Abeysundara N, Rasnitsyn A, Bahcheli A, Van Ommeren R, Juraschka K, Vladoiu M, Livingston B, de Antonellis P, Ly M, Lopez Holgado B, Sirbu O, Bahrampour S, Min HK, Fan J, Nor C, Visvanathan A, Zhang J, Wang H, Qin L, Huang N, Pallotta J, Douglas T, Mak E, Su H, Ng K, Zhang KY, Daniels C, Lucas CHG, Eberhart CG, Liu H, Jiang T, Notta F, Ramaswamy V, Reimand J, Gallo M, Rich JN, Wu X, Huang X, Taylor MD (2025) Metastatic medulloblastoma remodels the local leptomeningeal microenvironment to promote further metastatic colonization and growth. Nature Cell Biology 27: 863-874.
Abstract
Leptomeningeal metastases are the major source of morbidity and mortality for patients with medulloblastoma. The biology of the leptomeningeal metastases and the local tumour microenvironment are poorly characterized. Here we show that metastasis-associated meningeal fibroblasts (MB-MAFs) are transcriptionally distinct and signal extensively to tumour cells and the tumour microenvironment. Metastatic cells secrete platelet-derived growth factor (PDGF) ligands into the local microenvironment to chemotactically recruit meningeal fibroblasts. Meningeal fibroblasts are reprogrammed to become MB-MAFs, expressing distinct transcriptomes and secretomes, including bone morphogenetic proteins. Active bone morphogenetic protein signalling and co-implantation of tumour cells with MB-MAFs enhances the colonization of the leptomeninges by medulloblastoma cells and promotes the growth of established metastases. Furthermore, treatment of patient-derived xenograft mice with a PDGF-receptor-α neutralizing antibody enhances overall survival in vivo. Collectively, our results define a targetable intercellular communication cascade in the metastatic niche to treat leptomeningeal disease.
PMID: 40263572
The Terry Fox research Institute Marathon of Hope Cancer Centres Network (2025) The Terry Fox research Institute Marathon of Hope Cancer Centres Network: A pan-Canadian precision oncology initiative. Cancer Cell 43: 587-592.
Abstract
The Marathon of Hope Cancer Centres Network (MOHCCN), led by the Terry Fox Research Institute and the Terry Fox Foundation, unites researchers, clinicians, patients, funders, and other partners across Canada to accelerate precision oncology, promote collaboration and data sharing, and ultimately improve patient outcomes. This overview outlines the Network's goals, history, and challenges and opportunities. We also highlight progress toward the "MOHCCN Gold Cohort," a shared resource of clinical and genomic data from 15,000 patients.
PMID: 40185094.
Lee JYJ, Tao R, You Z, Haldipur P, Erickson AW, Farooq H, Hendriske LD, Abeysundara N, Richman CM, Wang EY, Gupta ND, Hadley J, Batts M, Mount CW, Wu X, Rasnitsyn A, Bailey S, Cavalli FMG, Morrissy S, Garzia L, Michealraj KA, Visvanathan A, Fong V, Palotta J, Suarez R, Livingston BG, Liu M, Luu B, Daniels C, Loukides J, Bendel1 A, French PJ, Kros JM, Korshunov A, Kool M, Ponce de León FC, Perezpeña-Diazconti M, Lach B, Singh SK, Leary S, Cho B-K, Kim S-K, Wang K-C, Lee J-Y, Tominaga T, Weiss WA, Phillips JJ, Dai S, Zadeh G, Saad AG, László B, Klekner A, Pollack IF, Hamilton RL, Ra Y, Grajkowska WA, Perek-Polnik M, Thompson RC, Kenney AM, Cooper MK, Mack SC, Jabado N, Lupien M, Gallo M, Ramaswamy V, Suva ML, Suzuki H, Millen K, L. Huang F, Northcott PA, Taylor MD (2024) ZIC1 is a context dependent medulloblastoma driver in the rhombic lip. Nature Genetics 57: 88-102.
