombic lip stem cells, and targeting PPHLN1 splicing reduces tumor growth, while enhancing differentiation and survival in vivo. CONCLUSIONS While splicing defects are commonly observed in adult cancers, the functional role of AS in the most aggressive MBs has never been explored. These findings identify OTX2-mediated AS as a major determinant of cell fate decisions and highlight AS events as novel therapeutic targets to drive Group 3 MB differentiation.

The OTX2 Gene Induces Tumor Growth and Triggers Leptomeningeal Metastasis by Regulating the mTORC2 Signaling Pathway in Group 3 Medulloblastomas.

Medulloblastoma (MB) encompasses diverse subgroups, and leptomeningeal disease/metastasis (LMD) plays a substantial role in associated fatalities. Despite extensive exploration of canonical genes in MB, the molecular mechanisms underlying LMD and the involvement of the orthodenticle homeobox 2 (OTX2) gene, a key driver in aggressive MB Group 3, remain insufficiently understood. Recognizing OTX2's pivotal role, we investigated its potential as a catalyst for aggressive cellular behaviors, including migration, invasion, and metastasis. OTX2 overexpression heightened cell growth, motility, and polarization in Group 3 MB cells. Orthotopic implantation of OTX2-overexpressing cells in mice led to reduced median survival, accompanied by the development of spinal cord and brain metastases. Mechanistically, OTX2 acted as a transcriptional activator of the Mechanistic Target of Rapamycin (mTOR) gene's promoter and the mTORC2 signaling pathway, correlating with upregulated downstream genes that orchestrate cell motility and migration. Knockdown of mTOR mRNA mitigated OTX2-mediated enhancements in cell motility and polarization. Analysis of human MB tumor samples (N = 952) revealed a positive correlation between OTX2 and mTOR mRNA expression, emphasizing the clinical significance of OTX2's role in the mTORC2 pathway. Our results reveal that OTX2 governs the mTORC2 signaling pathway, instigating LMD in Group 3 MBs and offering insights into potential therapeutic avenues through mTORC2 inhibition.

EXTH-46. SCHLAFEN11 IS A POWERFUL BIOMARKER OF CHEMOSENSITIVITY IN MEDULLOBLASTOMAS

Abstract Non-biased and biased screening for prognostic factors in medulloblastomas showed that Schlafen family member 11 (SLFN11) is a powerful prognostic marker. SLFN11 is a putative DNA/RNA helicase, whose expression improves response to DNA damaging agents in some cancers, but its role in medulloblastoma has not been reported. SLFN11 protein and mRNA level were found to be expressed exclusively in WNT-activated and a proportion of SHH-activated medulloblastoma. WNT pathway activation did not acutely induce SLFN11, rather, its expression correlated with a specific hypomethylation site on the SLFN11 promoter. Genetic introduction of SLFN11 into medulloblastoma cell lines with no baseline expression dramatically increased their response to the DNA damaging agents cisplatin and SN-38 in vitro. Intracranial xenograft studies also showed markedly increased sensitivity to cisplatin after induced SLFN11 overexpression in medulloblastoma cells. In addition, CRISPR-mediated deletion of SLFN11 in a medulloblastoma cell line with high baseline expression reduced the response to chemotherapy. SLFN11 could also be upregulated pharmacologically by the histone deacetylase-inhibitor RG2833, supporting the importance of epigenetic control. Importantly, markedly increased sensitivity to cisplatin and SN-38 was seen in initially SLFN11-negative medulloblastoma cells also treated with RG2833, suggesting an approach by which more aggressive medulloblastoma subtypes might be targeted. Our findings suggest a novel mechanism for the increased chemosensitivity of some medulloblastoma subtypes linked to a specific biomarker, as well as a novel combinatorial chemotherapeutic strategy for the treatment of more aggressive medulloblastoma subtypes (Groups 3 and 4) lacking SLFN11.

Research Trends in C-Terminal Domain Nuclear Envelope Phosphatase 1.

