Medulloblastoma Patients Research Articles

Rare cases of medulloblastoma with hypermutation.

BackgroundMedulloblastoma is the most common malignant brain tumor of childhood and is considered a tumor with low mutational burden (~1 Mut/Mb). Therefore, though the medulloblastoma genomes have been extensively characterized in literature, reports on potential hypermutations and underlying mutagenic processes in medulloblastomas are limited.AimIn this report, we studied the landscape of mutational burden in primary and recurrent medulloblastoma. Furthermore, we wanted to understand the differences in underlying mutagenic mechanisms in medulloblastoma with low and high mutational burdens.MethodsFifty‐three primary and recurrent medulloblastoma genomic sequence were downloaded from the European Genome Archive as BAM files. Thirty‐three cases were obtained from formalin‐fixed paraffin‐embedded tissues from pathology diagnostic archives of Spectrum Health and Cooperative Human Tissue Network. Somatic mutations were called using Mutect2, following best practices guidelines for Genome Analysis Toolkit V4. Mutational signatures were analyzed using deconstructSigs.ResultsWe identified nine medulloblastoma cases with high mutational burden (>5 Mut/Mb). Of them, five cases met the criteria of hypermutation (>10Mut/Mb), two of the five tumors had canonical mutations in the POLE proof‐reading domain, where a large proportion of mutations in these tumor genomes contributed to signature 10. The hypermutated cases also demonstrated mutational signatures 14, 15, and 21, indicating the role of mis match repair deficiency in their mutagenesis. Of the four known molecular subgroups in medulloblastoma–SHH, WNT, Group 3, and Group 4—both the POLE‐mutated cases belonged to the SHH subgroup. This report identifies rare cases of hypermutation in medulloblastoma driven by defects in DNA repair mechanisms.ConclusionHypermutation in medulloblastoma can impact therapeutic decisions, especially at recurrence in otherwise fatal high risk SHH‐medulloblastomas. A defect in DNA repair leading to SHH ‐medulloblastoma is yet another important mechanism that should be further investigated in the genesis of these tumors. Therefore, this report provides important scientific and clinical rationale for future research looking for incidence of hypermutation in large cohorts of medulloblastoma patients.

In-vivo proton range verification for reducing the risk of permanent alopecia in medulloblastoma treatment

ObjectiveThe purpose of this work is the clinical commissioning of a recently developed in-vivo range verification system for the head treatment of pediatric medulloblastoma patients. Inaccurate beam range for such treatment could lead to either inadequate dose coverage of the target volume or excessive dose to the head skin resulting permanent alopecia. MethodsThe in-vivo range verification system is designed to perform pre-treatment range verification and adjustment. An array of Si-diode detectors is to be placed on the patient immobilization mask in the exit direction of a whole-brain field; signal is analyzed, and the extracted water equivalent path length (WEPL) is compared to the expected one, revealing if a range correction is needed. The method was tested in solid water and anthropomorphic head phantom, with validation based on independent WEPL measurements. The measured WEPL were compared to those computed by the treatment planning system (TPS). ResultsThe accuracy for the WEPL measurements by the diode system in both solid water and anthropomorphic head phantom were on average within a millimeter from more accurate measurement by the dose-extinction technique, with the error range for the two phantoms as (0–1 ​mm) and (0–1.3 ​mm), respectively. When compared to the WEPL calculated by the treatment planning system, the measured values were on average within 1 ​% (range 0–3%) of the beam range. The accuracy of dose measurements by the diodes in the fall-off part of the depth dose profile was validated against the reference Markus chamber. No need for further correction (due to different beam parameters and detector dose ageing effects) was found. ConclusionsThe range verification workflow was successfully tested in the anthropomorphic head phantom. The performance of the in-vivo range verification system and related workflow meet the clinical requirements in terms of the needed WEPL accuracy for pretreatment range verification.

Exosomal miR-101-3p and miR-423-5p inhibit medulloblastoma tumorigenesis through targeting FOXP4 and EZH2.

