Medulloblastoma Subgroups Research Articles

MBRS-42. YB-1 - A NOVEL THERAPEUTIC TARGET IN HIGH-RISK MEDULLOBLASTOMA?

Medulloblastoma relapse occurs in 30–40% of patients and is typically fatal. The emergence of therapy resistant sub-clones likely plays a major role in a large proportion of recurrent medulloblastoma. Y-box binding protein 1 (YB-1) is a multifunctional transcription/translation factor and known onco-protein. Overexpression has been described in numerous cancers, where elevated expression and nuclear accumulation correlates with disease progression, metastasis and drug resistance. Genomic analysis of a large medulloblastoma cohort revealed YB-1 up-regulation across all subgroups of medulloblastoma, where elevated expression correlated with poor survival. Immunohistochemical staining of patient tissue microarrays displayed significant YB-1 expression, with a high proportion (83%) of patients exhibiting nuclear accumulation. High YB-1 expression was also observed at both protein and RNA level across medulloblastoma cell lines, with expression highest in Group 3 and 4. Hence, we hypothesised that YB-1 plays a role in medulloblastoma chemoresistance and progression. Treatment of Group 3 (HDMB-03 and D283MED) and SHH (DAOY) cell lines with vincristine and cisplatin and analysis of cellular localisation by nuclear/cytoplasmic fractionation and immunofluorescence demonstrated that YB-1 undergoes nuclear translocation in response to these standard medulloblastoma chemotherapy agents. Chromatin immunoprecipitation (ChIP) analysis of untreated Group 3 cell lines (D283MED and HDMB-03) demonstrated considerable YB-1 interaction with an inverted CCAAT box in the ATP-binding cassette subfamily B member 1 (ABCB1) promoter. RT-PCR analysis of ABCB1 following vincristine and cisplatin treatment revealed differences in transcript expression, indicative of different YB-1 promoter interactions dependent on chemotherapeutic treatment. Our results highlight YB-1 as a novel candidate chemoresistance driver in medulloblastoma.

MBCL-43. RECURRENT MEDULLOBLASTOMA – LONG-TERM SURVIVAL WITH A “MEMMAT” BASED ANTIANGIOGENIC APPROACH

INTRODUCTIONPatients with recurrent medulloblastoma have a poor prognosis with only around 8% of patients surviving at 5 years irrespective of salvage therapy used. We report on 29 patients from four institutions treated with a “MEMMAT” based antiangiogenic combination therapy.PATIENTS AND METHODSFrom 11/2006 to 06/2016, 29 patients were diagnosed with a recurrent medulloblastoma (19 first, 10 multiple recurrences). Median age at start of antiangiogenic therapy was 10 years (range 1–27). Subgroup of medulloblastoma was available in 18 patients and was group 3 or 4 in all except two (one WNT, one SHH-infant). For their current relapse patients received an antiangiogenic combination therapy consisting of bevacizumab, thalidomide, celecoxib, fenofibrate, and etoposide, alternating with cyclophosphamide and augmented with intraventricular therapy (etoposide and liposomal cytarabine).RESULTSAs of 01/2020, 8/29 patients are alive at a median of 44 months after recurrence. 6/8 surviving patients are currently in CCR between 66 and 134 months after recurrence that prompted MEMMAT therapy. Two patients are again in remission after intercurrent relapses 105 and 102 months after first starting MEMMAT therapy. Five patients died of another cause (accident, leukemia, septicemia). OS (median 44 months) was 44±10% at 5 years and 39±10% at 10 years, PFS was 33±10% at 5 years and 28 ±9% at 10 years. Therapy was well tolerated and toxicities were manageable.CONCLUSIONOur results suggest that antiangiogenic metronomic chemotherapy has clinical activity in recurrent medulloblastoma. Further investigation with an international phase II study is ongoing (MEMMAT; ClinicalTrials.gov Identifier: NCT01356290).

