Publications by authors named "Anthony T Papenfuss"

139 Publications

VIRUSBreakend: Viral Integration Recognition Using Single Breakends.

Bioinformatics 2021 May 11. Epub 2021 May 11.

Bioinformatics Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Australia.

Motivation: Integration of viruses into infected host cell DNA can cause DNA damage and disrupt genes. Recent cost reductions and growth of whole genome sequencing has produced a wealth of data in which viral presence and integration detection is possible. While key research and clinically relevant insights can be uncovered, existing software has not achieved widespread adoption, limited in part due to high computational costs, the inability to detect a wide range of viruses, as well as precision and sensitivity.

Results: Here, we describe VIRUSBreakend, a high-speed tool that identifies viral DNA presence and genomic integration. It utilizes single breakends, breakpoints in which only one side can be unambiguously placed, in a novel virus-centric variant calling and assembly approach to identify viral integrations with high sensitivity and a near-zero false discovery rate. VIRUSBreakend detects viral integrations anywhere in the host genome including regions such as centromeres and telomeres unable to be called by existing tools. Applying VIRUSBreakend to a large metastatic cancer cohort, we demonstrate that it can reliably detect clinically relevant viral presence and integration including HPV, HBV, MCPyV, EBV, and HHV-8.

Availability: VIRUSBreakend is part of the Genomic Rearrangement IDentification Software Suite (GRIDSS). It is available under a GPLv3 license from https://github.com/PapenfussLab/VIRUSBreakend.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btab343DOI Listing
May 2021

A single-cell RNA expression atlas of normal, preneoplastic and tumorigenic states in the human breast.

EMBO J 2021 May 5:e107333. Epub 2021 May 5.

ACRF Cancer Biology and Stem Cells Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Vic, Australia.

To examine global changes in breast heterogeneity across different states, we determined the single-cell transcriptomes of > 340,000 cells encompassing normal breast, preneoplastic BRCA1 tissue, the major breast cancer subtypes, and pairs of tumors and involved lymph nodes. Elucidation of the normal breast microenvironment revealed striking changes in the stroma of post-menopausal women. Single-cell profiling of 34 treatment-naive primary tumors, including estrogen receptor (ER) , HER2 , and triple-negative breast cancers, revealed comparable diversity among cancer cells and a discrete subset of cycling cells. The transcriptomes of preneoplastic BRCA1 tissue versus tumors highlighted global changes in the immune microenvironment. Within the tumor immune landscape, proliferative CD8 T cells characterized triple-negative and HER2 cancers but not ER tumors, while all subtypes comprised cycling tumor-associated macrophages, thus invoking potentially different immunotherapy targets. Copy number analysis of paired ER tumors and lymph nodes indicated seeding by genetically distinct clones or mass migration of primary tumor cells into axillary lymph nodes. This large-scale integration of patient samples provides a high-resolution map of cell diversity in normal and cancerous human breast.
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http://dx.doi.org/10.15252/embj.2020107333DOI Listing
May 2021

T-Cell Receptor Therapy in the Treatment of Ovarian Cancer: A Mini Review.

Front Immunol 2021 13;12:672502. Epub 2021 Apr 13.

School of Clinical Sciences, Monash University, Clayton, VIC, Australia.

Ovarian cancer, in particularly high-grade serous ovarian cancer (HGSOC) and ovarian carcinosarcoma (OCS), are highly aggressive and deadly female cancers with limited treatment options. These tumors are generally unresponsive to immune check-point inhibitor (ICI) therapy and are referred to as immunologically "cold" tumors. Cell-based therapy, in particular, adoptive T-cell therapy, is an alternative immunotherapy option that has shown great potential, especially chimeric antigen receptor T cell (CAR-T) therapy in the treatment of hematologic malignancies. However, the efficacy of CAR-T therapy in solid tumors has been modest. This review explores the potential of another cell-based therapy, T-cell receptor therapy (TCR-T) as an alternate treatment option for immunological "cold" OC and OCS tumors.
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http://dx.doi.org/10.3389/fimmu.2021.672502DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8076633PMC
April 2021

First Description of the Composition and the Functional Capabilities of the Skin Microbial Community Accompanying Severe Scabies Infestation in Humans.

Microorganisms 2021 Apr 23;9(5). Epub 2021 Apr 23.

Scabies Laboratory, Infectious Diseases Program, Biology Department, QIMR Berghofer Medical Research Institute, Brisbane 4006, Australia.

Epidemiological studies link infection and impetigo. Scabies mites can promote (Group A ) and infections by breaching the skin barrier and excreting molecules that inhibit host innate immune responses. However, little is known about the composition and the function of the scabies-associated microbiota. Here, high-throughput whole-metagenome sequencing was used to explore the scabies-associated microbiome. Scabies mites including their immediate microenvironments were isolated from two patients with severe scabies in Northern Australia. Two ~45-50 million paired-end reads Illumina libraries were generated of which ~2 (5.1%) and 0.7 million (1.3%) microbial reads were filtered out by mapping to human (hg19) and mite draft genomes. Taxonomic profiling revealed a microbial community dominated by the phylum Firmicutes (A: 79% and B: 59%) and genera that comprise , , and . Assembly of the metagenome reads resulted in genome bins representing reference genomes of , (Group C/G), and . The contigs contained genes relevant to pathogenicity and antibiotics resistance. Confocal microscopy of a patient skin sample confirmed , Streptococci and in scabies mite gut and faeces and the surrounding skin. The study provides fundamental evidence for the association of opportunistic pathogens with scabies infection.
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http://dx.doi.org/10.3390/microorganisms9050907DOI Listing
April 2021

Probabilistic outlier identification for RNA sequencing generalized linear models.