Exosomal microRNAs (miRNAs) have been implicated in the development and progression of a variety of tumors; however, whether they contribute to medulloblastoma (MB) tumorigenesis remains to be elucidated. To address this, we first characterized the miRNA profiles of circulating exosomes by miRNA sequencing to identify miRNAs differentially expressed between children with MB and healthy controls. Then, we conducted in vitro and in vivo functional assays with the identified miRNAs and their predicted targets. We found that, compared with healthy controls, 35 miRNAs were upregulated and 5 downregulated in exosomes isolated from the plasma of MB patients. We further found that the expression of miR-101-3p and miR-423-5p was significantly higher in plasma exosomes from MB patients than in healthy controls in an expanded cohort and these exosomal miRNAs could be delivered to tumor cells via exosomes. An in vitro functional analysis of miR-101-3p and miR-423-5p showed that treating MB cells with the corresponding mimics significantly inhibited the proliferation, colony-forming ability, migratory ability, and invasive capacity of tumor cells, and promoted cell apoptosis. Additionally, miR-101-3p and miR-423-5p were found to act as tumor suppressors by directly targeting a common gene, FOXP4, which encodes a transcription factor with a vital role in embryonic development and tumorigenesis. Moreover, miR-101-3p also targeted EZH2, a histone methyltransferase, to reinforce its tumor inhibitory effects. Using a xenograft nude mouse model of MB, we further identified that the overexpression of miR-101-3p and miR-423-5p inhibited tumorigenesis in vivo. Our findings provide novel insights into the functions of exosomal miRNAs in mediating MB progression and suggest a potential therapeutic approach for the treatment of children with MB.

Emergence and maintenance of actionable genetic drivers at medulloblastoma relapse.

BackgroundLess than 5% of medulloblastoma (MB) patients survive following failure of contemporary radiation-based therapies. Understanding the molecular drivers of medulloblastoma relapse (rMB) will be essential to improve outcomes. Initial genome-wide investigations have suggested significant genetic divergence of the relapsed disease.MethodsWe undertook large-scale integrated characterization of the molecular features of rMB—molecular subgroup, novel subtypes, copy number variation (CNV), and driver gene mutation. 119 rMBs were assessed in comparison with their paired diagnostic samples (n = 107), alongside an independent reference cohort sampled at diagnosis (n = 282). rMB events were investigated for association with outcome post-relapse in clinically annotated patients (n = 54).ResultsSignificant genetic evolution occurred over disease-course; 40% of putative rMB drivers emerged at relapse and differed significantly between molecular subgroups. Non-infant MBSHH displayed significantly more chromosomal CNVs at relapse (TP53 mutation-associated). Relapsed MBGroup4 demonstrated the greatest genetic divergence, enriched for targetable (eg, CDK amplifications) and novel (eg, USH2A mutations) events. Importantly, many hallmark features of MB were stable over time; novel subtypes (>90% of tumors) and established genetic drivers (eg, SHH/WNT/P53 mutations; 60% of rMB events) were maintained from diagnosis. Critically, acquired and maintained rMB events converged on targetable pathways which were significantly enriched at relapse (eg, DNA damage signaling) and specific events (eg, 3p loss) predicted survival post-relapse.ConclusionsrMB is characterised by the emergence of novel events and pathways, in concert with selective maintenance of established genetic drivers. Together, these define the actionable genetic landscape of rMB and provide a basis for improved clinical management and development of stratified therapeutics, across disease-course.

MOMC-5. Systems pharmacogenomics identifies novel targets and clinically actionable therapeutics for medulloblastoma

BackgroundMedulloblastoma (MB) is the most common malignant paediatric brain tumour and a leading cause of cancer-related mortality and morbidity. Existing treatment protocols are aggressive in nature resulting in significant neurological, intellectual and physical disabilities for the children undergoing treatment. Clearly, there is an urgent need for improved, targeted therapies that minimize these harmful side effects.MethodsWe identified candidate drugs for MB using a network-based systems-pharmacogenomics approach: based on results from a functional genomics screen, we identified a network of interactions implicated in human MB growth regulation. We then integrated drugs and their known mechanisms of action, along with gene expression data from a large collection of medulloblastoma patients to identify drugs with potential to treat MB.ResultsOur analyses identified drugs targeting CDK4, CDK6, and AURKA as strong candidates for MB; all of these genes are well validated as drug targets in other tumour types. We also identified non-WNT MB as a novel indication for drugs targeting TUBB, CAD, SNRPA, SLC1A5, PTPRS, P4HB and CHEK2. Based upon these analyses we subsequently demonstrated that one of these drugs, the new microtubule stabilizing agent, ixabepilone, blocked tumour growth in vivo in mice bearing Sonic Hedgehog and Group 3 patient-derived xenograft tumours, providing the first demonstration of its efficacy in MB.ConclusionsOur findings confirm that this data-driven systems pharmacogenomics strategy is a powerful approach for the discovery and validation of novel therapeutic candidates relevant to MB treatment, and along with data validating ixabepilone in PDX models of the two most aggressive subtypes of medulloblastoma, we present the network analysis framework as a resource for the field.