C-terminal domain nuclear envelope phosphatase 1 (CTDNEP1, formerly Dullard) is a member of the newly emerging protein phosphatases and has been recognized in neuronal cell tissues in amphibians. It contains the phosphatase domain in the C-terminal, and the sequences are conserved in various taxa of organisms. CTDNEP1 has several roles in novel biological activities such as neural tube development in embryos, nuclear membrane biogenesis, regulation of bone morphogenetic protein signaling, and suppression of aggressive medulloblastoma. The three-dimensional structure of CTDNEP1 and the detailed action mechanisms of CTDNEP1's functions have yet to be determined for several reasons. Therefore, CTDNEP1 is a protein phosphatase of interest due to recent exciting and essential works. In this short review, we summarize the presented biological roles, possible substrates, interacting proteins, and research prospects of CTDNEP1.

Loss of phosphatase CTDNEP1 potentiates aggressive medulloblastoma by triggering MYC amplification and genomic instability

MYC-driven medulloblastomas are highly aggressive childhood brain tumors, however, the molecular and genetic events triggering MYC amplification and malignant transformation remain elusive. Here we report that mutations in CTDNEP1, a CTD nuclear-envelope-phosphatase, are the most significantly enriched recurrent alterations in MYC-driven medulloblastomas, and define high-risk subsets with poorer prognosis. Ctdnep1 ablation promotes the transformation of murine cerebellar progenitors into Myc-amplified medulloblastomas, resembling their human counterparts. CTDNEP1 deficiency stabilizes and activates MYC activity by elevating MYC serine-62 phosphorylation, and triggers chromosomal instability to induce p53 loss and Myc amplifications. Further, phosphoproteomics reveals that CTDNEP1 post-translationally modulates the activities of key regulators for chromosome segregation and mitotic checkpoint regulators including topoisomerase TOP2A and checkpoint kinase CHEK1. Co-targeting MYC and CHEK1 activities synergistically inhibits CTDNEP1-deficient MYC-amplified tumor growth and prolongs animal survival. Together, our studies demonstrate that CTDNEP1 is a tumor suppressor in highly aggressive MYC-driven medulloblastomas by controlling MYC activity and mitotic fidelity, pointing to a CTDNEP1-dependent targetable therapeutic vulnerability.

LIN28 expression and function in medulloblastoma.

Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Current treatment modalities are not completely effective and can lead to severe neurological and cognitive adverse effects. In addition to urgently needing better treatment approaches, new diagnostic and prognostic biomarkers are required to improve the therapy outcomes of MB patients. The RNA-binding proteins, LIN28A and LIN28B, are known to regulate invasive phenotypes in many different cancer types. However, the expression and function of these proteins in MB had not been studied to date. This study identified the expression of LIN28A and LIN28B in MB patient samples and cell lines and assessed the effect of LIN28 inhibition on MB cell growth, metabolism and stemness. LIN28B expression was significantly upregulated in MB tissues compared to normal brain tissues. This upregulation, which was not observed in other brain tumors, was specific for the aggressive MB subgroups and correlated with patient survival and metastasis rates. Functionally, pharmacological inhibition of LIN28 activity concentration-dependently reduced LIN28B expression, as well as the growth of D283 MB cells. WhileLIN28 inhibition did not affect the levels of intracellular ATP, it reduced the expression of the stemness marker CD133 in D283 cellsand the sphere formation of CHLA-01R cells. LIN28B, which is highly expressed in the human cerebellum during the first few months after birth, subsequently decreased with age. The results of this study highlight the potential of LIN28B as a diagnostic and prognostic marker for MB and openthe possibility to utilize LIN28 as a pharmacological target to suppress MB cell growth and stemness.

A Transitional Cell Population Is Enriched in Aggressive Human Medulloblastoma.

A transitional progenitor population, enhanced in aggressive medulloblastoma, was identified in human fetal cerebellum.

Human fetal cerebellar cell atlas informs medulloblastoma origin and oncogenesis.