NAR Genom Bioinform 2021 Mar 1;3(1):lqab005. Epub 2021 Mar 1.

The Walter and Eliza Hall Institute, Parkville, Victoria, 3052, Australia.

Relative transcript abundance has proven to be a valuable tool for understanding the function of genes in biological systems. For the differential analysis of transcript abundance using RNA sequencing data, the negative binomial model is by far the most frequently adopted. However, common methods that are based on a negative binomial model are not robust to extreme outliers, which we found to be abundant in public datasets. So far, no rigorous and probabilistic methods for detection of outliers have been developed for RNA sequencing data, leaving the identification mostly to visual inspection. Recent advances in Bayesian computation allow large-scale comparison of observed data against its theoretical distribution given in a statistical model. Here we propose ppcseq, a key quality-control tool for identifying transcripts that include outlier data points in differential expression analysis, which do not follow a negative binomial distribution. Applying ppcseq to analyse several publicly available datasets using popular tools, we show that from 3 to 10 percent of differentially abundant transcripts across algorithms and datasets had statistics inflated by the presence of outliers.
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http://dx.doi.org/10.1093/nargab/lqab005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7936652PMC
March 2021

Ductal variant prostate carcinoma is associated with a significantly shorter metastasis-free survival.

Eur J Cancer 2021 May 5;148:440-450. Epub 2021 Mar 5.

Department of Surgery, University of Melbourne, Parkville, Victoria, Australia; Urology Unit, Royal Melbourne Hospital, Parkville, Victoria, Australia; Victorian Comprehensive Cancer Centre, Melbourne, Victoria, Australia; Department of Urology, Frankston Hospital, Frankston, Victoria, Australia.

Background: Ductal adenocarcinoma is an uncommon prostate cancer variant. Previous studies suggest that ductal variant histology may be associated with worse clinical outcomes, but these are difficult to interpret. To address this, we performed an international, multi-institutional study to describe the characteristics of ductal adenocarcinoma, particularly focussing on the effect of presence of ductal variant cancer on metastasis-free survival.

Methods: Patients with ductal variant histology from two institutional databases who underwent radical prostatectomies were identified and compared with an independent acinar adenocarcinoma cohort. After propensity score matching, the effect of the presence of ductal adenocarcinoma on time to biochemical recurrence, initiation of salvage therapy and the development of metastatic disease was determined. Deep whole-exome sequencing was performed for selected cases (n = 8).

Results: A total of 202 ductal adenocarcinoma and 2037 acinar adenocarcinoma cases were analysed. Survival analysis after matching demonstrated that patients with ductal variant histology had shorter salvage-free survival (8.1 versus 22.0 months, p = 0.03) and metastasis-free survival (6.7 versus 78.6 months, p < 0.0001). Ductal variant histology was consistently associated with RB1 loss, as well as copy number gains in TAP1, SLC4A2 and EHHADH.

Conclusions: The presence of any ductal variant adenocarcinoma at the time of prostatectomy portends a worse clinical outcome than pure acinar cancers, with significantly shorter times to initiation of salvage therapies and the onset of metastatic disease. These features appear to be driven by uncoupling of chromosomal duplication from cell division, resulting in widespread copy number aberration with specific gain of genes implicated in treatment resistance.
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http://dx.doi.org/10.1016/j.ejca.2020.12.030DOI Listing
May 2021

Evolution of late-stage metastatic melanoma is dominated by aneuploidy and whole genome doubling.

Nat Commun 2021 03 4;12(1):1434. Epub 2021 Mar 4.

Peter MacCallum Cancer Centre, Melbourne, VIC, Australia.

Although melanoma is initiated by acquisition of point mutations and limited focal copy number alterations in melanocytes-of-origin, the nature of genetic changes that characterise lethal metastatic disease is poorly understood. Here, we analyze the evolution of human melanoma progressing from early to late disease in 13 patients by sampling their tumours at multiple sites and times. Whole exome and genome sequencing data from 88 tumour samples reveals only limited gain of point mutations generally, with net mutational loss in some metastases. In contrast, melanoma evolution is dominated by whole genome doubling and large-scale aneuploidy, in which widespread loss of heterozygosity sculpts the burden of point mutations, neoantigens and structural variants even in treatment-naïve and primary cutaneous melanomas in some patients. These results imply that dysregulation of genomic integrity is a key driver of selective clonal advantage during melanoma progression.
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http://dx.doi.org/10.1038/s41467-021-21576-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7933255PMC
March 2021

Evolutionary analyses of the major variant surface antigen-encoding genes reveal population structure of Plasmodium falciparum within and between continents.

PLoS Genet 2021 Feb 25;17(2):e1009269. Epub 2021 Feb 25.

School of BioSciences, Bio21 Institute, The University of Melbourne, Melbourne, Australia.