Abstract 95: The landscape of extrachromosomal circular DNA in medulloblastoma subgroups

Abstract Extrachromosomal circular DNA (ecDNA) has been shown to be an important driver of particularly aggressive human cancers. However, it remains unknown to what extent and by which molecular mechanisms ecDNA promotes tumor development and progression in the different molecular subgroups of medulloblastoma (MB), the most common malignant pediatric brain tumor. To answer these questions, we have assembled a multi-institutional retrospective cohort of 472 MB patient samples with available whole genome sequencing (WGS) data, drawing from three non-overlapping public cancer genomic data repositories and covering all four MB subgroups (i.e. WNT, SHH, Group 3 and Group 4). Using genomic cloud computing platforms, local computing resources and recent computational methods for the detection and reconstruction of ecDNA, we find ecDNA in 66 patients (14%) and observe that the presence of ecDNA is associated with significantly poorer outcomes. By subgroup, ecDNA was found in 0/13 WNT (0%), 18/69 SHH (26%), 12/76 G3 (16%) and 14/123 G4 (11%) patients. Affected genomic loci harbor up to hundredfold amplification of oncogenes including MYC, MYCN, and TERT; as well as amplifications of novel putative driver genes involved in chromatin remodeling, DNA repair, and kinase signaling pathways. To investigate the frequency and properties of ecDNA in model systems of MB tumors, we further analyzed WGS data from 24 patient-derived xenografts (PDX) and four cell lines. ecDNA was substantially more frequent in these patient-derived models (17 of 29, 59%) than in our patient cohort. To elucidate the functional regulatory landscapes of ecDNAs in MB, we generated transcriptional (RNA-seq), accessible chromatin (ATAC-seq), and chromatin interaction (Hi-C) profiles from 6 MB tumor samples. In each case, we identify regulatory interactions that cross fusion breakpoints on the ecDNA, representing potential “enhancer rewiring” events that are likely to contribute to transcriptional activation of co-amplified oncogenes. To test this hypothesis, we are currently conducting in-vitro CRISPRi screens targeting regulatory regions on the ecDNA with the aim of testing whether enhancer-rewiring increases the transcription of co-amplified oncogenes and promotes proliferation and cell line growth. In summary, our study analyzes the frequency, diversity and functional relevance of regulatory enhancers co-amplified with oncogenes on ecDNA across MB subgroups and provides strong scientific justification for continued mechanistic studies of ecDNA in MB tumors and tumor models with the potential to uncover new therapeutic approaches. Citation Format: Owen S. Chapman, Shanqing Wang, Jens Luebeck, Alexandra Garancher, Jon D. Larson, Joshua Lange, John Crawford, Scott L. Pomeroy, Paul Mischel, Ernest Fraenkel, Robert J. Wechsler-Reya, Vineet Bafna, Jill P. Mesirov, Lukas Chavez. The landscape of extrachromosomal circular DNA in medulloblastoma subgroups [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 95.

Abstract 3023: B7 H3 and autophagy in medulloblastoma

Abstract B7-H3 and Autophagy in Medulloblastoma (MB) is the most common malignant pediatric brain tumor and we reported high expression of the immune checkpoint protein, B7-H3. We also showed that B7-H3 promotes exosome secretion, with a plausible cross-talk between exosomes and autophagy in tumor development. Autophagy promotes or suppresses tumorigenesis but its role in pediatric brain tumors has not be elucidated. As our recent research has shown, the elevated levels of B7-H3 in MB enhances exosome secretion that may regulate autophagy processes favoring tumorigenesis over tumor suppression. Therefore, the goal of this study is to investigate the relationship between B7-H3 and autophagy pathways in MB in efforts to improve treatment options and patient survival. Datamining studies conducted using MB patient datasets showed a negative correlation between B7-H3 and autophagy markers; Beclin1, LC3, and ATGs. Kaplan-Meier analysis further demonstrated an inverse relationship between autophagy markers and B7-H3 in the survival curves. Next, using human autophagy array, we observed significant reduction in the expression of Beclin1, LC3, and ATGs upon B7-H3 overexpression in D425 cells. In a separate experiment, we show that silencing B7-H3 using specific shRNA resulted in increased levels of LC3-II. Functional studies performed using FACs analyses and clonogenic assay on shB7-H3 treated- D425, UW228 cells revealed that inhibition of B7-H3 was associated with augmented apoptosis and reduction in colony formation, respectively. These results demonstrate the negative role of B7-H3 in MB autophagy regulation. Therefore, inhibition of B7-H3 may prove effective at promoting autophagy-mediated cell death in MB cells. Citation Format: Ayse E. Kesaf, Katherine Shishido, Kiran K. Velpula, Swapna Asuthkar. B7 H3 and autophagy in medulloblastoma [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 3023.