MBRS-63. THE ROLE OF THE SWI/SNF COMPLEX SUBUNIT SMARCD3 IN MEDULLOBLASTOMA

Medulloblastoma is the most common malignant brain tumor in children, with the Group 3 (G3) having the worst prognosis of the subgroups (WNT, SHH, and Group 4). We aimed to determine the underlying differences between G3 and the other subgroups, with an emphasis on genes that control the epigenome for developing effective treatments for patients with this disease. To this end, we found that G3 has elevated expression of the SWI/SNF subcomponent, SMARCD3 (P<0.001), which serves to guide the SWI/SNF complex to different genomic regions through interactions with various transcription factors. However, little is known about function of SMARCD3 in cancer, particularly in medulloblastoma. Clinically, elevated SMARCD3 mRNA resulted in a poorer prognosis in medulloblastoma patients (P<0.0001), which was further validated in 63 patient tumors by immunohistochemical staining for SMARCD3. Interestingly, tumors that had metastasized often had elevated expression of SMARCD3, in all subgroups of medulloblastomas (P<0.0001) and G3 only (P<0.01) based on analyzing multiple published databases. An orthotopic mouse model further supported that SMARCD3 is highly expressed in metastatic tumors compared to primary tumors. Importantly, CRISPR-CAS9-mediated SMARCD3 deletion decreased cell migration in medulloblastoma cell lines. Mechanistically, SMARCD3 deletion led to decreased H3K27me3, suggesting that SMARCD3 may cooperate with PRC2 in regulation of gene expression. Together, our results indicate that SMARCD3 plays a significant role in the development of metastatic dissemination in medulloblastoma, especially in the G3 subgroup. Thus, targeting the SMARCD3-containing SWI/SNF Complex may effectively prevent tumor dissemination and improve clinical outcomes in children with medulloblastoma.

MBRS-64. STUDY OF ARGININE METHYL TRANSFERASES IN MEDULLOBLASTOMA

Medulloblastoma is the most common malignant pediatric cancer and a leading cause of childhood cancer-related mortality. There is dire need for new therapies. Molecular sub-classification of medulloblastomas has identified chromatin modifiers as potential drivers of tumorigenesis. Expression of the RE1 Silencing Transcription Factor (REST), a hub for assembly of repressive chromatin remodelers and a known regulator of neurogenesis, is elevated in a subset of human sonic hedgehog (SHH) subgroup medulloblastomas, and is associated with poor prognosis. Using a novel transgenic mouse model, we showed REST to be a driver of medulloblastoma development. Surprisingly, our studies also revealed a role for REST in promoting proliferation of granule cell progenitors (GCPs), the cells of origin of SHH-driven medulloblastoma, and a concomitant loss of telomeres and increased genomic instability. We performed a gain of function screen using a library of chromatin modifiers to understand the mechanism by which proliferative potential was maintained, despite the loss of telomeres. This screen identified the Protein Arginine Methyltransferase 6 (PRMT6) as a high confidence hit. PRMT6 upregulation caused a reduction in CDKN2A, an important regulator of replicative senescence. Evasion of senescence is frequently implicated in tumor progression. Using a chemical screen, we also identified a novel, selective, reversible and competitive inhibitor of PRMT6. The consequence of genetic and pharmacological inhibition of PRMT6 on cell proliferation and senescence will be reported. Thus, our studies are the first to demonstrate a role for arginine methyl transferase family of chromatin modifiers in medulloblastoma genesis.

MBRS-45. TWIST1 AND ABCB1 ARE FUNCTIONAL DETERMINANTS OF METASTASIS IN MEDULLOBLASTOMA

Paediatric medulloblastomas (MB) are frequently metastatic, resulting in a poor prognosis for the patient. Of the four MB subgroups, group 3 patients present with the highest rates of metastasis and worst outcomes. The mechanisms behind the metastatic process are poorly understood, limiting our ability to develop novel therapeutic treatments. We hypothesised that the epithelial-mesenchymal transition (EMT) transcription factor TWIST1 and the multidrug efflux pump ABCB1 (ATP-binding cassette subfamily B member 1) synergistically drive MB metastasis. TWIST1 protein expression was analysed in patient tissue microarrays by immunohistochemistry. High TWIST1 expression was associated with metastatic patients (p=0.041). Physical and functional interactions between TWIST1 and ABCB1 were investigated using chromatin immunoprecipitation (ChIP) and a 3D migration and invasion model. ChIP analysis confirmed TWIST1 binding to the ABCB1 promoter in SHH (ONS-76) and group 3 (D283MED and HD-MB03) metastatic cell lines. TWIST1 and ABCB1 were inhibited in HDMB03 cells with harmine and vardenafil respectively, resulting in attenuated cell migration in the 3D model. Western blot and qRT-PCR analysis of harmine treated cells confirmed a reduction in ABCB1 protein and gene expression. Overall our data reveals TWIST1 and ABCB1 to be key targets for MB metastatic disease. Using bioinformatics analysis and ChIP sequencing, additional TWIST1 downstream targets are now being identified and compared across the metastatic cell lines (ONS-76, D283MED and HD-MB03). This data will provide a deeper insight into the pathways associated with MB metastases, enabling personalised treatment approaches for patients with metastatic disease.