Malaria remains a major public health problem in many countries. Unlike influenza and HIV, where diversity in immunodominant surface antigens is understood geographically to inform disease surveillance, relatively little is known about the global population structure of PfEMP1, the major variant surface antigen of the malaria parasite Plasmodium falciparum. The complexity of the var multigene family that encodes PfEMP1 and that diversifies by recombination, has so far precluded its use in malaria surveillance. Recent studies have demonstrated that cost-effective deep sequencing of the region of var genes encoding the PfEMP1 DBLα domain and subsequent classification of within host sequences at 96% identity to define unique DBLα types, can reveal structure and strain dynamics within countries. However, to date there has not been a comprehensive comparison of these DBLα types between countries. By leveraging a bioinformatic approach (jumping hidden Markov model) designed specifically for the analysis of recombination within var genes and applying it to a dataset of DBLα types from 10 countries, we are able to describe population structure of DBLα types at the global scale. The sensitivity of the approach allows for the comparison of the global dataset to ape samples of Plasmodium Laverania species. Our analyses show that the evolution of the parasite population emerging out of Africa underlies current patterns of DBLα type diversity. Most importantly, we can distinguish geographic population structure within Africa between Gabon and Ghana in West Africa and Uganda in East Africa. Our evolutionary findings have translational implications in the context of globalization. Firstly, DBLα type diversity can provide a simple diagnostic framework for geographic surveillance of the rapidly evolving transmission dynamics of P. falciparum. It can also inform efforts to understand the presence or absence of global, regional and local population immunity to major surface antigen variants. Additionally, we identify a number of highly conserved DBLα types that are present globally that may be of biological significance and warrant further characterization.
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http://dx.doi.org/10.1371/journal.pgen.1009269DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7906310PMC
February 2021

Molecular diagnosis of scabies using a novel probe-based polymerase chain reaction assay targeting high-copy number repetitive sequences in the Sarcoptes scabiei genome.

PLoS Negl Trop Dis 2021 Feb 24;15(2):e0009149. Epub 2021 Feb 24.

QIMR Berghofer Medical Research Institute, Brisbane, Australia.

Background: The suboptimal sensitivity and specificity of available diagnostic methods for scabies hampers clinical management, trials of new therapies and epidemiologic studies. Additionally, parasitologic diagnosis by microscopic examination of skin scrapings requires sample collection with a sharp scalpel blade, causing discomfort to patients and difficulty in children. Polymerase chain reaction (PCR)-based diagnostic assays, combined with non-invasive sampling methods, represent an attractive approach. In this study, we aimed to develop a real-time probe-based PCR test for scabies, test a non-invasive sampling method and evaluate its diagnostic performance in two clinical settings.

Methodology/principal Findings: High copy-number repetitive DNA elements were identified in draft Sarcoptes scabiei genome sequences and used as assay targets for diagnostic PCR. Two suitable repetitive DNA sequences, a 375 base pair microsatellite (SSR5) and a 606 base pair long tandem repeat (SSR6), were identified. Diagnostic sensitivity and specificity were tested using relevant positive and negative control materials and compared to a published assay targeting the mitochondrial cox1 gene. Both assays were positive at a 1:100 dilution of DNA from a single mite; no amplification was observed in DNA from samples from 19 patients with other skin conditions nor from house dust, sheep or dog mites, head and body lice or from six common skin bacterial and fungal species. Moderate sensitivity of the assays was achieved in a pilot study, detecting 5/7 (71.4% [95% CI: 29.0% - 96.3%]) of clinically diagnosed untreated scabies patients). Greater sensitivity was observed in samples collected by FLOQ swabs compared to skin scrapings.

Conclusions/significance: This newly developed qPCR assay, combined with the use of an alternative non-invasive swab sampling technique offers the possibility of enhanced diagnosis of scabies. Further studies will be required to better define the diagnostic performance of these tests.
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http://dx.doi.org/10.1371/journal.pntd.0009149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7939366PMC
February 2021

tidybulk: an R tidy framework for modular transcriptomic data analysis.

Genome Biol 2021 Jan 22;22(1):42. Epub 2021 Jan 22.

Bioinformatics Division, Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia.

Recently, efforts have been made toward the harmonization of transcriptomic data structures and workflows using the concept of data tidiness, to facilitate modularisation. We present tidybulk, a modular framework for bulk transcriptional analyses that introduces a tidy transcriptomic data structure paradigm and analysis grammar. Tidybulk covers a wide variety of analysis procedures and integrates a large ecosystem of publicly available analysis algorithms under a common framework. Tidybulk decreases coding burden, facilitates reproducibility, increases efficiency for expert users, lowers the learning curve for inexperienced users, and bridges transcriptional data analysis with the tidyverse. Tidybulk is available at R/Bioconductor bioconductor.org/packages/tidybulk .
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http://dx.doi.org/10.1186/s13059-020-02233-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7821481PMC
January 2021

Combined BRAF, MEK, and CDK4/6 Inhibition Depletes Intratumoral Immune-Potentiating Myeloid Populations in Melanoma.

Cancer Immunol Res 2021 Feb 10;9(2):136-146. Epub 2020 Dec 10.

Cancer Research Division, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia.