Systems pharmacogenomics identifies novel targets and clinically actionable therapeutics for medulloblastoma

BackgroundMedulloblastoma (MB) is the most common malignant paediatric brain tumour and a leading cause of cancer-related mortality and morbidity. Existing treatment protocols are aggressive in nature resulting in significant neurological, intellectual and physical disabilities for the children undergoing treatment. Thus, there is an urgent need for improved, targeted therapies that minimize these harmful side effects.MethodsWe identified candidate drugs for MB using a network-based systems-pharmacogenomics approach: based on results from a functional genomics screen, we identified a network of interactions implicated in human MB growth regulation. We then integrated drugs and their known mechanisms of action, along with gene expression data from a large collection of medulloblastoma patients to identify drugs with potential to treat MB.ResultsOur analyses identified drugs targeting CDK4, CDK6 and AURKA as strong candidates for MB; all of these genes are well validated as drug targets in other tumour types. We also identified non-WNT MB as a novel indication for drugs targeting TUBB, CAD, SNRPA, SLC1A5, PTPRS, P4HB and CHEK2. Based upon these analyses, we subsequently demonstrated that one of these drugs, the new microtubule stabilizing agent, ixabepilone, blocked tumour growth in vivo in mice bearing patient-derived xenograft tumours of the Sonic Hedgehog and Group 3 subtype, providing the first demonstration of its efficacy in MB.ConclusionsOur findings confirm that this data-driven systems pharmacogenomics strategy is a powerful approach for the discovery and validation of novel therapeutic candidates relevant to MB treatment, and along with data validating ixabepilone in PDX models of the two most aggressive subtypes of medulloblastoma, we present the network analysis framework as a resource for the field.

Immune cell landscape and immunotherapy of medulloblastoma.

ABSTRACTMedulloblastoma is the most common primary pediatric malignancy of the central nervous system. Recurrent and refractory patients account for approximately 30% of them. Immune cells are an important component of the brain tumor microenvironment, including tumor‐associated macrophages, T lymphocytes, natural killer cells, dendritic cells, neutrophils and B lymphocytes. Understanding how they behave and interact is important in the investigation of the onset and progression of medulloblastoma. Here, we overview the features and recent advances of each component of immune cells in medulloblastoma. Meanwhile, immunotherapy is a promising but also challenging treatment strategy for medulloblastoma. At present, there are a growing number of immunotherapeutic approaches under investigation including immune checkpoint inhibitors, oncolytic viruses, cancer vaccines, chimeric antigen receptor T cell therapies, and natural killer cells in recurrent and refractory medulloblastoma patients.

Proton therapy for adult medulloblastoma: Acute toxicity and disease control outcomes.

We report disease control, survival outcomes, and treatment-related toxicity among adult medulloblastoma patients who received proton craniospinal irradiation (CSI) as part of multimodality therapy. We reviewed 20 adults with medulloblastoma (≥ 22years old) who received postoperative proton CSI ± chemotherapy between 2008 and 2020. Patient, disease, and treatment details and prospectively obtained patient-reported acute CSI toxicities were collected. Acute hematologic data were analyzed. Median age at diagnosis was 27years; 45% of patients had high-risk disease; 75% received chemotherapy, most (65%) after CSI. Eight (40%) patients received concurrent vincristine with radiotherapy. Median CSI dose was 36GyE with a median tumor bed boost of 54GyE. Median duration of radiotherapy was 44days. No acute ≥ grade 3 gastrointestinal or hematologic toxicities attributable to CSI occurred. Grade 2 nausea and vomiting affected 25% and 5% of patients, respectively, while 36% developed acute grade 2 hematologic toxicity (36% grade 2 leukopenia and 7% grade 2 neutropenia). Those receiving concurrent chemotherapy with CSI had a 38% rate of grade 2 hematologic toxicity compared to 33% among those not receiving concurrent chemotherapy. Among patients receiving adjuvant chemotherapy (n = 13), 100% completed ≥ 4 cycles and 85% completed all planned cycles. With a median follow-up of 3.1years, 4-year actuarial local control, disease-free survival, and overall survival rates were 90%, 90%, and 95%, respectively. Proton CSI in adult medulloblastoma patients is very well tolerated and shows promising disease control and survival outcomes. These data support the standard use of proton CSI for adult medulloblastoma.