Abstract
Transcription factors are frequent cancer driver genes, exhibiting noted specificity based on the precise cell of origin. We demonstrate that ZIC1 exhibits loss-of-function (LOF) somatic events in group 4 (G4) medulloblastoma through recurrent point mutations, subchromosomal deletions and mono-allelic epigenetic repression (60% of G4 medulloblastoma). In contrast, highly similar SHH medulloblastoma exhibits distinct and diametrically opposed gain-of-function mutations and copy number gains (20% of SHH medulloblastoma). Overexpression of ZIC1 suppresses the growth of group 3 medulloblastoma models, whereas it promotes the proliferation of SHH medulloblastoma precursor cells. SHH medulloblastoma ZIC1 mutants show increased activity versus wild-type ZIC1, whereas G4 medulloblastoma ZIC1 mutants exhibit LOF phenotypes. Distinct ZIC1 mutations affect cells of the rhombic lip in diametrically opposed ways, suggesting that ZIC1 is a critical developmental transcriptional regulator in both the normal and transformed rhombic lip and identifying ZIC1 as an exquisitely context-dependent driver gene in medulloblastoma.
PMID: 39753768
Lee JYJ*, Johnston MJ*, Farooq H, Chen H-M, Younes ST, Suarez R, Zwaig M, Juretic N, Weiss WA, Ragoussis J, Jabado N, Taylor MD§, Gallo M§ (2024) 3D genome topology distinguishes molecular subgroups of medulloblastoma. American Journal of Human Genetics 111: 2720-2734. PMID: 39481374. *Co-first authors; §Co-corresponding authors.
Abstract
Four main medulloblastoma (MB) molecular subtypes have been identified based on transcriptional, DNA methylation, and genetic profiles. However, it is currently not known whether 3D genome architecture differs between MB subtypes. To address this question, we performed in situ Hi-C to reconstruct the 3D genome architecture of MB subtypes. In total, we generated Hi-C and matching transcriptome data for 28 surgical specimens and Hi-C data for one patient-derived xenograft. The average resolution of the Hi-C maps was 6,833 bp. Using these data, we found that insulation scores of topologically associating domains (TADs) were effective at distinguishing MB molecular subgroups. TAD insulation score differences between subtypes were globally not associated with differential gene expression, although we identified few exceptions near genes expressed in the lineages of origin of specific MB subtypes. Our study therefore supports the notion that TAD insulation scores can distinguish MB subtypes independently of their transcriptional differences.
Shahzad U, Nikolopoulos M, Li C, Johnston M, Wang JJ, Sabha N, Varn FS, Riemenschneider A, Krumholtz S, Krishnamurthy PM, Smith CA, Karamchandani J, Watts JK, Verhaak RGW, Gallo M, Rutka JT, Das S (2024) CASCADES, a novel SOX2 super-enhancer-associated long noncoding RNA, regulates cancer stem cell specification and differentiation in glioblastoma. Molecular Oncology 19: 764-784.
Abstract
Glioblastoma is the most common primary malignant brain tumor in adults, with a median survival of just over 1 year. The failure of available treatments to achieve remission in patients with glioblastoma (GBM) has been attributed to the presence of cancer stem cells (CSCs), which are thought to play a central role in tumor development and progression and serve as a treatment-resistant cell repository capable of driving tumor recurrence. In fact, the property of "stemness" itself may be responsible for treatment resistance. In this study, we identify a novel long noncoding RNA (lncRNA), cancer stem cell-associated distal enhancer of SOX2 (CASCADES), that functions as an epigenetic regulator in glioma CSCs (GSCs). CASCADES is expressed in isocitrate dehydrogenase (IDH)-wild-type GBM and is significantly enriched in GSCs. Knockdown of CASCADES in GSCs results in differentiation towards a neuronal lineage in a cell- and cancer-specific manner. Bioinformatics analysis reveals that CASCADES functions as a super-enhancer-associated lncRNA epigenetic regulator of SOX2. Our findings identify CASCADES as a critical regulator of stemness in GSCs that represents a novel epigenetic and therapeutic target for disrupting the CSC compartment in glioblastoma.
