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Background: Medulloblastoma (MB) is the most common solid malignant pediatric brain neoplasm. Group 3 (G3) MB, particularly MYC amplified G3 MB, is the most aggressive subgroup with the highest frequency of children presenting with metastatic disease, and is associated with a poor prognosis. To further our understanding of the role of MSI1 in MYC amplified G3 MB, we performed an unbiased integrative analysis of eCLIP binding sites, with changes observed at the transcriptome, the translatome, and the proteome after shMSI1 inhibition. Methods: Primary human pediatric MBs, SU_MB002 and HD-MB03 were kind gifts from Dr. Yoon-Jae Cho (Harvard, MS) and Dr. Till Milde (Heidelberg) and cultured for in vitro and in vivo experiments. eCLIP, RNA-seq, Polysome-seq, and TMT-MS were completed as previously described. Results:MSI1 is overexpressed in G3 MB. shRNA Msi1 interference resulted in a reduction in tumour burden conferring a survival advantage to mice injected with shMSI1 G3MB cells. Robust ranked multiomic analysis (RRA) identified an unconventional gene set directly perturbed by MSI1 in G3 MB. Conclusions: Our robust unbiased integrative analysis revealed a distinct role for MSI1 in the maintenance of the stem cell state in G3 MB through post-transcriptional modification of multiple pathways including identification of unconventional targets such as HIPK1.
Glioblastoma (GBM) is an aggressive brain tumor that is resistant to conventional radiation and cytotoxic chemotherapies. We hypothesize that brain tumor initiating cells (BTICs), a subpopulation of treatment-resistant cells with stem cell properties cause tumor relapse and a subset of neural stem cell genes regulate BTIC self-renewal, driving GBM recurrence. We adapted the existing treatment protocol for adults with primary GBM for in vivo treatment of immunocompromised mice engrafted with GBMs. Post-chemoradiotherapy, the recovered GFP+GBMs were profiled for self-renewal and expression of critical stem cell genes. Using invitro and invivo gain-of-function/loss-of-function experiments, we investigated the regulatory functions of Bmi1 in primary neural stem & progenitor cells (NSPCs) and GBM tumor formation. Finally, global RNA-Seq profiling was performed to understand the consequences of Bmi1 dysregulation on target gene expression. GBM cells showed an increase in Bmi1 levels post-chemoradiotherapy, suggesting the presence of a treatment-refractory BTICs. GFP+cells extracted from treated xenografts had higher self-renewal and BTIC marker expression. Although treated mice responded to therapy, we observed tumor relapse with increased Bmi1 expression. Knockdown of Bmi1 diminished self-renewal and proliferation of GBM cells and delayed tumorigenesis, highlighting a critical role for Bmi1 in tumor maintenance. Conversely, over-expressing Bmi1 in NSPCs failed to initiate tumor formation in vivo. Using high-throughput sequencing data, we generated a map of signaling pathways dysregulated in GBM that may lead to tumor recurrence. Our data confirms the existence of a rare treatment-refractory BTIC population with enhanced self-renewal capacity that escapes therapy and drives tumor relapse.
Medulloblastoma (MB) is the most common malignant pediatric brain tumour, and is categorized into four molecular subgroups, with Group 3 MB having the worst prognosis due to the highest rate of metastatic dissemination and relapse. In this work, we describe the epigenetic regulator Bmi1 as a novel therapeutic target for treatment of recurrent Group 3 MB. Through comparative profiling of primary and recurrent MB, we show that Bmi1 defines a treatment-refractory cell population that is uniquely targetable by a novel class of small molecule inhibitors. We have optimized an in vivo mouse-adapted therapy model that has the advantage of generating recurrent, human, treatment-refractory MBs. Our preliminary studies showed that although chemoradiotherapy administered to mice engrafted with human MB showed reduction in tumour size, Bmi1 expression was enriched in the post-treatment residual tumour. Furthermore, we found that knockdown of Bmi1 in human recurrent MB cells decreases proliferation and self-renewing capacities of MB cells in vitro as well as both tumour size and extent of spinal leptomeningeal metastases in vivo. Oral administration of a potent Bmi1 inhibitor, PTC 028, resulted in a marked reduction in tumour burden and an increased survival in treatment cohort. Bmi1 inhibitors showed high specificity for MB cells and spared normal human neural stem cells, when treated with doses relevant for MB cells. As Group 3 medulloblastoma is often metastatic and uniformly fatal at recurrence, with no current or planned trials of targeted therapy, an efficacious agent such as Bmi1 inhibitor could be rapidly transitioned to clinical trials.
