Browsing by Author "Gallo, Marco"
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Item Open Access Ancestry and frequency of genetic variants in the general population are confounders in the characterization of germline variants linked to cancer(2020-05-06) Bobyn, Anna; Zarrei, Mehdi; Zhu, Yuankun; Hoffman, Mary; Brenner, Darren; Resnick, Adam C; Scherer, Stephen W; Gallo, MarcoAbstract Background Pediatric high-grade gliomas (pHGGs) are incurable malignant brain cancers. Clear somatic genetic drivers are difficult to identify in the majority of cases. We hypothesized that this may be due to the existence of germline variants that influence tumor etiology and/or progression and are filtered out using traditional pipelines for somatic mutation calling. Methods In this study, we analyzed whole-genome sequencing (WGS) datasets of matched germlines and tumor tissues to identify recurrent germline variants in pHGG patients. Results We identified two structural variants that were highly recurrent in a discovery cohort of 8 pHGG patients. One was a ~ 40 kb deletion immediately upstream of the NEGR1 locus and predicted to remove the promoter region of this gene. This copy number variant (CNV) was present in all patients in our discovery cohort (n = 8) and in 86.3% of patients in our validation cohort (n = 73 cases). We also identified a second recurrent deletion 55.7 kb in size affecting the BTNL3 and BTNL8 loci. This BTNL3–8 deletion was observed in 62.5% patients in our discovery cohort, and in 17.8% of the patients in the validation cohort. Our single-cell RNA sequencing (scRNA-seq) data showed that both deletions result in disruption of transcription of the affected genes. However, analysis of genomic information from multiple non-cancer cohorts showed that both the NEGR1 promoter deletion and the BTNL3–8 deletion were CNVs occurring at high frequencies in the general population. Intriguingly, the upstream NEGR1 CNV deletion was homozygous in ~ 40% of individuals in the non-cancer population. This finding was immediately relevant because the affected genes have important physiological functions, and our analyses showed that NEGR1 expression levels have prognostic value for pHGG patient survival. We also found that these deletions occurred at different frequencies among different ethnic groups. Conclusions Our study highlights the need to integrate cancer genomic analyses and genomic data from large control populations. Failure to do so may lead to spurious association of genes with cancer etiology. Importantly, our results showcase the need for careful evaluation of differences in the frequency of genetic variants among different ethnic groups.Item Open Access Characterizing the Genomic Heterogeneity of Pediatric Glioblastoma(2018-09-05) Hoffman, Mary; Gallo, Marco; Chan, Jennifer A. W.; Grewal, Savraj S.; Cobb, Jennifer A.Brain tumors are currently the most common cause of cancer-related deaths among children. With a 5-year survival rate of 20%, pediatric glioblastoma (pGBM) is a lethal brain tumor with no effective treatment options. Although pathologically indistinct from its adult counterpart, recent work has shown that pGBMs diverge at both the genetic and transcriptional level from the adult malignancy. Genomic analyses have identified a recurrent mutation in H3F3A, but this lesion is only present in a fraction of patients and has not contributed to the advancement of effective therapies. Using a longitudinal collection of primary and recurrent pGBMs with matched germlines, I have described a molecularly heterogeneous disease with extreme tumoral evolution. Perhaps my most striking finding is the presence of potentially deleterious structural variants in the patient germlines. Together, these findings suggest a novel hereditary component to tumor etiology which has not been previously described in this malignancy.Item Open Access Construction and Application of an Epigenetic Atlas of the Human Heart(2019-12-19) Prasher, Dimple; Greenway, Steven C.; Gallo, Marco; Long, QuanEpigenetic modifications, including DNA methylation, regulate gene expression and contribute to the specialization of cells. Currently, there is limited knowledge of the epigenetic patterns of the human heart. To address this gap, we generated methylation profiles for all distinct regions of the human heart. We hypothesized that unique differentially methylated regions (DMRs) of DNA exist for each cardiac region and that changes in DNA methylation occur in diverse cardiac diseases such as heart failure (HF) and congenital heart disease (CHD). Using