PMID: 39323013
1) Johnston MJ*, Lee JJY*, Hu B*, Nikolic A*, Hasheminasabgorji E, Baguette A, Paik S, Chen H, Kumar S, Chen CCL, Jessa S, Balin P, Fong V, Zwaig M, MichealRaj A, Chen X, Zhang Y, Varadharajan S, Billon B, Juretic N, Daniels C, Nageswara Rao A, Giannini C, Thompson EM, Garami M, Hauser P, Pocza T, Shin Ra Y, Cho B-K, Kim S-K, Wang K-C, Lee JY, Grajkowska W, Perek-Polnik M, Agnihotri S, Mack S, Ellezam B, Weil A, Rich J, Bourque G, Chan JA, Yong VW, Lupien M, Ragoussis J, Kleinman C, Majewski J, Blanchette M, Jabado N§, Taylor MD§, Gallo M§ (2024) TULIPs decorate the three-dimensional genome of PFA ependymoma. Cell 187: 4926-4945. PMID: 38986619. *Co-first authors; §Corresponding authors
Abstract
Posterior fossa group A (PFA) ependymoma is a lethal brain cancer diagnosed in infants and young children. The lack of driver events in the PFA linear genome led us to search its 3D genome for characteristic features. Here, we reconstructed 3D genomes from diverse childhood tumor types and uncovered a global topology in PFA that is highly reminiscent of stem and progenitor cells in a variety of human tissues. A remarkable feature exclusively present in PFA are type B ultra long-range interactions in PFAs (TULIPs), regions separated by great distances along the linear genome that interact with each other in the 3D nuclear space with surprising strength. TULIPs occur in all PFA samples and recur at predictable genomic coordinates, and their formation is induced by expression of EZHIP. The universality of TULIPs across PFA samples suggests a conservation of molecular principles that could be exploited therapeutically.
PMID: 38986619
Sloan AR*, Silver DJ*, Kint S*, Gallo M‡, Lathia JD‡ (2024) Cancer stem cell hypothesis 2.0 in glioblastoma: Where are we now and where are we going? Neuro-Oncology. PMID: 38394444.
*Co-first authors. ‡Co-senior authors.
Abstract
Over the past 2 decades, the cancer stem cell (CSC) hypothesis has provided insight into many malignant tumors, including glioblastoma (GBM). Cancer stem cells have been identified in patient-derived tumors and in some mouse models, allowing for a deeper understanding of cellular and molecular mechanisms underlying GBM growth and therapeutic resistance. The CSC hypothesis has been the cornerstone of cellular heterogeneity, providing a conceptual and technical framework to explain this longstanding phenotype in GBM. This hypothesis has evolved to fit recent insights into how cellular plasticity drives tumor growth to suggest that CSCs do not represent a distinct population but rather a cellular state with substantial plasticity that can be achieved by non-CSCs under specific conditions. This has further been reinforced by advances in genomics, including single-cell approaches, that have used the CSC hypothesis to identify multiple putative CSC states with unique properties, including specific developmental and metabolic programs. In this review, we provide a historical perspective on the CSC hypothesis and its recent evolution, with a focus on key functional phenotypes, and provide an update on the definition for its use in future genomic studies.
PMID: 38394444
Mathur R, Wang Q, Schupp PG, Nikolic A, Hilz S, Hong C, Grishanina NR, Kwok D, Stevers NO, Jin Q, Youngblood MW, Stasiak LA, Hou Y, Wang J, Yamaguchi TN, Lafontaine M, Shai A, Smirnov IV, Solomon DA, Chang SM, Hervey-Jumper SL, Berger MS, Lupo JM, Okada H, Phillips JJ, Boutros PC, Gallo M, Oldman MC, Yue F, Costello JF (2024) Glioblastoma evolution and heterogeneity from a 3D whole-tumor perspective. Cell 187: 446-463. PMID: 38242087.
Abstract
Treatment failure for the lethal brain tumor glioblastoma (GBM) is attributed to intratumoral heterogeneity and tumor evolution. We utilized 3D neuronavigation during surgical resection to acquire samples representing the whole tumor mapped by 3D spatial coordinates. Integrative tissue and single-cell analysis revealed sources of genomic, epigenomic, and microenvironmental intratumoral heterogeneity and their spatial patterning. By distinguishing tumor-wide molecular features from those with regional specificity, we inferred GBM evolutionary trajectories from neurodevelopmental lineage origins and initiating events such as chromothripsis to emergence of genetic subclones and spatially restricted activation of differential tumor and microenvironmental programs in the core, periphery, and contrast-enhancing regions. Our work depicts GBM evolution and heterogeneity from a 3D whole-tumor perspective, highlights potential therapeutic targets that might circumvent heterogeneity-related failures, and establishes an interactive platform enabling 360° visualization and analysis of 3D spatial patterns for user-selected genes, programs, and other features across whole GBM tumors.
PMID: 38242087
Zwaig M, Johnston MJ, Lee JJ, Farook H, Gallo M, Jabado N, Taylor MD, Ragoussis J (2023) Linked-read based analysis of the medulloblastoma genome. Frontiers in Oncology 13.
