Medulloblastoma (MB) is the most common pediatric brain malignancy and is divided into four molecularly distinct subgroups: WNT, Sonic Hedgehog (SHHp53mut and SHHp53wt), Group 3, and Group 4. Previous reports suggest that SHH MB features a unique tumor microenvironment compared with other MB groups. To better understand how SHH MB tumor cells interact with and potentially modify their microenvironment, we performed cytokine array analysis of culture media from freshly isolated MB patient tumor cells, spontaneous SHH MB mouse tumor cells and mouse and human MB cell lines. We found that the SHH MB cells produced elevated levels of IGFBP2 compared to non-SHH MBs. We confirmed these results using ELISA, western blotting, and immunofluorescence staining. IGFBP2 is a pleiotropic member of the IGFBP super-family with secreted and intracellular functions that can modulate tumor cell proliferation, metastasis, and drug resistance, but has been understudied in medulloblastoma. We found that IGFBP2 is required for SHH MB cell proliferation, colony formation, and cell migration, through promoting STAT3 activation and upregulation of epithelial to mesenchymal transition markers; indeed, ectopic STAT3 expression fully compensated for IGFBP2 knockdown in wound healing assays. Taken together, our findings reveal novel roles for IGFBP2 in SHH medulloblastoma growth and metastasis, which is associated with very poor prognosis, and they indicate an IGFBP2-STAT3 axis that could represent a novel therapeutic target in medulloblastoma.

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

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

Glioblastoma multiforme (GBM) is the most common and fatal adult brain tumour. As radioresistance is a common occurrence and contributes to poor patient prognosis, exploiting the mechanistic basis of GBM radioresistance to develop new therapeutic approaches remains an unmet clinical need. Chromatin post-translational modifications (PTMs) can augment cancer radioresistance. Several chromatin PTMs are regulated by PI3K/AKT, which is frequently hyperactivated in GBM. Previous research demonstrated that the PI3K effector AKT phosphorylates histone H3 at threonine 45 (pH3T45) in response to DNA damage. To genetically dissect the function of H3T45 phosphorylation in the DNA damage response, we engineered the PI3K pathway-activated U87MG GBM cell line to stably overexpress wildtype H3, or a phospho-null H3T45A mutant. We show that in both the presence and absence of irradiation, cells harbouring the phospho-null H3T45A mutant decreases H3K36me3 on free and nucleosome-incorporated histones, which may suggest a deficiency in DNA repair protein recruitment. By surveying irradiation-induced 53BP1 foci formation, we demonstrate that loss of pH3T45 compromises damage repair through NHEJ by delaying the onset of repair signaling. We also observe a defect in DNA damage resolution, which enhances apoptosis to reduce cell clonogenicity and proliferative capacity. Irradiation does not further reduce clonogenicity and proliferation in the phospho-null H3T45A-expressing cells. Taken together, these data suggest a critical function for PI3K-directed H3T45 phosphorylation in DNA damage repair and that loss of pH3T45 may contribute to GBM radioresistance. We anticipate that further characterization of this phosphorylation event will inform the understanding of GBM radioresistance and may suggest novel therapeutic strategies.

Background Medulloblastoma (MB) is the most common malignant pediatric brain tumor that originates in the cerebellum and brainstem. Frequent somatic mutations and deregulated expression of epigenetic regulators in MB highlight the substantial role of epigenetic alterations. 5-hydroxymethylcytosine (5hmC) is a highly abundant cytosine modification in the developing cerebellum and is regulated by ten-eleven translocation (TET) enzymes. Results We investigate the alterations of 5hmC and TET enzymes in MB and their significance to cerebellar cancer formation. We show total abundance of 5hmC is reduced in MB, but identify significant enrichment of MB-specific 5hmC marks at regulatory regions of genes implicated in stem-like properties and Nanog-binding motifs. While TET1 and TET2 levels are high in MBs, only knockout of Tet1 in the smoothened ( SmoA1) mouse model attenuates uncontrolled proliferation, leading to a favorable prognosis. The pharmacological Tet1 inhibition reduces cell viability and platelet-derived growth factor signaling pathway-associated genes. Conclusions These results together suggest a potential key role of 5hmC and indicate an oncogenic nature for TET1 in MB tumorigenesis, suggesting it as a potential therapeutic target for MBs.

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