Medulloblastoma (MB) is the most common malignant childhood brain tumour1,2, yet the origin of the most aggressive subgroup-3 form remains elusive, impeding development of effective targeted treatments. Previous analyses of mouse cerebella3-5 have not fully defined the compositional heterogeneity of MBs. Here we undertook single-cell profiling of freshly isolated human fetal cerebella to establish a reference map delineating hierarchical cellular states in MBs. We identified a unique transitional cerebellar progenitor connecting neural stem cells to neuronal lineages in developing fetal cerebella. Intersectional analysis revealed that the transitional progenitors were enriched in aggressive MB subgroups, including group 3 and metastatic tumours. Single-cell multi-omics revealed underlying regulatory networks in the transitional progenitor populations, including transcriptional determinants HNRNPH1 and SOX11, which are correlated with clinical prognosis in group 3 MBs. Genomic and Hi-C profiling identified de novo long-range chromatin loops juxtaposing HNRNPH1/SOX11-targeted super-enhancers to cis-regulatory elements of MYC, an oncogenic driver for group 3 MBs. Targeting the transitional progenitor regulators inhibited MYC expression and MYC-driven group 3 MB growth. Our integrated single-cell atlases of human fetal cerebella and MBs show potential cell populations predisposed to transformation and regulatory circuitries underlying tumour cell states and oncogenesis, highlighting hitherto unrecognized transitional progenitor intermediates predictive of disease prognosis and potential therapeutic vulnerabilities.

MODL-18. Enhancing anti-CD47 mAb efficacy with radiotherapy for Group 3 paediatric medulloblastoma in preclinical models

Abstract Medulloblastoma is the most common paediatric brain cancer. Standard treatment approaches, including craniospinal irradiation (CSI), can result in severe lifelong side effects and have not changed for decades resulting in a stagnation of survival outcomes for children with aggressive medulloblastoma. Despite successes in other cancers, no immunotherapies have been approved for use in paediatric medulloblastoma. Unlike other solid tumours, medulloblastomas are myeloid dominant, and immunotherapies must be rationally designed with the tumour microenvironment in mind. Anti-CD47 antibody therapy activates macrophages against cancer cells by blocking anti-phagocytic signalling mediated by CD47-SIRPa ligation and has shown preclinical efficacy in brain cancer models. We have developed preclinical CSI protocols that mimic clinical treatment response using a small animal radiotherapy platform. We show that CSI depletes adaptive immune cells in the brain, increasing the proportional abundance of myeloid cells, suggesting an opportunity to combine radiation with myeloid-targeted immunotherapy. We show that anti-CD47 therapy is ineffective as a single agent against a patient-derived xenograft model of Group 3 medulloblastoma (SJ_MB002), and that while the CSI protocol causes temporary tumour regression, the combination of anti-CD47 with CSI results in marked and persistent tumour regression. To enhance our preclinical evaluation of CSI and anti-CD47, we have developed new mouse models that more accurately reflect the developing microenvironment of children and show that immune populations in paediatric brain are distinct from adult mouse brain. Future work will elucidate the mechanisms by which radiotherapy alters the medulloblastoma microenvironment to enhance the anti-tumour activity of myeloid immune cells in the brain. By evaluating this novel combination of immunotherapy with standard medulloblastoma treatments, in age-appropriate models, our research should facilitate the rational selection and rapid translation of optimised treatment combinations for future medulloblastoma clinical trials.

EMBR-13. NOVEL SYNERGISTIC APPROACHES FOR TARGETED THERAPY OF MYC-DRIVEN MEDULLOBLASTOMA USING CRISPR/CAS9 GENE EDITING

IntroductionResistance to chemotherapy is a common cause of treatment failure in cancer patients and a major problem facing current cancer research. Targeted modulation of oncogenic signaling pathways may be used to systematically characterize drug resistance mechanisms across tumor entities and may help to identify new therapeutic strategies. Since the transcription factor MYC is aberrantly activated in many cancers including pediatric malignant brain tumors, like medulloblastoma (MB), our study focused on MYC-related drug resistance. Methods and ResultsWe performed high-throughput drug screening using our in-house semi-automated platform and identified the HDAC inhibitor Entinostat as a drug that shows promising effects in MYC-driven MB. Investigating genome-wide dCas9-based transcriptional activation screening, potential drug response modulators, mainly TGFB1/Erk/MKNK1 signaling including neural EGFL like 2 (NELL2), were discovered. For further validation, we stably overexpressed NELL2 in MYC-driven MB cells and treated overexpressing cells and the corresponding control cells with Entinostat. Using PI staining, cell cycle status was tracked. Entinostat treatment led to modest induction of cell death in MYC-driven MB control cells but only slightly increased cell death rate in MYC-driven MB cells with NELL2-overexpression. ConclusionWe report that the combination of genetic and pharmacological approaches is a powerful approach to study drug resistance. Our data suggest that activation of the TGFB1/Erk/MKNK1 signaling pathway desensitizes MYC-driven MB cells to Entinostat. Synergistic targeting of TGFB1/Erk/MKNK1 signaling and MYC could therefore provide a novel therapeutic option in this aggressive MB subtype.