Abstract
Introduction: Medulloblastoma is the most common type of malignant pediatric brain tumor with group 4 medulloblastomas (G4 MBs) accounting for 40% of cases. However, the molecular mechanisms that underlie this subgroup are still poorly understood. Point mutations are detected in a large number of genes at low incidence per gene while the detection of complex structural variants in recurrently affected genes typically requires the application of long-read technologies.
Methods: Here, we applied linked-read sequencing, which combines the long-range genome information of long-read sequencing with the high base pair accuracy of short read sequencing and very low sample input requirements.
Results: We demonstrate the detection of complex structural variants and point mutations in these tumors, and, for the first time, the detection of extrachromosomal DNA (ecDNA) with linked-reads. We provide further evidence for the high heterogeneity of somatic mutations in G4 MBs and add new complex events associated with it.
Discussion: We detected several enhancer-hijacking events, an ecDNA containing the MYCNgene, and rare structural rearrangements, such a chromothripsis in a G4 medulloblastoma, chromoplexy involving 8 different chromosomes, a TERT gene rearrangement, and a PRDM6duplication.
PMID: 37576901
Nikolic A, Maule F, Bobyn A, Ellestad K, Paik S, Marhon S, Mehdipour P, Lun X, Huey-Miin Chen, Mallard C, Hay AJ, Johnston M, Gafuik CJ, Zemp FJ, Shen Y, Ninkovic N, Osz K, Labit E, Berger ND, Brownsey DK, Kelly JJ, Biernaskie J, Dirks PB, Derksen DJ, Jones SJM, Senger DL, Chan JA, Mahoney DJ, De Carvalho D, Gallo M (2023) macroH2A2 antagonizes epigenetic programs of stemness in glioblastoma. Nature Communications 14: 3062.
Abstract
Self-renewal is a crucial property of glioblastoma cells that is enabled by the choreographed functions of chromatin regulators and transcription factors. Identifying targetable epigenetic mechanisms of self-renewal could therefore represent an important step toward developing effective treatments for this universally lethal cancer. Here we uncover an epigenetic axis of self-renewal mediated by the histone variant macroH2A2. With omics and functional assays deploying patient-derived in vitro and in vivo models, we show that macroH2A2 shapes chromatin accessibility at enhancer elements to antagonize transcriptional programs of self-renewal. macroH2A2 also sensitizes cells to small molecule-mediated cell death via activation of a viral mimicry response. Consistent with these results, our analyses of clinical cohorts indicate that high transcriptional levels of this histone variant are associated with better prognosis of high-grade glioma patients. Our results reveal a targetable epigenetic mechanism of self-renewal controlled by macroH2A2 and suggest additional treatment approaches for glioblastoma patients.
PMID: 37244935
Mirzaei R, D’Mello C, Liu M, Nikolic A, Kumar M, Visser F, Bose P, Gallo M, Yong WV (2023) Single-cell spatial analysis identifies regulators of brain tumor initiating cells. Cancer Research 83: 1725-1741.
Abstract
Glioblastomas (GBM) are aggressive brain tumors with extensive intratumoral heterogeneity that contributes to treatment resistance. Spatial characterization of GBMs could provide insights into the role of the brain tumor microenvironment in regulating intratumoral heterogeneity. Here, we performed spatial transcriptomic and single-cell analyses of the mouse and human GBM microenvironment to dissect the impact of distinct anatomical regions of brains on GBM. In a syngeneic GBM mouse model, spatial transcriptomics revealed that numerous extracellular matrix (ECM) molecules, including biglycan, were elevated in areas infiltrated with brain tumor-initiating cells (BTIC). Single-cell RNA sequencing and single-cell assay for transposase-accessible chromatin using sequencing showed that ECM molecules were differentially expressed by GBM cells based on their differentiation and cellular programming phenotypes. Exogeneous biglycan or overexpression of biglycan resulted in a higher proliferation rate of BTICs, which was associated mechanistically with low-density lipoprotein receptor-related protein 6 (LRP6) binding and activation of the Wnt/β-catenin pathway. Biglycan-overexpressing BTICs developed into larger tumors and displayed mesenchymal phenotypes when implanted intracranially in mice. This study points to the spatial heterogeneity of ECM molecules in GBM and suggests that the biglycan-LRP6 axis could be a therapeutic target to curb tumor growth.