Combined inhibition of BRAF, MEK, and CDK4/6 is currently under evaluation in clinical trials for patients with melanoma harboring a mutation. While this triple therapy has potent tumor-intrinsic effects, the impact of this combination on antitumor immunity remains unexplored. Here, using a syngeneic melanoma model, we demonstrated that triple therapy promoted durable tumor control through tumor-intrinsic mechanisms and promoted immunogenic cell death and T-cell infiltration. Despite this, tumors treated with triple therapy were unresponsive to immune checkpoint blockade (ICB). Flow cytometric and single-cell RNA sequencing analyses of tumor-infiltrating immune populations revealed that triple therapy markedly depleted proinflammatory macrophages and cross-priming CD103 dendritic cells, the absence of which correlated with poor overall survival and clinical responses to ICB in patients with melanoma. Indeed, immune populations isolated from tumors of mice treated with triple therapy failed to stimulate T-cell responses While combined BRAF, MEK, and CDK4/6 inhibition demonstrates favorable tumor-intrinsic activity, these data suggest that collateral effects on tumor-infiltrating myeloid populations may impact antitumor immunity. These findings have important implications for the design of combination strategies and clinical trials that incorporate BRAF, MEK, and CDK4/6 inhibition with immunotherapy for the treatment of patients with melanoma.
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http://dx.doi.org/10.1158/2326-6066.CIR-20-0401DOI Listing
February 2021

Histone modifications associated with gene expression and genome accessibility are dynamically enriched at Plasmodium falciparum regulatory sequences.

Epigenetics Chromatin 2020 11 23;13(1):50. Epub 2020 Nov 23.

Department of Medicine, The University of Melbourne, Royal Melbourne Hospital, Parkville, VIC, 3050, Australia.

Background: The malaria parasite Plasmodium falciparum has an unusually euchromatic genome with poorly conserved positioning of nucleosomes in intergenic sequences and poorly understood mechanisms of gene regulation. Variant histones and histone modifications determine nucleosome stability and recruit trans factors, but their combinatorial contribution to gene regulation is unclear.

Results: Here, we show that the histone H3 acetylations H3K18ac and H3K27ac and the variant histone Pf H2A.Z are enriched together at regulatory sites upstream of genes. H3K18ac and H3K27ac together dynamically mark regulatory regions of genes expressed during the asexual life cycle. In contrast, H3K4me1 is depleted in intergenic sequence and dynamically depleted upstream of expressed genes. The temporal pattern of H3K27ac and H3K18ac enrichment indicates that they accumulate during S phase and mitosis and are retained at regulatory sequences until at least G1 phase and after cessation of expression of the cognate genes. We integrated our ChIPseq data with existing datasets to show that in schizont stages H3K18ac, H3K27ac and Pf H2A.Z colocalise with the transcription factor PfAP2-I and the bromodomain protein PfBDP1 and are enriched at stably positioned nucleosomes within regions of exposed DNA at active transcriptional start sites. Using transient transfections we showed that sequences enriched with colocalised H3K18ac, H3K27ac and Pf H2A.Z possess promoter activity in schizont stages, but no enhancer-like activity.

Conclusions: The dynamic H3 acetylations define P. falciparum regulatory sequences and contribute to gene activation. These findings expand the knowledge of the chromatin landscape that regulates gene expression in P. falciparum.
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http://dx.doi.org/10.1186/s13072-020-00365-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7682024PMC
November 2020

Unifying package managers, workflow engines, and containers: Computational reproducibility with BioNix.

Gigascience 2020 11;9(11)

Bioinformatics Division, Walter and Eliza Hall Institute of Medical Research, 1G Royal Pde., Parkville, VIC 3052, Australia.

Motivation: A challenge for computational biologists is to make our analyses reproducible-i.e. to rerun, combine, and share, with the assurance that equivalent runs will generate identical results. Current best practice aims at this using a combination of package managers, workflow engines, and containers.

Results: We present BioNix, a lightweight library built on the Nix deployment system. BioNix manages software dependencies, computational environments, and workflow stages together using a single abstraction: pure functions. This lets users specify workflows in a clean, uniform way, with strong reproducibility guarantees.

Availability And Implementation: BioNix is implemented in the Nix expression language and is released on GitHub under the 3-clause BSD license: https://github.com/PapenfussLab/bionix (biotools:BioNix) (BioNix, RRID:SCR_017662).
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http://dx.doi.org/10.1093/gigascience/giaa121DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7672450PMC
November 2020

High-quality nuclear genome for Sarcoptes scabiei-A critical resource for a neglected parasite.

PLoS Negl Trop Dis 2020 10 1;14(10):e0008720. Epub 2020 Oct 1.

Cell and Molecular Biology Department, Infectious Diseases Program, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia.

The parasitic mite Sarcoptes scabiei is an economically highly significant parasite of the skin of humans and animals worldwide. In humans, this mite causes a neglected tropical disease (NTD), called scabies. This disease results in major morbidity, disability, stigma and poverty globally and is often associated with secondary bacterial infections. Currently, anti-scabies treatments are not sufficiently effective, resistance to them is emerging and no vaccine is available. Here, we report the first high-quality genome and transcriptomic data for S. scabiei. The genome is 56.6 Mb in size, has a a repeat content of 10.6% and codes for 9,174 proteins. We explored key molecules involved in development, reproduction, host-parasite interactions, immunity and disease. The enhanced 'omic data sets for S. scabiei represent comprehensive and critical resources for genetic, functional genomic, metabolomic, phylogenetic, ecological and/or epidemiological investigations, and will underpin the design and development of new treatments, vaccines and/or diagnostic tests.
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http://dx.doi.org/10.1371/journal.pntd.0008720DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7591027PMC
October 2020

The Hippo pathway oncoprotein YAP promotes melanoma cell invasion and spontaneous metastasis.