Genomic and Transcriptomic Analyses Reveals ZNF124 as a Critical Regulator in Highly Aggressive Medulloblastomas.

Medulloblastoma (MB) is the most common malignant pediatric brain tumor, however, the mechanisms underlying tumorigenesis in different MB subgroups remain incompletely understood. Although previous studies of MB predisposition have been conducted in tertiary referral centers primarily in Caucasian cohorts, it is not unclear clear whether there exist population-specific genetic alterations in MBs. In this study, we investigated the contribution of genomic and transcriptomic alterations to the risk of malignant MB in the Chinese population (designated as the Asian cohort). We analyze the genomic and transcriptomic alterations of the Asian MB cohort by using a combination of whole-exome sequencing (WES) and RNA-deep-sequencing. In addition, we integrate publicly available data with the Asian MB cohort and identify a subset of potential MB-driving genes specifically enriched in each of the MB subgroups. We further characterize a newly identified group-3-enriched transcriptional regulator, ZNF124, and demonstrate that ZNF124 is critical for the growth of the most aggressive group-3 MB cells. Together, our analyses indicate conserved yet distinct genetic alterations and gene expression patterns of MBs between different ethnic groups. Our studies further provide an important resource for identifying potential tumor-driving factors in MBs, enhancing our understanding of the disease process for developing ethnically targeted therapies in patients with MB.

The AHR pathway represses TGF\u03b2-SMAD3 signalling and has a potent tumour suppressive role in SHH medulloblastoma

Sonic Hedgehog (SHH) medulloblastomas are brain tumours that arise in the posterior fossa. Cancer-propagating cells (CPCs) provide a reservoir of cells capable of tumour regeneration and relapse post-treatment. Understanding and targeting the mechanisms by which CPCs are maintained and expanded in SHH medulloblastoma could present novel therapeutic opportunities. We identified the aryl hydrocarbon receptor (AHR) pathway as a potent tumour suppressor in a SHH medulloblastoma mouse model. Ahr-deficient tumours and CPCs grown in vitro, showed elevated activation of the TGFβ mediator, SMAD3. Pharmacological inhibition of the TGFβ/SMAD3 signalling axis was sufficient to inhibit the proliferation and promote the differentiation of Ahr-deficient CPCs. Human SHH medulloblastomas with high expression of the AHR repressor (AHRR) exhibited a significantly worse prognosis compared to AHRRlow tumours in two independent patient cohorts. Together, these findings suggest that reduced AHR pathway activity promotes SHH medulloblastoma progression, consistent with a tumour suppressive role for AHR. We propose that TGFβ/SMAD3 inhibition may represent an actionable therapeutic approach for a subset of aggressive SHH medulloblastomas characterised by reduced AHR pathway activity.

NPI-0052 and gamma-radiation induce a synergistic apoptotic effect in the most aggressive medulloblastoma subgroup

Abstract Group 3 medulloblastoma (MB) is the most aggressive and least characterised of all MB subgroups. These tumours are highly metastatic and patients undergo aggressive treatments leaving survivors with severe side effects. The standard of care for these patients consists of surgical resection followed by gamma-radiation and chemotherapy. Whilst this conventional therapy is successful in the majority of cases, however, many survivors are left with lifelong severe neurocognitive and physical sequelae. Interestingly, the proteasome plays a vital role in the pathogenesis of different tumours, therefore proteasome inhibition can be used as a new strategy for treating MB. NPI-0052 is a proteasome inhibitor that can penetrate the blood-brain barrier with a good safety profile, making it an appealing treatment for brain tumors. In the present study, we evaluate the anticancer activity of NPI-0052 in a range of MB patient derived MB cells and cell lines. Our preclinical work demonstrated that NPI-0052 can inhibit proteasome activity and activate apoptosis in MB cells. We also show that the p53-family plays a substantial role in NPI-0052’s mechanism of action. Moreover, we observe that NPI-0052 has a synergistic apoptotic effect with gamma-radiation, a component of the current MB therapy, in both cells and tumour organoids. Our work underscores the use of NPI-0052 as a novel therapy for reducing the doses of gamma-radiation in G3-MB patients.