MBRS-65. FBXW7 ACTS A TUMOR SUPPRESSOR IN MYC-DRIVEN MEDULLOBLASTOMA BY CONTROLLING A FEED-FORWARD REGULATORY LOOP OF PLK1 AND MYC

Group 3 medulloblastoma (MB) is often accompanied by MYC amplification and has a higher rate of metastatic disease. So, it is critical to have more effective therapies for high MYC expressing sub-groups. Here we report that FBXW7, a substrate recognition component of the SKP1-CUL1-Fbox (SCF) E3 ligase, interacts with and targets c-MYC for polyubiquitination and proteasomal degradation. FBXW7 shows lower expression level in MYC-driven MB compared with other MB subgroups suggesting activity as a tumor suppressor. Genomic deletion or mutation of Fbxw7 has frequently been identified in many human cancers but not in MB. We demonstrate that overexpression of Fbxw7 in MB cells induces apoptosis and suppresses proliferation in vitro and in vivo. Both phospho-deficient (T205A) and phosphomimetic aspartic acid (T205D) mutants deactivate its tumor suppressor function suggesting a conformational change of its protein structure. Mechanistically, PLK1 kinase specifically phosphorylates FBXW7 and promotes its auto-polyubiquitination and proteasomal degradation, counteracting FBXW7-mediated degradation of oncogene substrates, including c-MYC and PLK1. Chip-Seq results show stabilized c-MYC in turn directly activates PLK1 and FBXW7 transcription, constituting a feedforward regulatory loop. Co-immunoprecipitation demonstrates that FBXW7 directly binds to PLK1 and c-MYC, facilitating their protein degradation by promoting the ubiquitination of both proteins. Furthermore, we show that FBXW7 protein can be stabilized by various kinase inhibitors, proposing a mechanism of kinase-targeted agents to treat MYC-driven MB. These results collectively demonstrate how kinase inhibition stabilizes the tumor suppressor FBXW7 in MYC-driven MB, thus revealing an important function of FBXW7 in suppressing MB progression.

MBRS-61. MOLECULAR SUB-GROUPING OF PEDIATRIC MEDULLOBLASTOMA: CORRELATION WITH CLINICAL AND HISTOLOGICAL FEATURES, A SINGLE INSTITUTIONAL STUDY

INTRODUCTIONMolecular subgroups of pediatric medulloblastomas are distinctive in infantile and non-infantile age-groups.METHODSReal-time quantitative PCR based GEP of customized 12 protein-coding genes was performed on 206 FFPE childhood medulloblastoma samples. FISH for MYC amplification, monosomy 6 and sequencing for CTNNB1 exon 3 mutation were done in relevant cases. H&E and reticulin-stained slides were used for histological subtyping. p53-protein immunoreactivity pattern was noted.RESULTSInfantile (n=33) comprised 57.6% SHH-activated (desmoplastic: 73.7%; MBEN: 15.8% and classic: 10.5%), 21.2% group 3 (large cell/anaplastic [LCA]: 28.6% and none were desmoplastic) and 12% group 4. 40% of group 3 patients died of disease and 21% of the SHH-activated (all desmoplastic) had subsequent local recurrence. Non-infantile (n=173) comprised 19.4% WNT-activated, 12.9% SHH-activated (15% classic, 30% desmoplastic, 10% paucinodular), 19.4% group 3 (63.3% classic & 26.7% LCA), 48.4% group 4 (73.3% classic, 5.3% desmoplastic, 10.7% paucinodular & 1.4% LCA), and non-WNT/non-SHH (NWNS), NOS (n=14,9%) and unclassified (n=4,2.6%). None of WNT-activated were desmoplastic/LCA histology. Non-infantile WNT-activated and group 3 MBs showed 90% monosomy 6 & CTNNB1 mutation, and 16.7% MYC-amplification respectively. 17.4% (13% spinal, 4.4% local) WNT-activated, 31% (12.5% local, 18.5% distant [spinal: 12.5%, intracranial:6%]) SHH-activated, 27% (18% both spinal and local, 9% spinal) group 3 and 31.5% (7.4% local, 5.5% intracranial, 11.2% spinal, 7.4% both spinal and local) group 4 showed metastases during follow up.CONCLUSIONSSHH-activated and group 3 are the common infantile subgroups but group 4 is not non-existent in infantile age. No desmoplastic (including paucinodular) histological subtype is of WNT- activated and group 3.