EMBR-04. BET INHIBITION TARGETS RADIOTHERAPY RESISTANCE IN H3K27ME3-DEFICIENT GROUP 3 MEDULLOBLASTOMA

Abstract Medulloblastoma has been categorized into four subgroups based on genetic, epigenetic and transcriptional profiling. However, molecular pathways determining radiotherapy response in this tumor remain elusive. Here, we investigated the role of the EZH2-dependent histone H3K27 tri-methylation in radiotherapy response in medulloblastoma. We demonstrate that 47.2% of group 3 and 4 medulloblastoma patients have H3K27me3-deficient tumors. Loss of H3K27me3 was associated with a radioresistant phenotype, high relapse rates and poor overall survival. We show that an epigenetic switch from H3K27me3 to H3K27ac occurs at specific genomic loci in H3K27me3-deficient medulloblastoma cells altering the transcriptional profile. The resulting up-regulation of EPHA2 (ephrin type-A receptor 2) stimulates an excessive activation of the pro-survival AKT signaling pathway leading to radiotherapy resistance. We show that BET inhibition targets radiation resistance in H3K27me3-deficient medulloblastoma by suppressing H3K27ac levels, blunting EPHA2 overexpression and mitigating the excessive AKT signaling. Additionally, BET inhibition sensitizes medulloblastoma cells to radiation by enhancing apoptotic response through suppression of Bcl-XL and up-regulation of Bim expression. Our work demonstrates a novel mechanism of radiation resistance in medulloblastoma and identifies an epigenetic marker predictive of radiotherapy response. Based on these findings we propose an epigenetically guided treatment approach targeting radiotherapy resistance in medulloblastoma patients.

EMBR-16. SMOOTHENED-ACTIVATING LIPIDS DRIVE RESISTANCE TO CDK4/6 INHIBITION IN HEDGEHOG-ASSOCIATED MEDULLOBLASTOMA

BackgroundMedulloblastoma is an aggressive pediatric brain tumor that is associated with misactivation of the Hedgehog (HH) pathway. Our lab has shown that CDK6, a critical activator of the cell cycle, is a direct transcriptional target of oncogenic HH signaling, and that inhibiting CDK6 blocks the growth of HH-associated medulloblastoma in mice. A clinical trial exploring the efficacy of CDK6 inhibition in medulloblastoma patients is underway, but prior attempts to target the HH pathway in medulloblastoma have been encumbered by resistance to molecular monotherapy. Thus, we sought to identify mechanisms of resistance to CDK6 inhibition in HH-associated medulloblastoma. MethodsWe performed orthogonal CRISPR and CRISPR interference screens in HH-associated medulloblastoma cells treated with pharmacologic inhibitors of CDK6 in vitro, and RNA-sequencing of HH-associated medulloblastomas with genetic deletion of CDK6 in vivo. Mechanistic and functional validation of resistance pathways was performed using CRISPR interference, immunoblotting, immunofluorescence, genetics, and pharmacology. Lipid quantification was carried out by ultra-high performance liquid chromatography-tandem mass spectrometry. ResultsOur results reveal that decreased ribosomal protein expression underlies resistance to CDK6 inhibition in HH-associated medulloblastoma, leading to endoplasmic reticular (ER) stress and activation of the unfolded protein response (UPR). We show that ER stress and the UPR increase the activity of enzymes producing Smoothened-activating sterol lipids that sustain oncogenic HH signaling in medulloblastoma despite CDK6 inhibition. These discoveries suggest that combination molecular therapy against CDK6 and HSD11ß2, an enzyme producing Smoothened-activating lipids, may be an effective treatment for HH-associated medulloblastoma. In support of this hypothesis, we demonstrate that concurrent genetic deletion or pharmacological inhibition of CDK6 and HSD11ß2 additively blocks the growth of multiple models of HH-associated medulloblastoma in mice. ConclusionsSmoothened-activating lipid biosynthesis underlies resistance to CDK6 inhibition in HH-associated medulloblastoma, revealing a novel combination therapy to treat the most common malignant brain tumor in children.