Oncogene 2020 07 19;39(30):5267-5281. Epub 2020 Jun 19.

Peter MacCallum Cancer Centre, 305 Grattan St, Melbourne, VIC, 3000, Australia.

Melanoma is a deadly form of skin cancer that accounts for a disproportionally large proportion of cancer-related deaths in younger people. Compared with most other skin cancers, a feature of melanoma is its high metastatic capacity, although the mechanisms that confer this are not well understood. The Hippo pathway is a key regulator of organ growth and cell fate that is deregulated in many cancers. To analyse the Hippo pathway in cutaneous melanoma, we generated a transcriptional signature of melanoma cells that overexpressed YAP, the key downstream Hippo pathway oncoprotein. YAP-mediated transcriptional activity varied in melanoma cell lines but did not cluster with known genetic drivers of melanomagenesis such as BRAF and NRAS mutations. Instead, it correlated strongly with published gene expression profiles linked to melanoma cell invasiveness and varied throughout the metastatic cascade in melanoma patient tumours. Consistent with this, YAP was both necessary and sufficient for melanoma cell invasion in vitro. In vivo, YAP promoted spontaneous melanoma metastasis, whilst the growth of YAP-expressing primary tumours was impeded. Finally, we identified the YAP target genes AXL, THBS1 and CYR61 as key mediators of YAP-induced melanoma cell invasion. These data suggest that YAP is a critical regulator of melanoma metastasis.
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http://dx.doi.org/10.1038/s41388-020-1362-9DOI Listing
July 2020

Determination of RNA structural diversity and its role in HIV-1 RNA splicing.

Nature 2020 06 6;582(7812):438-442. Epub 2020 May 6.

Whitehead Institute for Biomedical Research, Cambridge, MA, USA.

Human immunodeficiency virus 1 (HIV-1) is a retrovirus with a ten-kilobase single-stranded RNA genome. HIV-1 must express all of its gene products from a single primary transcript, which undergoes alternative splicing to produce diverse protein products that include structural proteins and regulatory factors. Despite the critical role of alternative splicing, the mechanisms that drive the choice of splice site are poorly understood. Synonymous RNA mutations that lead to severe defects in splicing and viral replication indicate the presence of unknown cis-regulatory elements. Here we use dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) to investigate the structure of HIV-1 RNA in cells, and develop an algorithm that we name 'detection of RNA folding ensembles using expectation-maximization' (DREEM), which reveals the alternative conformations that are assumed by the same RNA sequence. Contrary to previous models that have analysed population averages, our results reveal heterogeneous regions of RNA structure across the entire HIV-1 genome. In addition to confirming that in vitro characterized alternative structures for the HIV-1 Rev responsive element also exist in cells, we discover alternative conformations at critical splice sites that influence the ratio of transcript isoforms. Our simultaneous measurement of splicing and intracellular RNA structure provides evidence for the long-standing hypothesis that heterogeneity in RNA conformation regulates splice-site use and viral gene expression.
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http://dx.doi.org/10.1038/s41586-020-2253-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7310298PMC
June 2020

MTOR signaling orchestrates stress-induced mutagenesis, facilitating adaptive evolution in cancer.

Science 2020 06;368(6495):1127-1131

The Kinghorn Cancer Centre, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia.

In microorganisms, evolutionarily conserved mechanisms facilitate adaptation to harsh conditions through stress-induced mutagenesis (SIM). Analogous processes may underpin progression and therapeutic failure in human cancer. We describe SIM in multiple in vitro and in vivo models of human cancers under nongenotoxic drug selection, paradoxically enhancing adaptation at a competing intrinsic fitness cost. A genome-wide approach identified the mechanistic target of rapamycin (MTOR) as a stress-sensing rheostat mediating SIM across multiple cancer types and conditions. These observations are consistent with a two-phase model for drug resistance, in which an initially rapid expansion of genetic diversity is counterbalanced by an intrinsic fitness penalty, subsequently normalizing to complete adaptation under the new conditions. This model suggests synthetic lethal strategies to minimize resistance to anticancer therapy.
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http://dx.doi.org/10.1126/science.aau8768DOI Listing
June 2020

Tentacle Transcriptomes of the Speckled Anemone (Actiniaria: Actiniidae: Oulactis sp.): Venom-Related Components and Their Domain Structure.

Mar Biotechnol (NY) 2020 Apr 24;22(2):207-219. Epub 2020 Jan 24.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, Victoria, 3052, Australia.