IMMU-15. PROTEOGENOMIC DISCOVERY OF NOVEL TUMOR PEPTIDES AS NEOANTIGENS FOR PERSONALIZED T CELL IMMUNOTHERAPY IN MEDULLOBLASTOMA

T cell immunotherapies are promising new tools to combat high-risk subgroups of medulloblastoma without increasing the late effects burden. The ideal target antigen of an effective antitumor T-cell response is abundantly expressed by tumor cells but not by normal tissues, in order to limit off-target effects. Tumors translate a host of highly novel transcripts that are the result of aberrations in tumor DNA and the unmasking of alternative or novel exons. We developed a novel proteogenomic approach to identify tumor-restricted peptides and used them to select and expand T cells capable of mounting a tumor-specific cytotoxic immune response. Using RNA-seq and WGS data, we created personalized custom searchable databases containing predicted novel proteins from somatic mutations, novel junctions and fusion transcripts from 56 medulloblastoma tumors. By searching these databases with raw mass spectrometry data from paired medulloblastoma tumors, we identified tens of neoantigen peptides arising from the translation of tumor-specific transcripts; novel isoforms being the predominant source. We tested these peptides for their ability to select and expand autologous polyclonal populations of T cells and tested the immunogenicity of each individual peptide. Flow cytometry revealed populations of CD4+ and CD8+ cells with an activation profile marked by IFN-γ and TNF-α. Immunosuppressive marker profiles were also characterized. Using cytotoxicity assays, we demonstrated that tumor specific T cells can eliminate neoantigen bearing tumor cells. Thus, proteogenomics can identify immunogenic tumor specific peptides that can be used to create a personalized, targeted T cell therapy for children with high risk medulloblastoma.

MBRS-46. CHARTING NEOPLASTIC AND IMMUNE CELL HETEROGENEITY IN HUMAN AND GEM MODELS OF MEDULLOBLASTOMA USING scRNAseq

We explored cellular heterogeneity in medulloblastoma using single-cell RNA sequencing (scRNAseq), immunohistochemistry and deconvolution of bulk transcriptomic data. Over 45,000 cells from 31 patients from all main subgroups of medulloblastoma (2 WNT, 10 SHH, 9 GP3, 11 GP4 and 1 GP3/4) were clustered using Harmony alignment to identify conserved subpopulations. Each subgroup contained subpopulations exhibiting mitotic, undifferentiated and neuronal differentiated transcript profiles, corroborating other recent medulloblastoma scRNAseq studies. The magnitude of our present study builds on the findings of existing studies, providing further characterization of conserved neoplastic subpopulations, including identification of a photoreceptor-differentiated subpopulation that was predominantly, but not exclusively, found in GP3 medulloblastoma. Deconvolution of MAGIC transcriptomic cohort data showed that neoplastic subpopulations are associated with major and minor subgroup subdivisions, for example, photoreceptor subpopulation cells are more abundant in GP3-alpha. In both GP3 and GP4, higher proportions of undifferentiated subpopulations is associated with shorter survival and conversely, differentiated subpopulation is associated with longer survival. This scRNAseq dataset also afforded unique insights into the immune landscape of medulloblastoma, and revealed an M2-polarized myeloid subpopulation that was restricted to SHH medulloblastoma. Additionally, we performed scRNAseq on 16,000 cells from genetically engineered mouse (GEM) models of GP3 and SHH medulloblastoma. These models showed a level of fidelity with corresponding human subgroup-specific neoplastic and immune subpopulations. Collectively, our findings advance our understanding of the neoplastic and immune landscape of the main medulloblastoma subgroups in both humans and GEM models.

LINC-03. MOLECULAR CLASSIFICATION OF PAEDIATRIC MEDULLOBLASTOMA FROM FOUR TERTIARY CENTRES IN MALAYSIA: DIAGNOSTIC DILEMMA WITH CONVENTIONAL METHODS

OBJECTIVETo determine the prognostic significance of the four molecular subgroups of medulloblastoma (MB) among children in Malaysia.METHODSWe assembled MB samples of children < 18 years between January 1999 and July 2017 in University Malaya Medical Centre, Penang General Hospital, Sarawak General Hospital and Sabah Woman and Children’s Hospital. MB was sub-grouped using 850k DNA methylation profiling.RESULTSFifty-one tumour samples were retrieved. Histopathological subtypes were classic (n=12), MB extensive nodularity/desmoplastic (n=9) and 30 MB results without subtypes. Thirteen patients were M1-M4. Fourteen patients were stratified as standard-risk (SR,27.4%), 22 as high-risk (HR,43.2%) and 15 as high-risk children ≤ 3 years old (iHR,29.4%). Molecular subgrouping revealed 16 Group4, 11 SHH, 10 Group3 and 4 Wnt. In 8 patients, DNA methylation profiling identified a diagnosis other than MB and in 2 samples the DNA was inadequate. For patients >3 years old, the 5-year event-free survival (EFS) was 35.7%±13% in HR and 39.7%±20% in SR. The 5-year overall survival (OS) in these two groups was 43.4%±14% and 41.7±30% respectively. iHR had 5-year EFS and OS of 48.0%±16% and 60.0%±16% respectively. WNT tumours had the best 5y-OS of 66.7±22% of the cohort, albeit significantly lower than other reports, followed by SHH (56.8±17%), Group4 (44.3±17.6%) and Group3 (41.7±18%). Treatment abandonment rate was 20%.CONCLUSIONThe discrepancy in the histological diagnoses highlights the importance of DNA methylation profiling technique for accurate diagnosis. We observed poor OS across all the subgroups, in part due to treatment abandonment.