PMID: 37067922
Chen H-M, Nikolic A, Singhal D, Gallo M (2022) Roles of chromatin remodeling and molecular heterogeneity in therapy resistance in glioblastoma. Cancers 14: 4942.
Abstract
Cancer stem cells (CSCs) represent a therapy-resistant reservoir in glioblastoma (GBM). It is now becoming clear that epigenetic and chromatin remodelling programs link the stemlike behaviour of CSCs to their treatment resistance. New evidence indicates that the epigenome of GBM cells is shaped by intrinsic and extrinsic factors, including their genetic makeup, their interactions and communication with other neoplastic and non-neoplastic cells, including immune cells, and their metabolic niche. In this review, we explore how all these factors contribute to epigenomic heterogeneity in a tumour and the selection of therapy-resistant cells. Lastly, we discuss current and emerging experimental platforms aimed at precisely understanding the epigenetic mechanisms of therapy resistance that ultimately lead to tumour relapse. Given the growing arsenal of drugs that target epigenetic enzymes, our review addresses promising preclinical and clinical applications of epidrugs to treat GBM, and possible mechanisms of resistance that need to be overcome.
PMID: 36230865
Ding K, Barretto EC, Johnston MJ, Lee B, Gallo M, Grewal S (2022) Transcriptome analysis of FOXO-dependent hypoxia gene expression identifies Hipk as a regulator of low oxygen tolerance in Drosophila. G3 Genes|Genomes|Genetics 12: jkac263.
Abstract
When exposed to low oxygen or hypoxia, animals must alter their metabolism and physiology to ensure proper cell-, tissue- and whole-body level adaptations to their hypoxic environment. These alterations often involve changes in gene expression. While extensive work has emphasized the importance of the HIF-1 alpha transcription factor on controlling hypoxia gene expression, less is known about other transcriptional mechanisms. We previously identified the transcription factor FOXO as a regulator of hypoxia tolerance in Drosophila larvae and adults. Here we use an RNA-sequencing approach to identify FOXO-dependent changes in gene expression that are associated with these tolerance effects. We found that hypoxia altered the expression of over 2000 genes and that approximately 40% of these gene expression changes required FOXO. We discovered that hypoxia exposure led to a FOXO-dependent increase in genes involved in cell signaling, such as kinases, GTPase regulators, and regulators of the Hippo/Yorkie pathway. Among these, we identified homeodomain-interacting protein kinase (Hipk) as being required for hypoxia survival. We also found that hypoxia suppresses the expression of genes involved in ribosome synthesis and egg production, and we showed that hypoxia suppresses tRNA synthesis and mRNA translation and reduces female fecundity. Among the downregulated genes, we discovered that FOXO was required for suppression of many ribosomal protein genes and genes involved in oxidative phosphorylation, pointing to a role for FOXO in limiting energetically costly processes such as protein synthesis and mitochondrial activity upon hypoxic stress. This work uncovers a widespread role for FOXO in mediating hypoxia changes in gene expression.
PMID: 36200850
Mallard C, Johnston M, Bobyn A, Nikolic A, Argiropoulos B, Chan JA, Guilcher GMT, Gallo M (2022) Hi-C detects genomic structural variants in peripheral blood of pediatric leukemia patients. Cold Spring Harbor Molecular Case Studies 8: a006157
Abstract
B-cell acute lymphoblastic leukemia (B-ALL) is often driven by chromosome translocations that result in recurrent and well-studied gene fusions. Currently, fluorescent in-situ hybridization probes are employed to detect candidate translocations in bone marrow samples from B-ALL patients. Recently Hi-C, a sequencing-based technique originally designed to reconstruct the three-dimensional architecture of the nuclear genome, was shown to effectively recognize structural variants. Here, we demonstrate that Hi-C can be used as a genome-wide assay to detect translocations and other structural variants of potential clinical interest. Structural variants were identified in both bone marrow and peripheral blood samples, including an ETV6-RUNX1 translocation present in one pediatric B-ALL patient. Our report provides proof-of-principle that Hi-C could be an effective strategy to globally detect driver structural variants in B-ALL peripheral blood specimens, reducing the need for invasive bone marrow biopsies and candidate-based clinical tests.