Cnidarians are one of the oldest known animal lineages (ca. 700 million years), with a unique envenomation apparatus to deliver a potent mixture of peptides and proteins. Some peptide toxins from cnidarian venom have proven therapeutic potential. Here, we use a transcriptomic/proteomic strategy to identify sequences with similarity to known venom protein families in the tentacles of the endemic Australian 'speckled anemone' (Oulactis sp.). Illumina RNASeq data were assembled de novo. Annotated sequences in the library were verified by cross-referencing individuals' transcriptomes or protein expression evidence from LC-MS/MS data. Sequences include pore-forming toxins, phospholipases, peptidases, neurotoxins (sodium and potassium channel modulators), cysteine-rich secretory proteins and defensins (antimicrobial peptides). Fewer than 4% of the sequences in the library occurred across the three individuals examined, demonstrating high sequence variability of an individual's arsenal. We searched for actinoporins in Oulactis sp. to assess sequence similarity to the only described toxins (OR-A and -G) for this genus and examined the domain architecture of venom-related peptides and proteins. The novel putative actinoporin of Oulactis sp. has a greater similarity to other species in the Actiniidae family than to O. orientalis. Venom-related sequences have an architecture that occurs in single, repeat or multi-domain combinations of venom-related (e.g. ShK-like) and non-venom (e.g. whey acid protein) domains. This study has produced the first transcriptomes for an endemic Australian sea anemone species and the genus Oulactis, while identifying nearly 400 novel venom-related peptides and proteins for future structural and functional analyses and venom evolution studies.
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http://dx.doi.org/10.1007/s10126-020-09945-8DOI Listing
April 2020

Higher frequency of vertebrate-infecting viruses in the gut of infants born to mothers with type 1 diabetes.

Pediatr Diabetes 2020 03 7;21(2):271-279. Epub 2020 Jan 7.

School of Women's and Children's Health, University of New South Wales, Sydney, New South Wales, Australia.

Background: Microbial exposures in utero and early life shape the infant microbiome, which can profoundly impact on health. Compared to the bacterial microbiome, very little is known about the virome. We set out to characterize longitudinal changes in the gut virome of healthy infants born to mothers with or without type 1 diabetes using comprehensive virome capture sequencing.

Methods: Healthy infants were selected from Environmental Determinants of Islet Autoimmunity (ENDIA), a prospective cohort of Australian children with a first-degree relative with type 1 diabetes, followed from pregnancy. Fecal specimens were collected three-monthly in the first year of life.

Results: Among 25 infants (44% born to mothers with type 1 diabetes) at least one virus was detected in 65% (65/100) of samples and 96% (24/25) of infants during the first year of life. In total, 26 genera of viruses were identified and >150 viruses were differentially abundant between the gut of infants with a mother with type 1 diabetes vs without. Positivity for any virus was associated with maternal type 1 diabetes and older infant age. Enterovirus was associated with older infant age and maternal smoking.

Conclusions: We demonstrate a distinct gut virome profile in infants of mothers with type 1 diabetes, which may influence health outcomes later in life. Higher prevalence and greater number of viruses observed compared to previous studies suggests significant underrepresentation in existing virome datasets, arising most likely from less sensitive techniques used in data acquisition.
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http://dx.doi.org/10.1111/pedi.12952DOI Listing
March 2020

MaveDB: an open-source platform to distribute and interpret data from multiplexed assays of variant effect.

Genome Biol 2019 11 4;20(1):223. Epub 2019 Nov 4.

Bioinformatics Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia.

Multiplex assays of variant effect (MAVEs), such as deep mutational scans and massively parallel reporter assays, test thousands of sequence variants in a single experiment. Despite the importance of MAVE data for basic and clinical research, there is no standard resource for their discovery and distribution. Here, we present MaveDB ( https://www.mavedb.org ), a public repository for large-scale measurements of sequence variant impact, designed for interoperability with applications to interpret these datasets. We also describe the first such application, MaveVis, which retrieves, visualizes, and contextualizes variant effect maps. Together, the database and applications will empower the community to mine these powerful datasets.
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http://dx.doi.org/10.1186/s13059-019-1845-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6827219PMC
November 2019

Regulation of PRMT5-MDM4 axis is critical in the response to CDK4/6 inhibitors in melanoma.

Proc Natl Acad Sci U S A 2019 09 22;116(36):17990-18000. Epub 2019 Aug 22.

Department of Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030.

Cyclin-dependent kinase 4/6 (CDK4/6) inhibitors are an established treatment in estrogen receptor-positive breast cancer and are currently in clinical development in melanoma, a tumor that exhibits high rates of CDK4 activation. We analyzed melanoma cells with acquired resistance to the CDK4/6 inhibitor palbociclib and demonstrate that the activity of PRMT5, a protein arginine methyltransferase and indirect target of CDK4, is essential for CDK4/6 inhibitor sensitivity. By indirectly suppressing PRMT5 activity, palbociclib alters the pre-mRNA splicing of MDM4, a negative regulator of p53, leading to decreased MDM4 protein expression and subsequent p53 activation. In turn, p53 induces p21, leading to inhibition of CDK2, the main kinase substituting for CDK4/6 and a key driver of resistance to palbociclib. Loss of the ability of palbociclib to regulate the PRMT5-MDM4 axis leads to resistance. Importantly, combining palbociclib with the PRMT5 inhibitor GSK3326595 enhances the efficacy of palbociclib in treating naive and resistant models and also delays the emergence of resistance. Our studies have uncovered a mechanism of action of CDK4/6 inhibitors in regulating the MDM4 oncogene and the tumor suppressor, p53. Furthermore, we have established that palbociclib inhibition of the PRMT5-MDM4 axis is essential for robust melanoma cell sensitivity and provide preclinical evidence that coinhibition of CDK4/6 and PRMT5 is an effective and well-tolerated therapeutic strategy. Overall, our data provide a strong rationale for further investigation of novel combinations of CDK4/6 and PRMT5 inhibitors, not only in melanoma but other tumor types, including breast, pancreatic, and esophageal carcinoma.
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http://dx.doi.org/10.1073/pnas.1901323116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6731642PMC
September 2019

Two of a kind: transmissible Schwann cell cancers in the endangered Tasmanian devil (Sarcophilus harrisii).