LINC-30. A CLINICOPATHOLOGICAL STUDY OF IMMUNOGENICITY AND IMMUNE EVASION MECHANISMS AMONG MOLECULAR SUBGROUPS OF MEDULLOBLASTOMA

INTRODUCTIONMedulloblastomas have been well characterised in terms of genomics, epigenomics, transcriptomics and recently proteomics. However, there is limited knowledge regarding immunogenicity, immune-microenvironment and immune evasion mechanisms in different molecular subgroups of medulloblastoma. It is important to analyze these parameters to understand tumor progression, prognostic stratification as well as treatment response to available immunotherapeutic drugs.MATERIALS AND METHODSMolecular subgrouping performed by immunohistochemistry(IHC), Nanostring and 850k-methylation array. Immune profile by IHC for CD3, 20, CD8 [tumor infiltrating lymphocytes (TILs)], CD163 [tumor-associated macrophages (TAMs)], and PD-L1 and CTLA-4 [immune checkpoint proteins].RESULTSA total of 35 cases were analyzed with age-range from 1 to 54 years (77% pediatric and 23% adult). 82% cases were located in midline, while rest in cerebellar hemispheres. On molecular subgrouping, MBs were subdivided into 8 WNT, 10 SHH, 8 Group 3 and 9 Group 4. Twenty four cases had follow up, 12 with no evidence of disease while 12 with progressive disease or death. PD-L1 expression ranged from 0% to 20% and included 5SHH, 2WNT and 1Group 3. CTLA4 positive lymphocytes ranged from 0 to 33 in 4 cases: 1WNT, 3 SHH, 1Group4. TILs ranged from 0–220/mm2 with a median of 3. TAMs ranged from 0–60/mm2 with a median of 18. Both TILs and TAMs were significantly higher in SHH subgroup.CONCLUSIONPD-L1 positivity and number of TILs and TAMs were significantly more in SHH-subgroup tumors followed by WNT tumors. CTLA-4 expression did not correlate with subgroups. All parameters showed a positive trend with increasing age.

MBCL-13. CORRELATION OF HISTOPATHOLOGY, CHROMOSOMAL MICROARRAY, AND NANOSTRING BASED 22-GENE ASSAY FOR MEDULLOBLASTOMA SUBGROUP ASSIGNMENT ON “HEAD START” 4 CLINICAL TRIAL

“Head Start” 4 (HS 4) is a prospective randomized clinical trial that tailors treatment based on medulloblastoma molecular subgroups and response to induction chemotherapy to compare efficacy of one versus three (tandem) cycles of myeloablative chemotherapy. Advances in RNA and DNA profiling have identified four molecular subgroups of medulloblastoma with prognostic significance: Sonic Hedgehog (SHH) subtype, WNT subtype, Group 3, and Group 4. In HS 4 trial, we utilize a combination of histopathology and immunohistochemistry (pathology/IHC), as well as chromosomal microarray analysis (CMA) utilizing OncoScanTM (Thermo Fisher) to classify medulloblastoma samples into either SHH, WNT, or non-WNT/non-SHH (Group 3/4) subgroups at the time of diagnosis. NanoString based 22-gene assay is performed retrospectively to test concordance. We have pathology/IHC, CMA, and NanoString data on 26 infants and young children with medulloblastoma enrolled on HS 4. Pathology/IHC was able to assign samples to SHH, WNT, and non-WNT/non-SHH subgroups in all but two cases: one case was classified as Group 3, and the second as SHH by both CMA and NanoString. CMA was indeterminate in six cases, of which, pathology/IHC was able to assign all six samples aforementioned three subgroups. NanoString was indeterminate in two cases: one case was classified as SHH by CMA and pathology/IHC, and the second case was indeterminate by CMA but was assigned as non-WNT/non-SHH on pathology/IHC. There is excellent correlation between NanoString and combination of histopathology and CMA for core medulloblastoma subgrouping on HS 4. Methylation studies are ongoing.