Orally bioavailable glutamine antagonist prodrug JHU-083 penetrates mouse brain and suppresses the growth of MYC-driven medulloblastoma

A subset of poor-prognosis medulloblastoma has genomic amplification of MYC. MYC regulates glutamine metabolism in multiple cellular contexts. We modified the glutamine analog 6-diazo-5-oxo-l-norleucine (DON) to mask its carboxylate and amine functionalities, creating a prodrug termed JHU-083 with increased oral bioavailability. We hypothesized that this prodrug would kill MYC-expressing medulloblastoma. JHU-083 treatment caused decreased growth and increased apoptosis in human MYC-expressing medulloblastoma cell lines. We generated a mouse MYC-driven medulloblastoma model by transforming C57BL/6 mouse cerebellar stem and progenitor cells. When implanted into the brains of C57BL/6 mice, these cells formed large cell/anaplastic tumors that resembled aggressive medulloblastoma. A cell line derived from this model was sensitive to JHU-083 in vitro. Oral administration of JHU-038 led to the accumulation of micromolar concentrations of DON in the mouse brain. JHU-083 treatment significantly increased the survival of immune-competent animals bearing orthotopic tumors formed by the mouse cerebellar stem cell model as well as immune-deficient animals bearing orthotopic tumors formed by a human MYC-amplified medulloblastoma cell line. These data provide pre-clinical justification for the ongoing development and testing of orally bioavailable DON prodrugs for use in medulloblastoma patients.

Abstract 202: STAT1 regulates B7H3 mediated angiogenesis in medulloblastoma

Abstract B7-H3 (CD276), an immune checkpoint member of the B7/CD28 family, plays a key role in the repression of the immune response by cancer cells. B7-H3 is overexpressed on tumor and tumor-associated cells, making it an interesting therapeutic target. B7-H3 also appears to play a role outside of immune evasion, contributing to the progression of cancer via invasion/migration, angiogenesis, and metastasis. Our previous data has confirmed the presence of B7-H3 in highly aggressive Myc+ medulloblastoma (MB) tumors. Data-mining studies revealed that B7-H3 is correlated with Myc expression in Group 3 and 4 MBs, and is associated with worse patient outcomes. miR-29, a tumor suppressor, has been shown downregulating the expression of B7-H3 in various cancers. We have shown that miR-29 can reduce the angiogenesis abilities of D283 and D425 cells by downregulating B7-H3 in vitro. Subsequent RNA-sequencing analysis of miR-29-transfected D283 cells revealed upregulation of STAT1 activity. Immunoblot analysis and real-time PCR of D283 and D425 cells demonstrated increased expression of p-STAT1, when transfected with miR-29 plasmid. Treatment with all-trans retinoic acid induced p-STAT1 expression, with a concomitant decrease in the expression of B7-H3 and MMP-9. This preliminary data revealed a novel role for STAT1 in potentially regulating B7-H3, and consequently, angiogenesis and metastasis in MB. To date, the role of STAT1 in MB progression has not been fully investigated, and so our results provide a new avenue for developing potential treatments for aggressive MB tumors. Citation Format: Ian J. Purvis, Janardhan Avilala, Maheedhara R. Guda, Sujatha Venkataraman, Rajeev Vibhakar, Andrew J. Tsung, Kiran K. Velpula, Swapna Asuthkar. STAT1 regulates B7H3 mediated angiogenesis in medulloblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 202.