healthy cardiac tissues and reduced representation bisulfite sequencing on the Illumina platform, we generated genome-wide methylomes in triplicate for the human right atrium, left atrium, right ventricle, left ventricle, aorta, pulmonary artery, mitral valve, tricuspid valve, aortic valve and pulmonary valve. We also generated methylomes (in triplicate) from the left ventricle of adult patients with HF and children with CHD. DMRs, defined as regions with significantly different mean methylation differences, were identified using the software package Metilene. For each tissue we generated 10-20 million reads covering 8-10 million CpG methylation sites across the genome and identified 5-57 tissue-specific DMRs. We identified 290 unique DMRs in the HF group and 317 in the CHD group. Potential disease-associated genes identified from this analysis included FGFR2, TOM22, HEX3 for HF and ROCK1 and MSX1 for CHD. In conclusion, we have created a DNA methylation atlas of the human heart. This data provides a resource for future studies and may help to identify new disease biomarkers and increase our understanding of cardiac development.Item Open Access Deconvolution of Genetic Heterogeneity in Glioblastoma(2024-04-02) Gillmor, Aaron; Morrissy, Sorana; Gallo, Marco; Robbins, StephenGlioblastoma, an aggressive brain tumor, exhibits substantial genetic heterogeneity that contributes to functional variations and poor outcomes. To comprehensively understand this heterogeneity, I reconstructed phylogenies using multiple samples per patient. Phylogenies were predominantly branching and revealed that higher genetic diversity was positively associated with survival in glioblastoma and that standard therapy increased diversity. Surprisingly, recently diagnosed patients fared better, potentially linking advances in disease management to evolutionary adaptations. In a novel approach, I assessed the validity of phylogenies using long-range mutation phasing with linked reads, which supported most clones and the first branching point, thereby instilling confidence in findings derived from these phylogenies. Phylogenetic reconstruction incorporated a diverse array of sample types that reflected distinct selective pressures. Longitudinal samples capture changes in clonal composition in response to selective pressure of treatment. In contrast, phylogenies generated from multiregional and/or cell line, or xenograft models identified adaptations to microenvironmental differences or culture conditions. Through subsampling, I showed that two samples were the minimum required to capture most mutations, clones, and the initial branching point. Using the phylogenies, I implicated mutational signatures as drivers of clonal divergence events during the development of treatment resistance, including SBS11, previously associated with temozolomide resistance, and DNA mismatch repair (MMR) deficiency signatures SBS6 and SBS15. MMR signatures operated largely independently of each other during clonal evolution, affected different pathways, and had distinct associations with survival. SBS23, a signature of unknown etiology, was also significantly associated with recurrent disease and SBS11 and SBS6-relevant processes. Finally, to explore the relationship between genetic and functional diversity, I integrated expression from RNA and proteome data with genome-based phylogenies using a multi-omics approach. The resulting biological communities demonstrated normal cell infiltration and tumor cell states that broadly recapitulated glioblastoma subtypes and were associated with phylogenetic branching, glioblastoma subtypes, and survival length. Finally, I observed multiple communities annotated with diverse cell types and states linked to the mesenchymal subtype, suggesting diversity within this subtype. In summary, I developed a novel workflow that encompasses phylogenetic reconstruction of glioblastoma evolutionary trajectories and links cell state phenotypes through multi-omic integration.Item Open Access Deconvolution of spatiotemporal transcriptomic heterogeneity in the glioblastoma ecosystem(2024-09-23) Thoppey Manoharan, Varsha; Morrissy, Sorana; Gallo, Marco; Mahoney, Douglas; Neri, PaolaGlioblastoma (GBM) is the most common and lethal primary brain malignancy in adults, characterized by therapeutic resistance and inevitable relapse. Major clinical challenges are imposed by extensive intra-tumoral heterogeneity, diffuse infiltrative growth of the tumor, and bi-directional interactions with diverse non-malignant cell-types within the brain tumor microenvironment (TME). A better understanding of how the tumor cells organize spatially and interact with the TME to promote growth and invasion may reveal opportunities for improved therapeutic strategies. In this thesis, I explore transcriptional heterogeneity within the glioblastoma ecosystem using spatially profiled, temporal samples of GBM xenograft models. The species-specific distinction of the human tumor and mouse TME, coupled with spatial resolution, overcomes previous limitations in studying the invasive front and delineating co-existing non-malignant components within the tumor. By applying a novel computational framework based on unsupervised deconvolution, I characterize a compendium of 15 tumor cell gene expression programs set within the context of 90 mouse brain and TME cell types, cell activities, and anatomic structures. This approach reveals the spatial organization of tumor programs along an axis corresponding to tumor density and distinct colocalization patterns with spatiotemporally varying TME components. Notably, tumor-associated macrophages and reactive astrocytes colocalized with the tumor early in its growth, while an outward gradient of invasion programs, centered on hypoxia, was observed with tumor progression. Moreover, distinct tumor programs aligned with well-documented routes of GBM invasion including the white-matter tracts, perivasculature and parenchymal routes. Ligand-receptor analyses highlighted neuronal and extra-cellular matrix (ECM) signaling along these routes, and further analyses indicated that these routes could be distinguished by the expression of tumor and TME-derived ECM molecules. Lastly, using a network-graph of predicted protein-protein interactions, I identified sub-modules of genes serving as program network hubs that were highly prognostic in patient datasets. Taken together, spatial profiling of xenografts has revealed a granular repertoire of transcriptional programs and provides a basis for rational targeting of tumor and/or TME niches within the GBM ecosystem, paving the way for improved therapeutic interventions.Item Open Access Detection and genomic analysis of BRAF fusions in Juvenile Pilocytic Astrocytoma through the combination and integration of multi-omic data(2022-12-12) Zwaig, Melissa; Baguette, Audrey; Hu, Bo; Johnston, Michael; Lakkis, Hussein; Nakada, Emily M.; Faury, Damien; Juretic, Nikoleta; Ellezam, Benjamin; Weil, Alexandre G.; Karamchandani, Jason; Majewski, Jacek; Blanchette, Mathieu; Taylor, Michael D.; Gallo, Marco; Kleinman, Claudia L.; Jabado, Nada; Ragoussis, JiannisAbstract Background Juvenile Pilocytic Astrocytomas (JPAs) are one of the most common pediatric brain tumors, and they are driven by aberrant activation of the mitogen-activated protein kinase (MAPK) signaling pathway. RAF-fusions are the most common genetic alterations identified in JPAs, with the prototypical KIAA1549-BRAF fusion leading to loss of BRAF’s auto-inhibitory domain and subsequent constitutive kinase activation. JPAs are highly vascular and show pervasive immune infiltration, which can lead to low tumor cell purity in clinical samples. This can result in gene fusions that are difficult to detect with conventional omics approaches including RNA-Seq. Methods To this effect, we applied RNA-Seq as well as linked-read whole-genome sequencing and in situ Hi-C as new approaches to detect and characterize low-frequency gene fusions at the genomic, transcriptomic and spatial level. Results Integration of these datasets allowed the identification and detailed characterization of two novel BRAF fusion partners, PTPRZ1 and TOP2B, in addition to the canonical fusion with partner KIAA1549. Additionally, our Hi-C datasets enabled investigations of 3D genome architecture in JPAs which showed a high level of correlation in 3D compartment annotations between JPAs compared to other pediatric tumors, and high similarity to normal adult astrocytes. We detected interactions between BRAF and its fusion partners exclusively in tumor samples containing BRAF fusions. Conclusions We demonstrate the power of integrating multi-omic datasets to identify low frequency fusions and characterize the JPA genome at high resolution. We suggest that linked-reads and Hi-C could be used in clinic for the detection and characterization of JPAs.Item Open Access Epigenetic Investigation of PF(A) Ependymoma(2024-09-13) Hasheminasabgorji, Elham; Gallo, Marco; Nikolic, Ana; Bahlis, Nizar; Chan, JenniferPosterior Fossa type A (PFA) ependymoma in infants presents therapeutic challenges with a 60% survival rate and