MBCL-26. FACTORS ASSOCIATED WITH LONGER SURVIVAL AFTER FIRST RECURRENCE IN MEDULLOBLASTOMA BY MOLECULAR SUBGROUP AFTER RISK-BASED INITIAL THERAPY

OBJECTIVETo evaluate differences in time to recurrence among molecular subgroups of medulloblastoma treated on a single protocol and to identify factors associated with survival after first recurrence.METHODSTime to recurrence following SJMB03 treatment was compared across methylation subgroups among relapsed patients. Therapies received subsequent to relapse were noted. Kaplan-Meier methods and log-rank tests were used for statistical analyses.RESULTS74 of 330 medulloblastoma patients developed recurrence after initial therapy. (38 Standard-Risk; 36 High-Risk). The 2- and 5-year survival after first recurrence was 30.4% and 14.6% respectively. DNA methylation-based subgroups from initial diagnosis were SHH (n=14), Group 3 (n=24), Group 4 (n=26), and unclassified (n=8). None of the pts with WNT MB had recurrent disease. Median time to first recurrence was 1.23, 0.91, and 3.09 years in SHH, Group3, and Group 4 respectively. Group 4 patients had longer post-recurrence survival than others (p-value=0.0169). Clinical risk at diagnosis (p-value=0.337), anaplasia (p-value=0.4032), TP53 (p-value=0.1969), MYC (p-value=0.8967), and MYCN (p value = 0.9404) abnormalities were not associated with post progression survival. Patients who received any therapeutic modality (chemotherapy, re-radiation and second surgery) had longer survival and those who had all three (n=10) had the best outcome (p-value<0.0001).CONCLUSIONOutcome after recurrence in medulloblastoma is dismal, however, association with subgroups is still present. Group 4 patients had a longer time to recurrence and post progression survival. No other prognostic factor at initial diagnosis was associated with outcome after recurrence. Patients who received all 3 types of conventional therapy had better survival.

MBRS-31. COMBINING IRRADIATION AND ANTI-CD47 TO ENHANCE THE TREATMENT OF GROUP 3 MEDULLOBLASTOMA

Medulloblastoma (MB) is the most common malignant primary pediatric brain tumor. The Group 3 molecular subgroup of Medulloblastoma (Group 3 MB) is the deadliest with only 30% long term survival. Irradiation for Group 3 Medulloblastoma is required for long term survival of children. Methods to enhance the effect of irradiation against Group 3 MB are an active area of investigation. Immunotherapy using the anti-CD47 treatment has shown promise in treating Group 3 MB. We recently demonstrated that irradiation significantly enhanced anti-CD47-mediated phagocytosis of high-grade glioma cells in vitro. Furthermore, mice engrafted with human high-grade glioma that received anti-CD47 combined with irradiation showed a significant increase in the survival rate and a significant decrease in tumor growth than those that received a single treatment. We have now extended these studies to demonstrate the enhancement of anti-CD47-dependent phagocytosis of human Group 3 MB with irradiation. We also analyzed normal human neural stem cells exposed to the same treatments to assess for the potential toxicity that uniquely exists with this treatment combination.

DDEL-15. NANOTHERAPEUTIC TARGETING OF TUMOR ENDOTHELIUM FOR ENHANCING DRUG DELIVERY PAST THE BLOOD-BRAIN BARRIER

OBJECTIVEThe Sonic Hedgehog (SHH) medulloblastoma subgroup accounts for ~25% of all cases and has an intermediate prognosis. Current therapies result in devastating morbidities including intellectual disability and secondary malignancies. Although molecularly targeted agents against the SHH pathway have demonstrated efficacy, on-target bone toxicities suggest new therapeutic approaches are needed.METHODSWe investigated the SHH pathway inhibitor, vismodegib, packaged in a fucoidan-based nanoparticle (Fi-Vis) that targets P-selectin expressed on endothelial cells and induced by low-dose ionizing radiation (XRT) in a time- and dose-dependent manner. This P-selectin targeting nanoparticle shows selectivity toward tumor and not normal brain vasculature in a GEM SHH medulloblastoma model as assessed by ex vivo infrared imaging and molecular studies.RESULTSQuantitative RT-PCR analysis of SHH medulloblastoma following single dose XRT and Fi-Vis treatment (10mg/kg) showed synergistic reduction of Gli1 expression (>90% target inhibition). We demonstrate that low-dose XRT (0.25Gy) can induce P-selectin expression specifically on medulloblastoma tumor endothelium and synergize with low-dose Fi-Vis (10mg/kg) to significantly enhance mouse survival (p<0.01) compared to radiation or Fi-Vis alone. Assessment of bone toxicity using micro-CT and histological analysis following Fi-Vis administration in postnatal (P10) mice shows no bone toxicity when compared to free vismodegib. Finally, in vitro studies using bEnd.3 brain endothelial cells and in vivo studies using Cav1 knockout mice suggest a caveolin-1 mediated transcytosis mechanism for nanoparticle entry across the blood-brain barrier.CONCLUSIONSThese data suggest applicability of combined XRT and tumor vasculature-targeted nanotherapeutic dose de-escalation strategies for SHH medulloblastoma with implications for other pediatric brain tumors.

