Publications

Indigenous Canadians experience a disproportionate burden of chronic atherosclerotic diseases, including peripheral artery disease (PAD). Despite an estimated prevalence of 800 000 patients with PAD in Canada, the burden of the disease among Indigenous Canadians is unclear. Available evidence suggests that this population has a higher prevalence of several major risk factors associated with PAD (diabetes, smoking and kidney disease). Unique socioeconomic, geographic and systemic obstacles affecting Indigenous Canadians’ health and health care access may worsen chronic disease outcomes. Little is known about the cardiovascular and limb outcomes of Indigenous peoples with PAD. A novel approach via multidisciplinary vascular health teams engaging Indigenous communities in a culturally competent manner may potentially provide optimal vascular care to this population. Further research into the prevalence and outcomes of PAD among Indigenous Canadians is necessary to define the problem and allow development of more ffective initiatives to alleviate the disease burden in this marginalized group.

Genome sequencing yields the sequence of many short snippets of DNA (reads) from a genome. Genome assembly attempts to reconstruct the original genome from which these reads were derived. This task is difficult due to gaps and errors in the sequencing data, repetitive sequence in the underlying genome, and heterozygosity. As a result, assembly errors are common. In the absence of a reference genome, these misassemblies may be identified by comparing the sequencing data to the assembly and looking for discrepancies between the two. Once identified, these misassemblies may be corrected, improving the quality of the assembled sequence. Although tools exist to identify and correct misassemblies using Illumina paired-end and mate-pair sequencing, no such tool yet exists that makes use of the long distance information of the large molecules provided by linked reads, such as those offered by the 10x Genomics Chromium platform. We have developed the tool Tigmint to address this gap.

Diffuse large B-cell lymphoma (DLBCL) is an aggressive cancer originating from mature B-cells. Prognosis is strongly associated with molecular subgroup, although the driver mutations that distinguish the two main subgroups remain poorly defined. Through an integrative analysis of whole genomes, exomes, and transcriptomes, we have uncovered genes and non-coding loci that are commonly mutated in DLBCL. Our analysis has identified novel cis-regulatory sites, and implicates recurrent mutations in the 3' UTR of NFKBIZ as a novel mechanism of oncogene deregulation and NF-κB pathway activation in the activated B-cell (ABC) subgroup. Small amplifications associated with over-expression of FCGR2B (the Fcγ receptor protein IIB), primarily in the germinal centre B-cell (GCB) subgroup, correlate with poor patient outcomes suggestive of a novel oncogene. These results expand the list of subgroup driver mutations that may facilitate implementation of improved diagnostic assays and could offer new avenues for the development of targeted therapeutics.

Virotherapies are maturing in the clinical setting. Adenoviruses (Ad) are excellent vectors for the manipulability and tolerance of transgenes. Poor tumor selectivity, off-target sequestration, and immune inactivation hamper clinical efficacy. We sought to completely redesign Ad5 into a refined, tumor-selective virotherapy targeted to αvβ6 integrin, which is expressed in a range of aggressively transformed epithelial cancers but nondetectable in healthy tissues. Ad5{{sub}}NULL{{/sub}}-A20 harbors mutations in each major capsid protein to preclude uptake via all native pathways. Tumor-tropism via αvβ6 targeting was achieved by genetic insertion of A20 peptide (NAVPNLRGDLQVLAQKVART) within the fiber knob protein. The vector's selectivity and was assessed. The tropism-ablating triple mutation completely blocked all native cell entry pathways of Ad5{{sub}}NULL{{/sub}}-A20 via coxsackie and adenovirus receptor (CAR), αvβ3/5 integrins, and coagulation factor 10 (FX). Ad5{{sub}}NULL{{/sub}}-A20 efficiently and selectively transduced αvβ6{{sup}}+{{/sup}} cell lines and primary clinical ascites-derived EOC , including in the presence of preexisting anti-Ad5 immunity. biodistribution of Ad5{{sub}}NULL{{/sub}}-A20 following systemic delivery in non-tumor-bearing mice was significantly reduced in all off-target organs, including a remarkable 10{{sup}}7{{/sup}}-fold reduced genome accumulation in the liver compared with Ad5. Tumor uptake, transgene expression, and efficacy were confirmed in a peritoneal SKOV3 xenograft model of human EOC, where oncolytic Ad5{{sub}}NULL{{/sub}}-A20-treated animals demonstrated significantly improved survival compared with those treated with oncolytic Ad5. Oncolytic Ad5{{sub}}NULL{{/sub}}-A20 virotherapies represent an excellent vector for local and systemic targeting of αvβ6-overexpressing cancers and exciting platforms for tumor-selective overexpression of therapeutic anticancer modalities, including immune checkpoint inhibitors. .