significant treatment-associated neurocognitive sequalae. Our research revealed a distinctive 3D genomic architecture — Type B Ultra-Long Interactions in PFA (TULIPs) - universally present in PFA samples and occurring at recurrent regions of the genome. Here, we investigate the molecular origins of TULIPs in PFA ependymoma. We hypothesized that EZHIP expression may underlie TULIP formation, given its widespread expression in virtually all PFAs. To test this hypothesis, EZHIP was expressed in human neural progenitor cell (hNPC) cultures, and we validated the models using immunofluorescence and western blot assays. Our results demonstrated the nuclear localization of EZHIP and its association with chromatin. Moreover, the expression of EZHIP induced profound alterations in the conformation and localization of heterochromatin in the cells, consistent with the induction of TULIPs. Additionally, functional EdU incorporation assays provided evidence that the expression of EZHIP positively influences cell proliferation. Based on our immunofluorescence results, TULIPs appear as large H3K9me3-rich foci in the nucleus of cells. In various cell types, regions of heterochromatin marked by H3K9me3 are typically associated with HP1 proteins. Considering that HP1 proteins are involved in maintaining the liquid-liquid phase separation (LLPS) state of H3K9me3, we assessed whether TULIPs were in fact condensates. hNPCs expressing EZHIP were treated with 1,6-hexanediol, a compound widely used to disrupt liquid condensates. Our results indicated that 1,6-hexanediol treatment caused disruption of the H3K9me3 configuration, suggesting that TULIPs are in fact in LLPS states. In conclusion, our study provides valuable insights into the molecular origins of TULIPs in PFA ependymoma, elucidating the role of EZHIP expression and its impact on chromatin conformation and cell proliferation. While our data suggest that TULIPs may exist in an LLPS state, further investigation is required to confirm this. These findings enrich our understanding of the molecular landscape of PFA ependymomas and offer potential therapeutic targets.Item Open Access Exploring brain cancer pathogenesis in a novel mouse model of glioblastoma (GBM)(2018-06-08) Bohm, Alexandra Katerina; Cairncross, J. Gregory; Goodarzi, Aaron A.; Gallo, Marco; Cobb, Jennifer A.The molecular architecture of glioblastoma (GBM) has been well documented, but the cause of the disease, especially the primary adult form, remains elusive. Although it has been hypothesized that amplification of platelet-derived growth factor A (PDGFA) and p53 mutations are early alterations, how these orchestrate tumour initiation has yet to be discerned. Considering the poor prognosis and minimal benefit received from current treatment, understanding the early events of tumour initiation may be the only way to find more effective or preventative treatment strategies. It is well known that p53 has multiple functions as a tumour suppressor, but the outcome of its loss and the role this aberration plays in GBM remains unknown. Similarly, as a growth factor, PDGFA is hypothesized to promote rapid proliferation leading to cellular transformation, but this has yet to be demonstrated or modelled in GBM. Using a mouse model of GBM developed in the Cairncross lab, I show that, unlike most growth factors, subventricular zone (SVZ) cells struggle to proliferate in PDGFA. When p53 is present, PDGFA-cultured SVZ cells undergo apoptosis, eventually resulting in a total loss of cell culture viability. When p53 is absent, cells undergo a crisis-like period, and eventually transform. Detailed characterization of the crisis period in PDGFA demonstrates the cells undergo abnormal mitosis, likely due to abnormal centrosome numbers, and sustain gross chromosomal alterations that lead to alterations in pathways similar to those observed in human GBM. These analyses clarify how PDGFA and the absence of p53 promote gliomagenesis: PDGFA supports attenuated proliferation concurrent to inducing chromosomal instability, while the absence of p53 enables cells to evade apoptosis and continue through the crisis period until transformation is achieved.Item Open Access Interrogating the Epigenetic Determinants of State Heterogeneity in Adult Glioblastoma(2023-01-24) Nikolic, Ana; Gallo, Marco; Chan, Jennifer; Weiss, Samuel; Goodarzi, Aaron; Hawkins, CynthiaGlioblastoma is the most common malignant brain tumour in adults, and prognosis remains poor, with most patients dying in a year or two despite aggressive treatment. These tumours have high degrees