OMIC-01. THE LANDSCAPE OF EXTRACHROMOSOMAL CIRCULAR DNA IN MEDULLOBLASTOMA SUBGROUPS

Extrachromosomal circular DNA (ecDNA) is an important driver of particularly aggressive human cancers. However, the prevalence of ecDNA, and its role in tumor development and progression in the different molecular subgroups of medulloblastoma (MB), remain unknown. To answer these questions, we have assembled a multi-institutional retrospective cohort of 472 MB patients with available whole genome sequencing (WGS) data, drawing from three cancer genomic data repositories and covering all MB subgroups (WNT, SHH, Group 3 and Group 4). Using recent computational methods to detect and reconstruct ecDNA, we find ecDNA in 66 patients (14%) and observe that the presence of ecDNA is associated with significantly poorer outcomes. By subgroup, ecDNA was found in 0/24 WNT (0%), 22/109 SHH (20%), 15/107 Group 3 (14%) and 20/181 Group 4 (11%) patients. Affected genomic loci harbor up to hundredfold amplification of oncogenes including MYC, MYCN, TERT, and other novel putative oncogenes. We further analyzed 24 patient-derived xenograft (PDX) and four cell line models of MB tumors. ecDNA was substantially more frequent in patient-derived models (17 of 29, 59%) than in our patient cohort. To elucidate the functional regulatory landscapes of ecDNAs in MB, we generated transcriptional (RNA-seq), accessible chromatin (ATAC-seq), and chromatin interaction (Hi-C) profiles of 6 MB tumor samples. In each case, we identify regulatory interactions that cross fusion breakpoints on the ecDNA, representing potential “enhancer rewiring” events which may contribute to transcriptional activation of co-amplified oncogenes. To test this hypothesis, we are currently conducting in-vitro CRISPRi screens targeting regulatory regions on the ecDNA of a MB cell line to determine whether these enhancers promote proliferation. In summary, our study analyzes the frequency, diversity and functional relevance of ecDNA across MB subgroups and provides strong justification for continued mechanistic studies of ecDNA in MB with the potential to uncover new therapeutic approaches.

Cryptic developmental events determine medulloblastoma radiosensitivity and cellular heterogeneity without altering transcriptomic profile

It is unclear why medulloblastoma patients receiving similar treatments experience different outcomes. Transcriptomic profiling identified subgroups with different prognoses, but in each subgroup, individuals remain at risk of incurable recurrence. To investigate why similar-appearing tumors produce variable outcomes, we analyzed medulloblastomas triggered in transgenic mice by a common driver mutation expressed at different points in brain development. We genetically engineered mice to express oncogenic SmoM2, starting in multipotent glio-neuronal stem cells, or committed neural progenitors. Both groups developed medulloblastomas with similar transcriptomic profiles. We compared medulloblastoma progression, radiosensitivity, and cellular heterogeneity, determined by single-cell transcriptomic analysis (scRNA-seq). Stem cell-triggered medulloblastomas progressed faster, contained more OLIG2-expressing stem-like cells, and consistently showed radioresistance. In contrast, progenitor-triggered MBs progressed slower, down-regulated stem-like cells and were curable with radiation. Progenitor-triggered medulloblastomas also contained more diverse stromal populations, with more Ccr2+ macrophages and fewer Igf1+ microglia, indicating that developmental events affected the subsequent tumor microenvironment. Reduced mTORC1 activity in M-Smo tumors suggests that differential Igf1 contributed to differences in phenotype. Developmental events in tumorigenesis that were obscure in transcriptomic profiles thus remained cryptic determinants of tumor composition and outcome. Precise understanding of medulloblastoma pathogenesis and prognosis requires supplementing transcriptomic/methylomic studies with analyses that resolve cellular heterogeneity.

Epigenetic-Based Therapy-A Prospective Chance for Medulloblastoma Patients' Recovery.

Medulloblastoma (MB) is one of the most frequent and malignant brain tumors in children. The prognosis depends on the advancement of the disease and the patient’s age. Current therapies, which include surgery, chemotherapy, and irradiation, despite being quite effective, cause significant side effects that influence the central nervous system’s function and cause neurocognitive deficits. Therefore, they substantially lower the quality of life, which is especially severe in a developing organism. Thus, there is a need for new therapies that are less toxic and even more effective. Recently, knowledge about the epigenetic mechanisms that are responsible for medulloblastoma development has increased. Epigenetics is a phenomenon that influences gene expression but can be easily modified by external factors. The best known epigenetic mechanisms are histone modifications, DNA methylation, or noncoding RNAs actions. Epigenetic mechanisms comprehensively explain the complex phenomena of carcinogenesis. At the same time, they seem to be a potential key to treating medulloblastoma with fewer complications than past therapies. This review presents the currently known epigenetic mechanisms that are involved in medulloblastoma pathogenesis and the potential therapies that use epigenetic traits to cure medulloblastoma while maintaining a good quality of life and ensuring a higher median overall survival rate.