MBRS-16. MYC REGULATED LONG NONCODING RNA LNC-HLX-2–7 IS A PUTATIVE MOLECULAR MARKER AND A THERAPEUTIC TARGET FOR GROUP 3 MEDULLOBLASTOMAS IN CHILDREN

Medulloblastoma (MB), a central nervous system tumor that predominantly affects children, requires aggressive therapy. Recent advances in the noncoding RNA genome could contribute to the sub-classification of medulloblastoma. The focus of this study is to identify novel long noncoding RNAs (lncRNAs) as molecular markers and potential therapeutic targets within each subgroup of MBs, in particular within Group 3. We analyzed publicly available 175 RNA-seq datasets to identify a group of putative lncRNA signatures that may be able to differentiate medulloblastoma subgroups accurately. Among those, lncRNA lnc-HLX-2–7 was highly upregulated in Group 3 MB cell lines, patient-derived xenografts, FFPE samples compared to other groups. CRISPR/Cas9 deletion of the lnc-HLX-2–7 followed by the fluorescence-activated sorting and generating monoclonal Group 3 MB cells significantly reduced the cell growth and 3-D colony formation together with the induction of apoptosis. Intracranial injection to mouse cerebellum using lnc-HLX-2–7 deleted cells resulted in reduced tumor growth compared to parental cells, and tumors were further characterized by single-cell sequencing. We identified that oncogene MYC regulates lnc-HLX-2–7 and its expression can be controlled by the small molecule JQ1, a BET-bromodomain (BRD4) inhibitor that disrupts interactions with MYC. RNA-FISH analysis using FFPE, PDX, and tissue microarrays revealed that lnc-HLX-2–7 expression is specific to Group 3 MB compared to other groups. We present supporting evidence that lnc-HLX-2–7 is a novel molecular marker and a potential therapeutic target for Group 3 MBs in children.

MODL-03. ADAPTING PALBOCICLIB FOR MEDULLOBLASTOMA THERAPY BY IMPROVING DRUG DELIVERY AND ADDRESSING RESISTANCE

CDK4/6 inhibition may be a promising therapy for medulloblastoma. All medulloblastoma subgroups show D-cyclin/CDK4/RB pathway activity, suggesting broad potential for efficacy. To address drug delivery and systemic toxicity limitations, we developed a nanoparticle formulation of CDK 4/6 inhibitor, palbociclib, in poly (2-oxazoline) micelles (POx-palbo). POx-palbo showed reduced systemic toxicity in transgenic mice engineered to develop medulloblastoma, allowing for higher dosing. Pharmacodynamic studies showed POx-palbo suppressed RB phosphorylation acutely and after 24hrs, the effect diminished. This inhibition produced a longer lasting suppression of SHH pathway activity, demonstrated by Gli-luc reporter tumor mice. Importantly, POx-palbo therapy, administered daily, reduced tumor growth and improved the survival of mice with medulloblastoma. While POx-palbo was clearly effective as a single agent, all mice treated with POx-palbo eventually developed progressive disease, as resistant populations of tumors cells emerged. To understand the mechanisms of resistance, we compared tumors early and late in the course of therapy. We found that after 5 days of treatment, palbociclib altered cell cycle progression to produce an extended period of S-phase and that the fractions of cell expressing the stem cell marker Olig2 were markedly increased. Based on these data, we propose that tumors respond to the initial suppressive effect of palbociclib by increasing the pool of Olig2+ stem cells, that these cells show discernably different cell cycle kinetics and are resistant to CDK4/6 inhibition. Combining POx-palbo with additional therapies that target Olig2+ stem cells, by disrupting their prolonged S-phase, or by disrupting Olig2 function, may lead to newly effective medulloblastoma treatment.

Activated leukocyte cell adhesion molecule expression correlates with the WNT subgroup in medulloblastoma and is involved in regulating tumor cell proliferation and invasion.

Cluster of differentiation (CD) 166 or activated leukocyte cell adhesion molecule (ALCAM) is a transmembrane molecule known to be an intercellular adhesion factor. The expression and function of ALCAM in medulloblastoma (MB), a pediatric brain tumor with highly advanced molecular genetics, remains unclear. Therefore, this study aimed to clarify the significance and functional role of ALCAM expression in MB. ALCAM expression in 45 patients with MB was evaluated by immunohistochemical analysis of formalin-fixed paraffin-embedded clinical specimens and the relationship between ALCAM expression and pathological type/molecular subgroup, such as WNT, SHH, Group 3, and Group 4, was examined. Eight ALCAM positive (18%), seven partially positive (16%), and 30 negative (67%) cases were detected. All seven cases of the WNT molecular subgroup were ALCAM positive and ALCAM expression strongly correlated with this subgroup (P < 0.0001). In addition, functional studies using MB cell lines revealed ALCAM expression affected proliferation and migration as a positive regulator in vitro. However, ALCAM silencing did not affect survival or the formation of leptomeningeal dissemination in an orthotopic mouse model, but did induce a malignant phenotype with increased tumor cell invasion at the dissemination sites (P = 0.0029). In conclusion, our results revealed that ALCAM exhibited highly specific expression in the WNT subgroup of MB. Furthermore, we demonstrated that the cell kinetics of MB cell lines can be altered by the expression of ALCAM.