PMID: 34819303
Nikolic A, Singhal D, Ellestad K, Johnston MJ, Shen Y, Gillmor A, Morrissy S, Cairncross JG, Jones S, Lupien M, Chan JA, Neri P, Bahlis N, Gallo M (2021) Copy-scAT: Deconvoluting single-cell chromatin accessibility of genetic subclones in cancer. Science Advances 7: abg6045.
Abstract
Single-cell epigenomic assays have tremendous potential to illuminate mechanisms of transcriptional control in functionally diverse cancer cell populations. However, application of these techniques to clinical tumor specimens has been hampered by the current inability to distinguish malignant from nonmalignant cells, which potently confounds data analysis and interpretation. Here, we describe Copy-scAT, an R package that uses single-cell epigenomic data to infer copy number variants (CNVs) that define cancer cells. Copy-scAT enables studies of subclonal chromatin dynamics in complex tumors like glioblastoma. By deploying Copy-scAT, we uncovered potent influences of genetics on chromatin accessibility profiles in individual subclones. Consequently, some genetic subclones were predisposed to acquire stem-like or more differentiated molecular phenotypes, reminiscent of developmental paradigms. Copy-scAT is ideal for studies of the relationships between genetics and epigenetics in malignancies with high levels of intratumoral heterogeneity and to investigate how cancer cells interface with their microenvironment.
PMID: 34644115
Inoue Y, Nikolic A, Farnsworth D, Shi R, Johnson FD, Liu A, Ladanyi M, Somwar R, Gallo M, Lockwood WW (2021) Extracellular signal-regulated kinase mediates chromatin rewiring and lineage transformation in lung cancer. Elife 10: e66524.
Abstract
Lineage transformation between lung cancer subtypes is a poorly understood phenomenon associated with resistance to treatment and poor patient outcomes. Here, we aimed to model this transition to define underlying biological mechanisms and identify potential avenues for therapeutic intervention. Small cell lung cancer (SCLC) is neuroendocrine in identity and, in contrast to non-SCLC (NSCLC), rarely contains mutations that drive the MAPK pathway. Likewise, NSCLCs that transform to SCLC concomitantly with development of therapy resistance downregulate MAPK signaling, suggesting an inverse relationship between pathway activation and lineage state. To test this, we activated MAPK in SCLC through conditional expression of mutant KRAS or EGFR, which revealed suppression of the neuroendocrine differentiation program via ERK. We found that ERK induces the expression of ETS factors that mediate transformation into a NSCLC-like state. ATAC-seq demonstrated ERK-driven changes in chromatin accessibility at putative regulatory regions and global chromatin rewiring at neuroendocrine and ETS transcriptional targets. Further, ERK-mediated induction of ETS factors as well as suppression of neuroendocrine differentiation were dependent on histone acetyltransferase activities of CBP/p300. Overall, we describe how the ERK-CBP/p300-ETS axis promotes a lineage shift between neuroendocrine and non-neuroendocrine lung cancer phenotypes and provide rationale for the disruption of this program during transformation-driven resistance to targeted therapy.
PMID: 34121659
Paik S, Maule F, Gallo M (2021) ¬¬Dysregulation of chromatin organization in pediatric and adult brain tumors: Oncoepigenomic contributions to tumorigenesis and cancer stem cell properties. Genome 64: 326-336
Abstract
The three-dimensional (3D) organization of the genome is a crucial enabler of cell fate, identity, and function. In this review, we will focus on the emerging role of altered 3D genome organization in the etiology of disease, with a special emphasis on brain cancers. We discuss how different genetic alterations can converge to disrupt the epigenome in childhood and adult brain tumours, by causing aberrant DNA methylation and by affecting the amounts and genomic distribution of histone post-translational modifications. We also highlight examples that illustrate how epigenomic alterations have the potential to affect 3D genome architecture in brain tumours. Finally, we will propose the concept of "epigenomic erosion" to explain the transition from stem-like cells to differentiated cells in hierarchically organized brain cancers.
PMID: 33075237
Guilhamon P, Chesnelong C, Kushida MM, Nikolic A, Singhal D, MacLeod G, Madani Tonekaboni SA, Cavalli FMG, Arlidge C, Rajakulendran N, Rastegar N, Hao X, Hassam R, Smith LJ, Whetstone H, Coutinho FJ, Nadorp B, Ellestad K, Luchman HA, Chan JA, Shoichet MS, Taylor MD, Haibe-Kains B, Weiss S, Angers S, Gallo M, Dirks PB, Lupien M (2021) Single-cell chromatin accessibility profiling of glioblastoma identifies an invasive cancer stem cell population associated with lower survival. eLife 10: e64090.