Cell Mol Life Sci 2020 May 2;77(9):1847-1858. Epub 2019 Aug 2.

Menzies Institute for Medical Research, University of Tasmania, 17 Liverpool Street, Hobart, TAS, 7000, Australia.

Devil facial tumour disease (DFTD) comprises two genetically distinct transmissible cancers (DFT1 and DFT2) endangering the survival of the Tasmanian devil (Sarcophilus harrisii) in the wild. DFT1 first arose from a cell of the Schwann cell lineage; however, the tissue-of-origin of the recently discovered DFT2 cancer is unknown. In this study, we compared the transcriptome and proteome of DFT2 tumours to DFT1 and normal Tasmanian devil tissues to determine the tissue-of-origin of the DFT2 cancer. Our findings demonstrate that DFT2 expresses a range of Schwann cell markers and exhibits expression patterns consistent with a similar origin to the DFT1 cancer. Furthermore, DFT2 cells express genes associated with the repair response to peripheral nerve damage. These findings suggest that devils may be predisposed to transmissible cancers of Schwann cell origin. The combined effect of factors such as frequent nerve damage from biting, Schwann cell plasticity and low genetic diversity may allow these cancers to develop on rare occasions. The emergence of two independent transmissible cancers from the same tissue in the Tasmanian devil presents an unprecedented opportunity to gain insight into cancer development, evolution and immune evasion in mammalian species.
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http://dx.doi.org/10.1007/s00018-019-03259-2DOI Listing
May 2020

Comprehensive evaluation and characterisation of short read general-purpose structural variant calling software.

Nat Commun 2019 07 19;10(1):3240. Epub 2019 Jul 19.

Bioinformatics Division, Walter and Eliza Hall Institute of Medical Research, 1G Royal Pde, Parkville, VIC, 3052, Australia.

In recent years, many software packages for identifying structural variants (SVs) using whole-genome sequencing data have been released. When published, a new method is commonly compared with those already available, but this tends to be selective and incomplete. The lack of comprehensive benchmarking of methods presents challenges for users in selecting methods and for developers in understanding algorithm behaviours and limitations. Here we report the comprehensive evaluation of 10 SV callers, selected following a rigorous process and spanning the breadth of detection approaches, using high-quality reference cell lines, as well as simulations. Due to the nature of available truth sets, our focus is on general-purpose rather than somatic callers. We characterise the impact on performance of event size and type, sequencing characteristics, and genomic context, and analyse the efficacy of ensemble calling and calibration of variant quality scores. Finally, we provide recommendations for both users and methods developers.
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http://dx.doi.org/10.1038/s41467-019-11146-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6642177PMC
July 2019

A 4-cyano-3-methylisoquinoline inhibitor of Plasmodium falciparum growth targets the sodium efflux pump PfATP4.

Sci Rep 2019 07 16;9(1):10292. Epub 2019 Jul 16.

La Trobe University, Melbourne, Victoria, 3086, Australia.

We developed a novel series of antimalarial compounds based on a 4-cyano-3-methylisoquinoline. Our lead compound MB14 achieved modest inhibition of the growth in vitro of the human malaria parasite, Plasmodium falciparum. To identify its biological target we selected for parasites resistant to MB14. Genome sequencing revealed that all resistant parasites bore a single point S374R mutation in the sodium (Na) efflux transporter PfATP4. There are many compounds known to inhibit PfATP4 and some are under preclinical development. MB14 was shown to inhibit Na dependent ATPase activity in parasite membranes, consistent with the compound targeting PfATP4 directly. PfATP4 inhibitors cause swelling and lysis of infected erythrocytes, attributed to the accumulation of Na inside the intracellular parasites and the resultant parasite swelling. We show here that inhibitor-induced lysis of infected erythrocytes is dependent upon the parasite protein RhopH2, a component of the new permeability pathways that are induced by the parasite in the erythrocyte membrane. These pathways mediate the influx of Na into the infected erythrocyte and their suppression via RhopH2 knockdown limits the accumulation of Na within the parasite hence protecting the infected erythrocyte from lysis. This study reveals a role for the parasite-induced new permeability pathways in the mechanism of action of PfATP4 inhibitors.
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http://dx.doi.org/10.1038/s41598-019-46500-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6635429PMC
July 2019

Targeting enhancer switching overcomes non-genetic drug resistance in acute myeloid leukaemia.

Nat Commun 2019 06 20;10(1):2723. Epub 2019 Jun 20.

Cancer Research Division, Peter MacCallum Cancer Centre, Melbourne, VIC, Australia.