TAMI-58. A NOVEL ROLE FOR REST IN THE CONTROL OF THE MEDULLOBLASTOMA MICROENVIRONMENT

Abstract The REI Silensing Transcription Factor (REST) is a transcriptional repressor and a canonical regulator of neurogenesis. Its expression is elevated in human sonic hedgehog (SHH) subgroup of medulloblastomas (MBs), where functional studies shown its elevated expression to promote proliferation and block neuronal specification. A role for REST in the control of the MB tumor microenvironment (TME) has not been described previously. Here, we demonstrate that REST also controls the MB-TME, and specifically vascular remodeling. Using our unique RESTTG mouse model, we show that conditional expression of human REST transgene in cerebellar granule neuron progenitors (CGNPs), the cell of origin of a subset of SHH MBs, promoted increased vascular growth. In the context of constitutive activation of SHH signaling, a key driver of SHH-MB development, REST elevation drove tumor progression by altering the tumor vasculature. These findings were validated in mouse orthotopic models of human MB cell lines and through analyses of publicly available transcriptomic database of human MB samples. A strong positive correlation between REST and that of endothelial genes CD31/VEGFR1/ETS1 was seen in samples from patients with SHH-MBs subtypes that are associated with the worst prognosis. Proteomic analyses identified increased secretion of a number of pro-angiogenic factors in the context of upregulated REST expression in MB cells. Unexpectedly, in vitro and in vivo studies showed that MB cells expressed these endothelial markers and co-localized with endothelial cells suggesting that REST elevation may have altered the fate of cells that were destined to become neurons. Finally, ETS1 knockdown in MB cell lines not only downregulated VEGFR1 levels in these cells, and blocked tube formation in vitro, but also caused a reduction in tumor cell co-localization with endothelial cells. Collectively, these data suggest that REST elevation remodels MB vasculature through cell-intrinsic and cell-extrinsic mechanisms.

CSIG-21. BAF60C/SMARCD3 REGULATES TUMOR CELL DISSEMINATION IN MEDULLOBLASTOMA

Abstract Medulloblastoma, the most common malignant brain tumor in children, is a diverse and heterogeneous disease. Molecular characterization of MB revealed four major subgroups, WNT, SHH, Group 3, and Group 4. Although surgical resection, radiotherapy, and chemotherapy are effective at eliminating some forms, patients with the aggressive tumors cannot be cured with conventional therapies, particularly in Group 3. Group 3 is the most aggressive subtype of this disease, accounting for about 25%-30% of cases, and characterized by frequent metastasis at diagnosis and the worst prognosis among all the subgroups. Metastatic dissemination is a major treatment challenge and strongly associated with poor prognosis in patients with medulloblastoma. However, the molecular mechanisms of tumor cell early dissemination and late metastasis remain largely unknown. Here, we found that Group 3 has elevated expression of BAF60C/SMARCD3, a subcomponent of SWI/SNF complex, which significantly mobilizes nucleosomes and remodels chromatin. Clinically, elevated SMARCD3 mRNA resulted in a poorer prognosis in medulloblastoma patients, which was further validated in 63 patient tumors by immunohistochemical staining for SMARCD3. Interestingly, tumors that had metastasized often had elevated expression of SMARCD3, in all subgroups of medulloblastomas and Group 3 only. An orthotopic mouse model further supported that SMARCD3 is highly expressed in metastatic tumors compared to primary tumors. Importantly, CRISPR-Cas9-mediated SMARCD3 deletion decreased cell migration and invasion in multiple medulloblastoma cell lines. Mechanistically, SMARCD3 deletion led to decreased H3K27me3, suggesting that SMARCD3 may cooperate with PRC2 in regulation of gene expression. Strikingly, SMARCD3 deletion downregulated Reelin signaling pathway, an essential role in neuronal positioning during neurodevelopment, which indicates that tumors with highly expressed SMARCD3 hijack a developmental signaling to promote their dissemination and aggressiveness. Together, our results suggest that inhibition of SMARCD3 controlling downstream signaling may effectively prevent tumor dissemination and improve clinical outcomes in children with medulloblastoma.