Abstract
Chromatin accessibility discriminates stem from mature cell populations, enabling the identification of primitive stem-like cells in primary tumours, such as glioblastoma (GBM) where self-renewing cells driving cancer progression and recurrence are prime targets for therapeutic intervention. We show, using single-cell chromatin accessibility, that primary human GBMs harbour a heterogeneous self-renewing population whose diversity is captured in patient-derived glioblastoma stem cells (GSCs). In-depth characterization of chromatin accessibility in GSCs identifies three GSC states: Reactive, Constructive, and Invasive, each governed by uniquely essential transcription factors and present within GBMs in varying proportions. Orthotopic xenografts reveal that GSC states associate with survival, and identify an invasive GSC signature predictive of low patient survival, in line with the higher invasive properties of Invasive state GSCs compared to Reactive and Constructive GSCs as shown by in vitro and in vivo assays. Our chromatin-driven characterization of GSC states improves prognostic precision and identifies dependencies to guide combination therapies.
PMID: 33427645
Copy-scAT: Deconvoluting Single-Cell Chromatin Accessibility of Genetic Subclones in Cancer
Published: September 21, 2020 in bioRxiv
Abstract
Single-cell epigenomic assays have tremendous potential to illuminate mechanisms of transcriptional control in functionally diverse cancer cell populations. However, application of these techniques to clinical tumour specimens has been hampered by the current inability to distinguish malignant from non-malignant cells, which potently confounds data analysis and interpretation. Here we describe Copy-scAT, an R package that uses single-cell epigenomic data to infer copy number variants (CNVs) that define cancer cells. Copy-scAT enables studies of subclonal chromatin dynamics in complex tumours like glioblastoma. By deploying Copy-scAT, we uncovered potent influences of genetics on chromatin accessibility profiles in individual subclones. Consequently, some genetic subclones were predisposed to acquire stem-like or more differentiated molecular phenotypes, reminiscent of developmental paradigms. Copy-scAT is ideal for studies of the relationships between genetics and epigenetics in malignancies with high levels of intratumoral heterogeneity and to investigate how cancer cells interface with their microenvironment.
bioRχiv doi: https://doi.org/10.1101/2020.09.21.305516.
Footnotes: https://github.com/spcdot/CopyscAT
Microbiome-Derived Inosine Modulates Response to Checkpoint Inhibitor Immunotherapy
Published: September 18, 2020
Abstract
Several species of intestinal bacteria have been associated with enhanced efficacy of checkpoint blockade immunotherapy, but the underlying mechanisms by which the microbiome enhances antitumor immunity are unclear. In this study, we isolated three bacterial species-Bifidobacterium pseudolongum, Lactobacillus johnsonii, and Olsenella species-that significantly enhanced efficacy of immune checkpoint inhibitors in four mouse models of cancer. We found that intestinal B. pseudolongum modulated enhanced immunotherapy response through production of the metabolite inosine. Decreased gut barrier function induced by immunotherapy increased systemic translocation of inosine and activated antitumor T cells. The effect of inosine was dependent on T cell expression of the adenosine A2A receptor and required costimulation. Collectively, our study identifies a previously unknown microbial metabolite immune pathway activated by immunotherapy that may be exploited to develop microbial-based adjuvant therapies.
PMID: 32792462
Bobyn A§, Zarrei M§, Zhu Y, Hoffman M, Brenner D, Resnick AC, Scherer SW*, Gallo M* (2020) Ancestry and frequency of genetic variants in the general population are confounders in the characterization of germline variants linked to cancer. BMC Medical Genetics 21: 92. PMID: 32375678. §These authors contributed equally to this study. *Corresponding authors.
Abstract
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 tumour 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 tumour 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% of 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.
