Paul A. Northcott - US grants

PROJECT SUMMARY Medulloblastoma (MB) is the most common malignant brain tumor in children. Although aggressive treatments have improved outcomes, many MB patients still die of their disease, and survivors suffer severe long-term side effects from therapy. Thus, more effective and less toxic treatments are desperately needed. Genomics has established that MB is not a single entity, but more accurately a collection of biologically and clinically distinct diseases designated as subgroups: WNT, SHH, Group 3, and Group 4. In contrast to WNT and SHH subgroup MBs, the molecular basis of Group 3 and Group 4, the most common and aggressive forms of MB, remains only partially understood. We recently discovered a series of recurrent structural genomic alterations that relocate normally distal highly active enhancers proximal to the genes encoding GFI1 and GFI1B, resulting in profound GFI1/GFI1B over- expression in affected Group 3 and Group 4 MBs. The remarkable but complex nature of this genetic- epigenetic interplay mitigated by structural alterations leading to misappropriation of enhancer activity and oncogene deregulation, prompted us to designate this phenomenon `enhancer hijacking'. Intensive sequencing efforts have determined that Group 3 and Group 4 MBs exhibit a paucity of recurrent gene- level mutations, yet often harbor extensive structural alterations of unknown significance. In light of these findings, we hypothesize that enhancer hijacking plays a prominent role in the etiology of Group 3 and Group 4 and strategies aimed to systematically identify and mechanistically characterize these events will advance our understanding of these poorly defined subgroups. To test this hypothesis, we propose to: (i) systematically investigate the spectrum and prevalence of enhancer hijacking in MB subgroups; (ii) elucidate the mechanistic basis of prominent enhancer hijacking events contributing to MB, including the role of 3-dimensional genome organization; and (iii) functionally recapitulate MB-associated enhancer hijacking in relevant cellular contexts. The results from these studies will extend beyond poorly understood MB subgroups and aim to yield essential insights into the molecular mechanisms governing oncogene deregulation in cancer and provide a deeper understanding into how noncoding genomic variation contributes to malignancy.

PROJECT SUMMARY Medulloblastoma (MB) is among the most common malignant brain tumors in children. Despite advances in multi-modal patient care and understanding of tumor biology, one-third of affected children still succumb to their disease. Efforts to improve patient outcome have been hindered by the lack of sensitive biomarkers to stratify treatment response and predict relapse. In relapsing patients, potential tumor evolution with treatment implies that capturing genomic profiles at recurrence might be required for identifying actionable therapeutic vulnerabilities. There is thus a dire need for novel, sensitive biomarker-driven assays to (i) complement conventional imaging-based disease evaluation and (ii) inform molecular targets at relapse. Liquid biopsies have recently shown promise for detecting and tracking tumor-specific genomic alterations, including targeted sequencing of tumor-derived cell-free DNA (cfDNA) collected from the cerebrospinal fluid (CSF) of brain tumor patients. However, the utility of cfDNA analysis for children with MB remains understudied. We recently devised and an experimental pipeline for inferring somatic copy number variants (CNVs) in CSF-derived cfDNA based on low-coverage WGS (lcWGS). In pilot studies, we demonstrated feasibility of inferring genome-wide CNVs based on lcWGS generated from sub-nanogram cfDNA inputs. Tumor-associated CNVs were detected in 70% of cfDNA samples obtained at baseline, with higher detectability in MB patients with metastatic disease than in those without. Analysis of serial cfDNA samples from CSF during treatment and follow-up indicated an association between CNV detectability and disease course. Re-emergence of somatic CNVs was observed in patients who progressed, identifiable >3 months before relapse was diagnosed radiographically. In addition, divergent CNVs were observed in selected patient-matched primary and relapse pairs. Based on these findings, we hypothesize that the detection of tumor-specific somatic alterations in CSF-derived cfDNA will correlate with patient outcomes in an expanded cohort of children with MB, and longitudinal profiling of such will enhance understanding of mechanisms underlying tumor evolution and recurrence. To test these hypotheses, we propose to (i) establish the utility of CSF-derived cfDNA profiling for correlation with disease burden and prediction of progression in a derivation cohort of prospectively treated children with MB; (ii) validate the algorithm of cfDNA analysis in an independent MB trial cohort; and (iii) investigate tumor evolution in MB through comparison of somatic alterations in longitudinal cfDNA samples. These studies will be conducted in an unprecedented cohort of serial CSF samples collected from children enrolled on two prospective, multi-institutional MB trials (n=140 patients; n>600 CSF samples). The proposed research is anticipated to establish the use of CSF as a minimally invasive, sensitive, and robust form of routine liquid biopsy for MB patients with the potential of revolutionizing risk stratification, disease monitoring, and intervention for affected children.

PROJECT SUMMARY Medulloblastoma (MB) is among the most common malignant childhood brain tumors. Although aggressive treatments have improved outcomes, too many affected children still die of their disease, and survivors often suffer from severe long-term side effects of therapy. Extensive molecular and biological heterogeneity underlying MB has been described, culminating in the recognition of consensus molecular subgroups ? WNT, SHH, Group 3, and Group 4 ? each of which is associated with divergent genomic landscapes, patient demographics, and clinical outcomes. Although somatically altered genes and biological pathways are well annotated, comprehensive understanding of genetic predisposition to MB has lagged behind. We recently investigated germline loss-of-function (LoF) across all protein-coding genes in a series of >1,000 MB patients. This unbiased approach uncovered highly significant deleterious germline variants in ELP1 that were specific to childhood SHH-MB patients and twice as common as pathogenic variants affecting known MB-associated genes. ELP1 encodes a scaffolding subunit of Elongator, a multi-subunit protein complex (ELP1-6) that chemically modifies wobble U34 uridines in the anticodon loop of tRNAs to enable efficient translational elongation and maintenance of physiological protein folding dynamics. ELP1-associated tumors exhibited frequent co-occurrence of somatic PTCH1 mutations and amplifications of PPM1D and MDM4, suggesting germline ELP1 LoF variants cooperate with constitutive activation of SHH and/or TP53 signaling to promote MB development. ELP1-associated SHH-MBs were characterized by a destabilized Elongator complex, loss of Elongator-dependent tRNA modifications, codon-dependent translational reprogramming, and induction of the unfolded protein response, consistent with loss of protein homeostasis. Based on these findings, we hypothesize that ELP1 is a novel cancer predisposition gene and aim to functionally elucidate the developmental, biochemical, and molecular mechanisms by which pathogenic ELP1 LoF promotes SHH-MB. To test this hypothesis, we propose to (i) evaluate the requirement for Elp1 during cerebellar development; (ii) validate the tumor suppressive role of Elp1 in SHH-MB; and (iii) determine the impact of MB-associated Elp1 LoF on translation and the proteome. These studies will be conducted in a series of novel Elp1+/- transgenic mice, primary cells derived from the developing mouse cerebellum, and genetically faithful SHH-MB patient-derived xenografts. Successful execution of this research program will effectively link germline ELP1 LoF to the biochemical and molecular mechanisms governing SHH-MB pathogenesis. Outcomes of the proposed research will be of broad interest, extending to scientists and clinicians with an interest in cancer predisposition, as well as basic researchers studying the fundamentals of translational regulation and protein homeostasis and their role in human disease.