A novel anthelminthic drug suppresses the growth of medulloblastoma tumors by inhibiting PKA/Gli1 signaling axis

Pediatric brain tumor is one of the most malignant solid tumors in children and have profound impact on the morbidity and mortality in these patients. Statistically, brain tumors are one of the leading cause of cancer-related deaths in patients from 0-19 years of age. Medulloblastoma (MB) is one of the most common pediatric brain tumors occurring in children. Sonic hedgehog (Shh) activated subgroup of MB is considered to be highly aggressive and metastatic in nature. Shh-MB is characterized by mutations in PTCH1, SMO and SuFu along with amplified activation of Gli1, a major transcription factor of this signaling pathway. In the current study, we have evaluated the anti-cancer effects of an anthelminthic drug ‘moxidectin’. Several MB cell lines such as Daoy, UW426, UW228, ONS76, and PFSK1 were treated with moxidectin in a concentration and time dependent manner. Our results demonstrated that moxidectin treatment resulted in significantly reduced proliferation of MB cells. The IC50 of moxidectin in all the MB cell lines ranged 10-17μM after 24, 48 and 72 hours of treatment. Moreover, moxidectin was able to induce 3-4 fold increase of apoptosis in all the MB cell lines as evaluated by AnnexinV-FITC/PI assay, and increased cleavage of caspase 3 and PARP. Western blotting analysis demonstrated that moxidectin treatment significantly reduced non-canonical activation of Gli1 and its downstream effector molecules such as Pax-6, Oct-4, Sox-2 and Nanog. To our knowledge, this study for the first time focuses on GABAA receptor agonist mediated PKA inhibition and ultimately suppression of non-canonical Gli1 activation. Efficacy of moxidectin was evaluated in an in vivo tumor model by subcutaneously and intracranically implanting human Daoy MB cells. Our results demonstrated that 2.5 mg/kg moxidectin by oral administration everyday suppressed the growth of Daoy tumors by 50-55% in both subcutaneous and intracranial tumor models. Conclusively, our results indicate that moxidectin effectively reduces the growth of MB tumors by inhibiting PKA/Gli1 signaling. Most importantly, moxidectin is FDA approved drug and is already in clinical use for the treatment of river blindness in humans with an established safety record, therefore any positive findings from our studies will prompt further clinical investigation into repositioning moxidectin for the treatment of MB patients.

Molecular Characterization of Medulloblastoma in a Patient with Neurofibromatosis Type 1: Case Report and Literature Review

Brain tumors are the most common solid neoplasms of childhood. They are frequently reported in children with Neurofibromatosis type 1 (NF1). The most frequent central nervous system malignancies described in NF1 are optic pathway gliomas and brainstem gliomas. Medulloblastoma (MB) in NF1 patients is extremely rare, and to our knowledge, only 10 cases without molecular characterization are described in the literature to date. We report the case of a 14-year-old girl with NF1 that came to our attention for an incidental finding of a lesion arising from cerebellar vermis. The mass was completely resected, revealing a localized classic medulloblastoma (MB), subgroup 4. She was treated as a standard-risk MB with a dose-adapted personalized protocol. The treatment proved to be effective, with minor toxicity. Brain and spine MRI one year after diagnosis confirmed the complete remission of the disease. To our knowledge, this is the only case of MB reported in a patient with NF1 with molecular characterization by the methylation profile. The association between NF1 and MB, although uncommon, may not be an accidental occurrence.

DDRE-02. SMOOTHENED-ACTIVATING LIPIDS DRIVE RESISTANCE TO CDK4/6 INHIBITION IN HEDGEHOG-ASSOCIATED MEDULLOBLASTOMA

BACKGROUNDMedulloblastoma is an aggressive pediatric brain tumor that is associated with misactivation of the Hedgehog (HH) pathway. Our lab has shown that CDK6, a critical activator of the cell cycle, is a direct transcriptional target of oncogenic HH signaling, and that inhibiting CDK6 blocks the growth of HH-associated medulloblastoma in mice. A clinical trial exploring the efficacy of CDK6 inhibition in medulloblastoma patients is underway, but prior attempts to target the HH pathway in medulloblastoma have been encumbered by resistance to molecular monotherapy. Thus, we sought to identify mechanisms of resistance to CDK6 inhibition in HH-associated medulloblastoma.METHODSWe performed orthogonal CRISPR and CRISPR interference screens in HH-associated medulloblastoma cells treated with pharmacologic inhibitors of CDK6 in vitro, and RNA-sequencing of HH-associated medulloblastomas with genetic deletion of CDK6 in vivo. Mechanistic and functional validation of resistance pathways was performed using CRISPR interference, immunoblotting, immunofluorescence, genetics, and pharmacology. Lipid quantification was carried out by ultra-high performance liquid chromatography-tandem mass spectrometry.RESULTSOur results reveal that decreased ribosomal protein expression underlies resistance to CDK6 inhibition in HH-associated medulloblastoma, leading to endoplasmic reticular (ER) stress and activation of the unfolded protein response (UPR). We show that ER stress and the UPR increase the activity of enzymes producing Smoothened-activating sterol lipids that sustain oncogenic HH signaling in medulloblastoma despite CDK6 inhibition. These discoveries suggest that combination molecular therapy against CDK6 and HSD11ß2, an enzyme producing Smoothened-activating lipids, may be an effective treatment for HH-associated medulloblastoma. In support of this hypothesis, we demonstrate that concurrent genetic deletion or pharmacological inhibition of CDK6 and HSD11ß2 additively blocks the growth of multiple models of HH-associated medulloblastoma in mice.CONCLUSIONSSmoothened-activating lipid biosynthesis underlies resistance to CDK6 inhibition in HH-associated medulloblastoma, revealing a novel combination therapy to treat the most common malignant brain tumor in children.