PMID: 32375678
High-Resolution Structural Genomics Reveals New Therapeutic Vulnerabilities in Glioblastoma
Published: August 29, 2019
Abstract
We investigated the role of 3D genome architecture in instructing functional properties of glioblastoma stem cells (GSCs) by generating sub-5-kb resolution 3D genome maps by in situ Hi-C. Contact maps at sub-5-kb resolution allow identification of individual DNA loops, domain organization, and large-scale genome compartmentalization. We observed differences in looping architectures among GSCs from different patients, suggesting that 3D genome architecture is a further layer of inter-patient heterogeneity for glioblastoma. Integration of DNA contact maps with chromatin and transcriptional profiles identified specific mechanisms of gene regulation, including the convergence of multiple super-enhancers to individual stemness genes within individual cells. We show that the number of loops contacting a gene correlates with elevated transcription. These results indicate that stemness genes are hubs of interaction between multiple regulatory regions, likely to ensure their sustained expression. Regions of open chromatin common among the GSCs tested were poised for expression of immune-related genes, including CD276 We demonstrate that this gene is co-expressed with stemness genes in GSCs and that CD276 can be targeted with an antibody-drug conjugate to eliminate self-renewing cells. Our results demonstrate that integrated structural genomics data sets can be employed to rationally identify therapeutic vulnerabilities in self-renewing cells.
PMID: 31249064
Hoffman M, Gillmor AH, Kunz DJ, Johnston MJ, Nikolic A, Karta K, Zarrei M, King J, Ellestad K, Dang NH, Cavalli FMG, Kushida MM, Coutinho FJ, Zhu Y, Luu B, Ma Y, Mungall AJ, Moore R, Marra MA, Taylor MD, Pugh TJ, Dirks PB, Strother D, Lafay-Cousin L, Resnick AC, Scherer SW, Senger DL, Simons BD, Chan JA, Morrissy AS, Gallo M. (2019) Intratumoral genetic and functional heterogeneity in pediatric glioblastoma. Cancer Research 79: 2111-2123. PMID:30877103.
Our paper was featured on the cover of the May 2019 issue of the journal.
Abstract
Pediatric glioblastoma (pGBM) is a lethal cancer with no effective therapies. To understand the mechanisms of tumour evolution in this cancer, we performed whole-genome sequencing with linked reads on longitudinally resected pGBM samples. Our analyses showed that all diagnostic and recurrent samples were collections of genetically diverse subclones. Clonal composition rapidly evolved at recurrence, with less than 8% of nonsynonymous single-nucleotide variants being shared in diagnostic-recurrent pairs. To track the origins of the mutational events observed in pGBM, we generated whole-genome datasets for two patients and their parents. These trios showed that genetic variants could be (i) somatic, (ii) inherited from a healthy parent, or (iii) de novo in the germlines of pGBM patients. Analysis of variant allele frequencies supported a model of tumour growth involving slow-cycling cancer stem cells that give rise to fast-proliferating progenitor-like cells and to nondividing cells. Interestingly, radiation and antimitotic chemotherapeutics did not increase overall tumour burden upon recurrence. These findings support an important role for slow-cycling stem cell populations in contributing to recurrences, because slow-cycling cell populations are expected to be less prone to genotoxic stress induced by these treatments and therefore would accumulate few mutations. Our results highlight the need for new targeted treatments that account for the complex functional hierarchies and genomic heterogeneity of pGBM. SIGNIFICANCE: This work challenges several assumptions regarding the genetic organization of pediatric GBM and highlights mutagenic programs that start during early prenatal development.
PMID: 30877103
DNA Hypermethylation Within Tert Promoter Upregulates Tert Expression in Cancer
Published: January 02, 2019
Abstract
Replicative immortality is a hallmark of cancer cells governed by telomere maintenance. Approximately 90% of human cancers maintain their telomeres by activating telomerase, driven by the transcriptional upregulation of telomerase reverse transcriptase (TERT). Although TERT promoter mutations (TPMs) are a major cancer-associated genetic mechanism of TERT upregulation, many cancers exhibit TERT upregulation without TPMs. In this study, we describe the TERT hypermethylated oncological region (THOR), a 433-bp genomic region encompassing 52 CpG sites located immediately upstream of the TERT core promoter, as a cancer-associated epigenetic mechanism of TERT upregulation. Unmethylated THOR repressed TERT promoter activity regardless of TPM status, and hypermethylation of THOR counteracted this repressive function. THOR methylation analysis in 1,352 human tumours revealed frequent (>45%) cancer-associated DNA hypermethylation in 9 of 11 (82%) tumour types screened. Additionally, THOR hypermethylation, either independently or along with TPMs, accounted for how approximately 90% of human cancers can aberrantly activate telomerase. Thus, we propose that THOR hypermethylation is a prevalent telomerase-activating mechanism in cancer that can act independently of or in conjunction with TPMs, further supporting the utility of THOR hypermethylation as a prognostic biomarker.
PMID: 30358567