Abstract 2865: AsiDNATM, a novel DNA repair inhibitor to sensitize aggressive medulloblastoma subtypes

Abstract Purpose: Medulloblastoma is paediatric tumor of the cerebellum. It represents the most frequent malignant brain tumor in childhood. It can be classified into four disparate molecular subgroups but current therapies are not yet tailored to their specificities. This is particularly important for subgroups with worse prognosis as Group3. Patients who survive often present severe treatment-related morbidity, which can be largely attributed to brain radiation in ages of development. It is therefore important to improve treatment efficacy in more aggressive subgroups as well as reduce treatment-related morbidity across all subgroups. We investigated whether AsiDNATM, a radiosensitizer in clinical trials which works through DNA repair inhibition, could be used to improve radiotherapy efficacy with no additional toxicity allowing radiation dose de-escalation in groups with better prognosis and improved radiotherapy efficacy in those with high-risk disease. Experimental design: The cytotoxic effect of AsiDNATM was determined in a panel of 4 human medulloblastoma cell lines from different subgroups and different P53 status. We analyzed the ability of AsiDNATM to reach brain tissue and toxicity of treatments with or without irradiation in pups (10 days old mice). Distribution of AsiDNA was estimated by quantification of Cy-5-AsiDNA in brain tissues. Radiation toxicity was estimated by survival to high doses, monitoring of circulating growth hormone (GH), weight loss, bones size at adult age and neuro histology analysis. We assessed the AsiDNATM radio-enhancing efficacy in Group3 xenografted preclinical models using X-RAD 320 (PXI Inc) X-ray irradiator. Local brain irradiation was performed with an image-guided X-ray system (SARPP, Xstrahl Ldt), in which dosimetry and animal setting were controlled at each treatment session. Results: AsiDNATM treatments exhibit an additive effect to radiation in all cell lines, leading to an approximate 2-fold reduction of the radiation dose to reach 50% survival. Cy5-AsiDNA distributed to pup brain but not to adult brain devoid of tumors. However, in adults AsiDNATM distributed in tumors in brain through permeability of tumor vessels. Irradiation toxicity in pups was confirmed by a significant decrease in GH secretion and associated reduction of bone size. The lethal dose after irradiation was 20 Gy. No increase of irradiation toxicity was observed with AsiDNATM. In vivo, AsiDNATM alone significantly enhances survival rates (p=0.005) and increases radiotherapy efficacy. When combined with radiotherapy, AsiDNATM led to delay in tumor growth and survival improvement as compared to radiotherapy alone. Conclusion: Overall, our current results show AsiDNATM as an attractive candidate to improve radiation therapy in medulloblastoma in both standard- and high-risk patients with no indication of additional toxicity in healthy developing brain tissues. Citation Format: Sofia Ferreira, Chloe Foray, Sophie Heinrich, Celio Pouponnot, Marie Dutreix. AsiDNATM, a novel DNA repair inhibitor to sensitize aggressive medulloblastoma subtypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2865.

MEDU-26. LATENT SOX9-POSITIVE CELLS RESPONSIBLE FOR MYC-DRIVEN MEDULLOBLASTOMA RECURRENCE

Abstract Tumor recurrence is the leading cause of death among children with medulloblastoma, the most common type of malignant pediatric brain tumors. The mechanisms behind medulloblastoma recurrence are not fully understood. We previously showed that the transcription factor SOX9 promotes cisplatin treatment resistance in medulloblastoma. Here we show that SOX9 levels correlate with poor prognosis in Group 3 tumors. By studying paired primary-recurrent medulloblastoma samples and patient-derived xenograft (PDX) models we further identified rare SOX9-positive slow-cycling, therapy-resistant tumor cells that accumulate in relapses and in leptomenigeal metastases of Group 3 and Group 4 patients. By using an inducible Tet-OFF transgenic (GTML) mouse model for malignant MYCN-driven Group 3 tumors we identified rare SOX9-positive, quiescent brain tumor cells that are more resistant to cisplatin. Dox treatment normally cures GTML transgenic animals that developed aggressive medulloblastoma by turning MYCN off. However, when crossing the Tet-OFF GTML model with a Tet-ON rtTA-Sox9 model we can redirect MYCN expression to the Sox9 promoter ultimately driving brain tumor recurrence from rare SOX9-positive cells with 100% penetrance. In this novel animal model, recurrent tumors were actively disseminating from the hindbrain to the spinal cord and into the forebrain similar to distant relapses found in patients. By overexpressing SOX9 in human Group 3 tumor cells, MYC was directly inhibited and cell proliferation was decreased. PDX models of Group 3 tumors further showed increased levels of SOX9-positivity and less proliferative cells in metastatic compartments. Expression profiling revealed that recurrences were more inflammatory, metastatic, immune evasive and showed elevated MGMT methyltransferase levels which depleted recurrent cells and sensitized them for chemotherapy when using the MGMT inhibitor lomeguatrib. To summarize, our data clarify important and complex mechanisms by which latent medulloblastoma cells fail to respond to standard therapy and generate relapses.