Despite aggressive multimodal therapy, human glioblastoma (hGBM), a highly malignant grade IV astrocytic tumour, remains incurable and inevitably relapses. Recent data has implicated intratumoral heterogeneity as the driver of therapy resistance and tumour relapse in hGBM. Thus models that capture the evolving hGBM biology in response to chemoradiotherapy will allow for the identification of cellular pathways that govern GBM therapy failure. In this study, we have developed a novel model to profile the clonal evolution of treatment naïve brain tumour initiating cell (BTIC) enriched hGBMs through chemoradiotherapy using: stem cell assays, BTIC marker expression and transcriptome analysis, immunohistochemistry, and cellular DNA barcoding technology. We report that treatment of hGBM BTICs leads to increased self-renewal capacity and higher transcript expression of stem cell genes Bmi1 and Sox2. Based on global transcriptome analysis of the in vitro treated hGBM, we also identify a hyper-aggressive form of glioma. Using our therapy-adapted hGBM-mouse xenograft model, we discover that despite tumour regression and increased mouse survival post-therapy, tumour relapse remains inevitable. The treatment-refractory cells again have increased self-renewal capacity and higher expression of Bmi1 and Sox2. Furthermore, by combining cellular DNA barcoding technology, which barcodes hGBM at single cell resolution, with our novel in vitro and in vivo therapy models, we are able to determine whether a pre-existing or a therapy driven subpopulation(s) seeds hGBM tumour relapse. Profiling the dynamic nature of heterogeneous hGBM subpopulations through disease progression and treatment may lead to the identification of novel therapeutic targets for the treatment of recurrent hGBM.
Medulloblastoma (MB), the most common malignant pediatric brain tumor, is categorized into four molecular subgroups. Given the high rate of metastatic dissemination at diagnosis and recurrence in Group 3 MBs, these patients have the worst clinical outcome with a 5-year survivorship of approximately 50%. By adapting the existing COG (Children’s Oncology Group) Protocol for children with newly diagnosed high-risk MB, for treatment of immuno-deficient mice intracranially engrafted with human MB brain tumour initiating cells we aim to identify and characterize the treatment-refractory cell population in Group 3 MBs. Mice were sacrificed at multiple time points during the course of tumor development and therapy: (i) at engraftment; (ii) post-radiation; (iii) post-radiation and chemotherapy; and (iv) at MB recurrence. MB cell populations recovered separately from brains and spines were comprehensively profiled for gene expression analysis, stem cell and molecular features to generate a global, comparative profile of MB cells through therapy. We report a higher expression of CD133, Sox2 and Bmi1 in addition to increased self-renewal capacity following chemoradiotherapy treatment. The enrichment map constructed from global gene expression analysis showed an increase in pathways regulating self-renewal, DNA repair and chemoresistance post-therapy despite the apparent decrease in tumour size and vascularity. Additionally, from gene expression at MB recurrence, we identified a list of genes that negatively correlate with survival in patients diagnosed with Group 3 MB. A differential genomic profile of the “treatment-responsive” tumors against those that fail therapy may contribute to discovery of novel therapeutic approaches for the most aggressive subgroup of MB.
Brain Metastases (BM) represent a leading cause of cancer mortality. While metastatic lesions contain subclones derived from their primary lesion, their functional characterization has been limited by a paucity of preclinical models accurately recapitulating the stages of metastasis. This work describes the isolation of a unique subset of metastatic stem-like cells from primary human patient samples of BM, termed brain metastasis initiating cells (BMICs). Utilizing these BMICs we have established a novel patient-derived xenograft (PDX) model of BM that recapitulates the entire metastatic cascade, from primary tumor initiation to micro-metastasis and macro-metastasis formation in the brain. We then comprehensively interrogated human BM to identify genetic regulators of BMICs using in vitro and in vivo RNA interference screens, and validated hits using both our novel PDX model as well as primary clinical BM specimens. We identified SPOCK1 and TWIST2 as novel BMIC regulators, where in our model SPOCK1 regulated BMIC self-renewal and tumor initiation, and TWIST2 specifically regulated cell migration from lung to brain. A prospective cohort of primary lung cancer specimens was used to establish that SPOCK1 and TWIST2 were only expressed in patients who ultimately developed BM, thus establishing both clinical and functional utility for these gene products. This work offers the first comprehensive preclinical model of human brain metastasis for further characterization of therapeutic targets, identification of predictive biomarkers, and subsequent prophylactic treatment of patients most likely to develop BM. By blocking this process, metastatic lung cancer would effectively become a localized, more manageable disease.
Brain tumours represent the leading cause of childhood cancer mortality, of which medulloblastoma (MB) is the most frequent malignant pediatric brain tumour. Current molecular Nsubgroups of MB recognize distinct disease entities of which activated Wnt signaling (monosomy 6, exon 3 mutations in CTNNB1, and Wnt gene signature) is associated with a distinct subgroup and the best overall outcome. In contrast, only non-Wnt MBs are characterized by metastatic disease, increased rate of recurrence, and poor overall survivorship. Given the excellent clinical outcome in patients with Wnt-driven MB, we aimed to convert treatment-resistant MB subgroups into an ostensibly benign tumour through selective targeting by small molecule Wnt agonists (Wnt3A), GSK3 inhibitors (CHIR99021), and transgenic lines containing a stabilized beta-catenin mutant. Activated Wnt signaling resulted in decreased in vitro self-renewal and promoted differentiation within primary human MB stem cells. The clinical relevance of these findings were demonstrated with an in vivo survival advantage in mice containing orthotopic injections of cells containing a stabilized beta-catenin mutant representative of constitutively active Wnt signaling. Xenografts generated from Wnt-activated tumours were much smaller in size, maintained a much lower rate of proliferation, and reduction in key MB stem cell self-renewal genes (Bmi1, Sox2, Msi1, FoxG1). Our work establishes activated Wnt signaling as a novel treatment paradigm in childhood MB, while providing evidence for the context-specific tumour suppressive function of the canonical Wnt pathway.
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