Observed-to-expected incidence ratios of second malignant neoplasms after radiation therapy for medulloblastoma: A Surveillance, Epidemiology, and End Results analysis.

The authors analyzed the incidence and types of second malignant neoplasms (SMNs) in patients treated for medulloblastoma. The authors compared the incidence of SMNs after radiotherapy (RT) for medulloblastoma in patients treated in 1973-2014 with the incidence in the general population with the multiple primary-standardized incidence ratio function of Surveillance, Epidemiology, and End Results 9. Observed-to-expected incidence (O/E) ratios and 95% confidence intervals (CIs) were reported for the entire cohort and by disease site according to age at diagnosis, treatment era, and receipt of chemotherapy. P values < .05 were considered statistically significant. Of the 1294 patients with medulloblastoma who received RT, 68 developed 75 SMNs. The O/E ratio for SMNs among all patients was 4.49 (95% CI, 3.53-5.62; P < .05). The site at highest risk was the central nervous system (CNS; O/E, 40.62; 95% CI, 25.46-61.51), which was followed by the endocrine system (O/E, 15.95; 95% CI, 9.12-25.91), bone (O/E, 14.45; 95% CI, 1.75-52.21), soft tissues (O/E, 9.01; 95% CI, 1.09-32.56), the digestive system (O/E, 5.03; 95% CI, 2.51-9.00), and the lymphatic/hematopoietic system (O/E, 3.37; 95% CI, 1.35-6.94). The O/E ratio was higher for patients given chemotherapy and RT (O/E, 5.52; 95% CI, 3.75-7.83) than for those given RT only (O/E, 3.96; 95% CI, 2.88-5.32). Patients with medulloblastoma are at elevated risk for SMNs in comparison with the general population. Variations in O/E for SMNs by organ systems were found for treatment modality, age at diagnosis, and time of diagnosis. The most common site, the CNS, was involved more often in younger patients and those given chemotherapy with RT.

An intrathecal limited postoperative chemotherapy regimen for the treatment of young children with nodular/desmoplastic medulloblastoma and medulloblastoma with extensive nodularity

Therapy for medulloblastoma in patients < 4years old omits radiotherapy due to anticipated neurocognitive deficits. The German Pediatric Brain Tumor Study Group described a chemotherapy regimen (HIT-SKK' 92 and HIT-SKK 2000) without radiation which yielded a 5-year progression-free survival (PFS) rate of 85% in children with nodular/desmoplastic medulloblastoma (NDMB) and medulloblastoma with extensive nodularity (MBEN). We modified the HIT-SKK regimen to reduce the total number of intrathecal methotrexate (IT MTX) doses from 12 to 2 doses/cycle and obviate Ommaya reservoir implantation through the use of lumbar administration. We report the outcomes of five patients treated with our approach. IT MTX was eliminated altogether on weeks when high-dose intravenous methotrexate was administered. On weeks when no systemic methotrexate was administered, a single dose of lumbar-administered IT MTX was substituted in place of multiple intra-Ommaya doses. Cumulative dosing of MTX was 16-24mg/cycle (age-based) compared to 24mg/cycle in the HIT-SKK regimen. Following chemotherapy, patients were monitored with interval imaging, observation for acute and late effects, and survival. Four children remained in remission 3, 5, 9, and 10years post-treatment respectively, without observed learning difficulties. One child had recurrent tumor and metastasis 6months post-treatment. She failed the attempted salvage regimen and continued to deteriorate, dying of disease at 3years old. Review of existing literature supported our modifications well. While this report is limited by the small number of children treated, we believe there is encouraging evidence that our approach warrants further evaluation in a larger population of young children with NDMB and MBEN.