of intratumoral heterogeneity, with a subpopulation of cells having stem cell properties. The drivers of this stem cell phenotype are complex, and include a large number of epigenetic and genetic factors. In this thesis, I explore glioblastoma heterogeneity in two distinct ways: I use single-cell chromatin accessibility data to explore influences of copy number alterations on the glioblastoma epigenome, and I apply functional experimental techniques and genomics to characterize macroH2A2, an epigenetic regulator in this disease. In the first part of my thesis, I develop a tool for calling copy number alterations in single-cell ATAC-seq data, called CopyscAT, and show that glioblastoma tumours have limited intratumoral genetic heterogeneity. In addition, genetic subclones share similar epigenetic profiles, but with slightly different predispositions to acquiring distinct cellular states. In the second section of the thesis, I focus on the role of macroH2A2, a histone variant involved in development and cancer. I find that expression of macroH2A2 is associated with improved prognosis in glioblastoma patients, especially in patients receiving treatment. Examining existing datasets, I find that macroH2A2 expression is enriched in neural progenitor-like cells and repressed at the leading edge of tumours, and confirm this in primary patient specimens. MacroH2A2 is associated with reduced self-renewal in vitro and in vivo and knockdown of the protein increases self-renewal markers, reduces the proportion of CD44-positive cells, and shortens mouse survival. Genome-wide, macroH2A2 knockdown results in altered accessibility, with loss of early response (FOS/AP-1) motifs, which I confirm in patient-derived single-cell ATAC-seq data. Chromatin immunoprecipitation confirms that direct effects of macroH2A2 are largely repressive in nature. Lastly, a screen of epigenetic drugs identifies a compound, MI-3, capable of increasing macroH2A2 levels, and shows that macroH2A2 repression impairs sensitivity to this compound. This work contributes novel ideas to the regulation of cell states and fates in glioblastoma.Item Open Access Molecular Pathways to Initiation and Progression in Gliomas(2017) Chesnelong, Charles; Weiss, Samuel; Cairncross, J. Gregory; Schuurmans, Carol; Chan, Jennifer; Gallo, MarcoGliomas are the most common primary adult brain tumors. Despite current treatment, including surgery radio- and chemotherapy, these tumors continue to progress and recur with a more aggressive and resistant phenotype. Recurrence is believed to be, in no small part, due to the presence within the tumors of Brain Tumor Initiating Cells (BTICs). These cells have stem-like properties, are resistant to therapeutic effort and demonstrate enhanced tumorigenic potential. The key molecular alterations sustaining growth and therapeutic resistance in gliomas are now well described. However, targeted therapies have so far failed to make significant impact in the clinic, perhaps due to the heterogeneity and rapid evolution characteristic of these tumors. A better understanding of the players driving recurrence and the molecular pathways to initiation and progression in gliomas is hence essential to developing novel therapeutic strategies and improve clinical outcome. Isocitrate dehydrogenase 1 or 2 (IDH1/2) mutation is found in grade II-III astrocytomas, oligodendrogliomas and in secondary glioblastomas (GBMs), but is completely absent from primary GBMs, thus reflecting a different origin and evolution. In the first section of this thesis, we study the IDH1/2 mutation in glioma and its contradictory roles during tumor initiation and progression. More specifically, we show that the IDH1/2 mutation is directly involved in downregulating glycolytic genes such as LDHA. We also report the selection of large chr2 deletions resulting in the loss of either the IDH1 wild-type or mutant allele. Although essential during initiation, we argue that the IDH1/2 mutation may be a limiting factor of tumor progression at later stages of tumorigenesis. Often considered a different disease, primary GBMs are the most aggressive and lethal primary brain tumors. In the second section of this thesis, we focus on these tumors through the study of primary GBM-derived BTICs. We report the existence of distinct precursor states (stem-like and progenitor-like) in BTICs predictive of survival and associated with GBM subtypes. Further, we highlight a STAT3-driven EMT-like process in progenitor-like BTICs, which may drive the progression towards more aggressive and resistant recurrent GBM.