Inhibition of enhancer of zest homologue 2 is a potential therapeutic target for high-MYC medulloblastoma.

MYC amplification is common in Group 3 medulloblastoma and is associated with poor survival. Group 3 and Group 4 medulloblastomas are also known to have elevated levels of histone H3-lysine 27-tri-methylation (H3K27me3), at least in part due to high expression of the H3K27 methyltransferase enhancer of zest homologue 2 (EZH2), which can be regulated by MYC. We therefore examined whether MYC expression is associated with elevated EZH2 and H3K27me3 in medulloblastoma, and if high-MYC medulloblastomas are particularly sensitive to pharmacological EZH2 blockade. Western blot analysis of low (DAOY, UW228, CB SV40) and high (DAOY-MYC, UW228-MYC, CB-MYC, D425) MYC cell lines showed that higher levels of EZH2 and H3K27me3 were associated with elevated MYC. In fixed medulloblastoma samples examined using immunohistochemistry, most MYC positive tumors also had high H3K27me3, but many MYC negative ones did as well, and the correlation was not statistically significant. All high MYC lines tested were sensitive to the EZH2 inhibitor EPZ6438. Many low MYC lines also grew more slowly in the presence of EPZ6438, although DAOY-MYC cells responded more strongly than parent DAOY cultures with lower MYC levels. We find that higher MYC levels are associated with increased EZH2, and pharmacological blockade of EZH2 is a potential therapeutic strategy for aggressive medulloblastoma with elevated MYC.

Embryonic Stem Cell Models of Human Brain Tumors.

Utilization of human embryonic stem cells (hESCs) as a model system to study highly malignant pediatric cancers has led to significant insight into the molecular mechanisms governing tumor progression and has revealed novel therapeutic targets for these devastating diseases. Here, we describe a method for generating heterogeneous populations of neural precursors from both normal and neoplastic hESCs and the subsequent injection of neoplastic human embryonic neural cells (hENs) into intracerebellar or intracranial xenograft models. Histopathologically, neural tumors derived from neoplastic hENs exhibit features similar to more aggressive medulloblastoma, the most common malignant primary pediatric brain tumor. In this chapter, we will outline the detailed methods for culturing normal and neoplastic neural precursor cells in both adherent and tumorsphere format and the full characterization of the brain tumors generated from these cells in non-obese diabetic severe combined immunodeficiency (NOD SCID) mice.

The FDA approved PI3K inhibitor GDC-0941 enhances invitro the anti-neoplastic efficacy of Axitinib against c-myc-amplified high-risk medulloblastoma.

Aberrant receptor kinase signalling and tumour neovascularization are hallmarks of medulloblastoma development and are both considered valuable therapeutic targets. In addition to VEGFR1/2, expression of PDGFR α/β in particular has been documented as characteristic of metastatic disease correlating with poor prognosis. Therefore, we have been suggested that the clinically approved multi‐kinase angiogenesis inhibitor Axitinib, which specifically targets these kinases, might constitute a promising option for medulloblastoma treatment. Indeed, our results delineate anti‐neoplastic activity of Axitinib in medulloblastoma cell lines modelling the most aggressive c‐myc‐amplified Non‐WNT/Non‐SHH and SHH‐TP53‐mutated tumours. Exposure of medulloblastoma cell lines to Axitinib results in marked inhibition of proliferation and profound induction of cell death. The differential efficacy of Axitinib is in line with target expression of medulloblastoma cells identifying VEGFR 1/2, PDGFR α/β and c‐kit as potential markers for drug application. The high specificity of Axitinib and the consequential low impact on the haematopoietic and immune system render this drug ideal multi‐modal treatment approaches. In this context, we demonstrate that the clinically available PI3K inhibitor GDC‐0941 enhances the anti‐neoplastic efficacy of Axitinib against c‐myc‐amplified medulloblastoma. Our findings provide a rational to further evaluate Axitinib alone and in combination with other therapeutic agents for the treatment of most aggressive medulloblastoma subtypes.