RNA-seq is a powerful and cost-effective technology for molecular diagnostics of cancer and other diseases, and it can reach its full potential when coupled with validated clinical-grade informatics tools. Despite recent advances in long-read sequencing, transcriptome assembly of short reads remains a useful and cost-effective methodology for unveiling transcript-level rearrangements and novel isoforms. One of the major concerns for adopting the proven de novo assembly approach for RNA-seq data in clinical settings has been the analysis turnaround time. To address this concern, we have developed a targeted approach to expedite assembly and analysis of RNA-seq data.

The cysteine protease ATG4B is a key component of the autophagy machinery, acting to proteolytically prime and recycle its substrate MAP1LC3B. The roles of ATG4B in cancer and other diseases appear to be context dependent but are still not well understood. To help further explore ATG4B functions and potential therapeutic applications, we employed a chemical biology approach to identify ATG4B inhibitors. Here, we describe the discovery of 4-28, a styrylquinoline identified by a combined computational modeling, in silico screening, high content cell-based screening and biochemical assay approach. A structure-activity relationship study led to the development of a more stable and potent compound LV-320. We demonstrated that LV-320 inhibits ATG4B enzymatic activity, blocks autophagic flux in cells, and is stable, non-toxic and active in vivo. These findings suggest that LV-320 will serve as a relevant chemical tool to study the various roles of ATG4B in cancer and other contexts.

RUNX1 is frequently mutated in T-cell acute lymphoblastic leukemia (T-ALL). The spectrum of RUNX1 mutations has led to the notion that it acts as a tumor suppressor in this context; however, other studies have placed RUNX1, along with transcription factors TAL1 and NOTCH1, as core drivers of an oncogenic transcriptional program. To reconcile these divergent roles, we knocked down RUNX1 in human T-ALL cell lines and deleted Runx1 or Cbfb in primary mouse T-cell leukemias. RUNX1 depletion consistently resulted in reduced cell proliferation and increased apoptosis. RUNX1 upregulated variable sets of target genes in each cell line, but consistently included a core set of oncogenic effectors including insulin-like growth factor 1 receptor (IGF1R) and NRAS. Our results support the conclusion that RUNX1 has a net positive effect on cell growth in the context of established T-ALL.

Increasing evidence of functional and transcriptional heterogeneity in phenotypically similar cells examined individually has prompted interest in obtaining parallel methylome data. We describe the development and application of such a protocol to index-sorted murine and human hematopoietic cells that are highly enriched in their content of functionally defined stem cells. Utilizing an optimized single-cell bisulfite sequencing protocol, we obtained quantitative DNA methylation measurements of up to 5.7 million CpGs in single hematopoietic cells. In parallel, we developed an analytical strategy (PDclust) to define single-cell DNA methylation states through pairwise comparisons of single-CpG methylation measurements. PDclust revealed that a single-cell epigenetic state can be described by a small (<1%) stochastically sampled fraction of CpGs and that these states are reflective of cell identity and state. Using relationships revealed by PDclust, we derive near complete methylomes for epigenetically distinct subpopulations of hematopoietic cells enriched for functional stem cell content.

The eukaryotic endomembrane system is a complex series of interconnected membranous organelles that play important roles in responding to stress and maintaining cell homeostasis during health and disease. Two components of this system, exosome biogenesis and autophagy, are linked by the endolysosomal pathway. Exosomes are cargo-laden extracellular vesicles that arise from endosome-derived multivesicular bodies, and autophagy is a lysosomal-dependent degradation and recycling pathway. Recent studies have revealed shared molecular machinery between exosome biogenesis and autophagy, as well as substantial crosstalk between these two processes. In this Review, we first describe the classic view of exosome biogenesis and autophagy, including their links to the endolysosomal pathway. We then present the evidence for autophagy-related proteins in exosome biogenesis, the emerging roles of amphisomes and the evolving models of exosome-autophagy pathway interactions. Finally, we discuss the implications of exosome and autophagy interplay in the context of neurodegeneration and cancer.

In this White Paper, we discuss the current state of microbial cancer therapy. This paper resulted from a meeting ('Microbial Based Cancer Therapy') at the US National Cancer Institute in the summer of 2017. Here, we define 'Microbial Therapy' to include both oncolytic viral therapy and bacterial anticancer therapy. Both of these fields exploit tumor-specific infectious microbes to treat cancer, have similar mechanisms of action, and are facing similar challenges to commercialization. We designed this paper to nucleate this growing field of microbial therapeutics and increase interactions between researchers in it and related fields. The authors of this paper include many primary researchers in this field. In this paper, we discuss the potential, status and opportunities for microbial therapy as well as strategies attempted to date and important questions that need to be addressed. The main areas that we think will have the greatest impact are immune stimulation, control of efficacy, control of delivery, and safety. There is much excitement about the potential of this field to treat currently intractable cancer. Much of the potential exists because these therapies utilize unique mechanisms of action, difficult to achieve with other biological or small molecule drugs. By better understanding and controlling these mechanisms, we will create new therapies that will become integral components of cancer care.