Non-genetic drug resistance is increasingly recognised in various cancers. Molecular insights into this process are lacking and it is unknown whether stable non-genetic resistance can be overcome. Using single cell RNA-sequencing of paired drug naïve and resistant AML patient samples and cellular barcoding in a unique mouse model of non-genetic resistance, here we demonstrate that transcriptional plasticity drives stable epigenetic resistance. With a CRISPR-Cas9 screen we identify regulators of enhancer function as important modulators of the resistant cell state. We show that inhibition of Lsd1 (Kdm1a) is able to overcome stable epigenetic resistance by facilitating the binding of the pioneer factor, Pu.1 and cofactor, Irf8, to nucleate new enhancers that regulate the expression of key survival genes. This enhancer switching results in the re-distribution of transcriptional co-activators, including Brd4, and provides the opportunity to disable their activity and overcome epigenetic resistance. Together these findings highlight key principles to help counteract non-genetic drug resistance.
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http://dx.doi.org/10.1038/s41467-019-10652-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6586637PMC
June 2019

Gut microbiome dysbiosis and increased intestinal permeability in children with islet autoimmunity and type 1 diabetes: A prospective cohort study.

Pediatr Diabetes 2019 08 20;20(5):574-583. Epub 2019 May 20.

Department of Diabetes and Endocrinology, Women's and Children's Hospital, North Adelaide, South Australia, Australia.

Aims/hypothesis: To investigate the longitudinal relationship between the gut microbiome, circulating short chain fatty acids (SCFAs) and intestinal permeability in children with islet autoimmunity or type 1 diabetes and controls.

Methods: We analyzed the gut bacterial microbiome, plasma SCFAs, small intestinal permeability and dietary intake in 47 children with islet autoimmunity or recent-onset type 1 diabetes and in 41 unrelated or sibling controls over a median (range) of 13 (2-34) months follow-up.

Results: Children with multiple islet autoantibodies (≥2 IA) or type 1 diabetes had gut microbiome dysbiosis. Anti-inflammatory Prevotella and Butyricimonas genera were less abundant and these changes were not explained by differences in diet. Small intestinal permeability measured by blood lactulose:rhamnose ratio was higher in type 1 diabetes. Children with ≥2 IA who progressed to type 1 diabetes (progressors), compared to those who did not progress, had higher intestinal permeability (mean [SE] difference +5.14 [2.0], 95% confidence interval [CI] 1.21, 9.07, P = .006), lower within-sample (alpha) microbial diversity (31.3 [11.2], 95% CI 9.3, 53.3, P = .005), and lower abundance of SCFA-producing bacteria. Alpha diversity (observed richness) correlated with plasma acetate levels in all groups combined (regression coefficient [SE] 0.57 [0.21], 95% CI 0.15, 0.99 P = .008).

Conclusions/interpretation: Children with ≥2 IA who progress to diabetes, like those with recent-onset diabetes, have gut microbiome dysbiosis associated with increased intestinal permeability. Interventions that expand gut microbial diversity, in particular SCFA-producing bacteria, may have a role to decrease progression to diabetes in children at-risk.
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http://dx.doi.org/10.1111/pedi.12865DOI Listing
August 2019

CNspector: a web-based tool for visualisation and clinical diagnosis of copy number variation from next generation sequencing.

Sci Rep 2019 04 23;9(1):6426. Epub 2019 Apr 23.

Department of Pathology, Peter MacCallum Cancer Centre, Parkville, VIC, Australia.

Next Generation Sequencing is now routinely used in the practice of diagnostic pathology to detect clinically relevant somatic and germline sequence variations in patient samples. However, clinical assessment of copy number variations (CNVs) and large-scale structural variations (SVs) is still challenging. While tools exist to estimate both, their results are typically presented separately in tables or static plots which can be difficult to read and are unable to show the context needed for clinical interpretation and reporting. We have addressed this problem with CNspector, a multi-scale interactive browser that shows CNVs in the context of other relevant genomic features to enable fast and effective clinical reporting. We illustrate the utility of CNspector at different genomic scales across a variety of sample types in a range of case studies. We show how CNspector can be used for diagnosis and reporting of exon-level deletions, focal gene-level amplifications, chromosome and chromosome arm level amplifications/deletions and in complex genomic rearrangements. CNspector is a web-based clinical variant browser tailored to the clinical application of next generation sequencing for CNV assessment. We have demonstrated the utility of this interactive software in typical applications across a range of tissue types and disease contexts encountered in the context of diagnostic pathology. CNspector is written in R and the source code is available for download under the GPL3 Licence from https://github.com/PapenfussLab/CNspector . A server running CNspector loaded with the figures from this paper can be accessed at https://shiny.wehi.edu.au/jmarkham/CNspector/index.html .
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http://dx.doi.org/10.1038/s41598-019-42858-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6478945PMC
April 2019

Tasmanian devils with contagious cancer exhibit a constricted T-cell repertoire diversity.

Commun Biol 2019 13;2:99. Epub 2019 Mar 13.

School of Life and Environmental Sciences, The University of Sydney, Sydney, NSW, 2006, Australia.

The Tasmanian devil () is threatened by a contagious cancer, known as Devil Facial Tumour Disease (DFTD). A highly diverse T-cell receptor (TCR) repertoire is crucial for successful host defence against cancers. By investigating TCR beta chain diversity in devils of different ages, we show that the T-cell repertoire in devils constricts in their second year of life, which may explain the higher DFTD prevalence in older devils. Unexpectedly, we also observed a pronounced decline in TCR diversity and T cell clonal expansion in devils after DFTD infection. These findings overturned the previous assumption that DFTD did not directly impact host immunity.
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http://dx.doi.org/10.1038/s42003-019-0342-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6416256PMC
April 2020