Publications by authors named "Gordon K Smyth"

204 Publications

High dimensional mass cytometry identifies T cell and B cell signatures predicting reduced risk of Plasmodium vivax malaria.

JCI Insight 2021 Jun 15. Epub 2021 Jun 15.

Infectious Disease and Immune Defense, The Walter and Eliza Hall Institute of Medical Research, Parkville, Australia.

IFN-γ-driven responses to malaria have been shown to modulate the development and function of T follicular helper (TFH) cells and memory B cells (MBCs), with conflicting evidence in their involvement in the induction of antibody responses required to achieve clinical immunity and their association with disease outcomes. Using high-dimensional single cell mass cytometry, we identified distinct populations of TH1-polarized CD4+ T cells and MBCs expressing the TH1-defining transcription factor T-bet, associated with either increased or reduced risk of Plasmodium vivax malaria, demonstrating that inflammatory responses to malaria are not universally detrimental for infection. Furthermore, we found that whereas class-switched but not IgM+ MBCs were associated with reduced risk of symptomatic malaria, populations of TH1 cells with a stem central memory phenotype, TH17 cells and T regulatory cells were associated with protection from asymptomatic infection, suggesting that activation of cell mediated immunity might be also required to control persistent P. vivax infection of low parasite burden.
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http://dx.doi.org/10.1172/jci.insight.148086DOI Listing
June 2021

Procalcitonin and Interleukin-10 May Assist in Early Prediction of Bacteraemia in Children With Cancer and Febrile Neutropenia.

Front Immunol 2021 20;12:641879. Epub 2021 May 20.

Department of Infectious Diseases, Peter MacCallum Cancer Centre, Melbourne, VIC, Australia.

Objectives: Febrile neutropenia (FN) causes treatment disruption and unplanned hospitalization in children with cancer. Serum biomarkers are infrequently used to stratify these patients into high or low risk for serious infection. This study investigated plasma abundance of cytokines in children with FN and their ability to predict bacteraemia.

Methods: Thirty-three plasma cytokines, C-reactive protein (CRP) and procalcitonin (PCT) were measured using ELISA assays in samples taken at FN presentation (n = 79) and within 8-24 h (Day 2; n = 31). Optimal thresholds for prediction of bacteraemia were identified and the predictive ability of biomarkers in addition to routinely available clinical variables was assessed.

Results: The median age of included FN episodes was 6.0 years and eight (10%) had a bacteraemia. On presentation, elevated PCT, IL-10 and Mip1-beta were significantly associated with bacteraemia, while CRP, IL-6 and IL-8 were not. The combination of PCT (≥0.425 ng/ml) and IL-10 (≥4.37 pg/ml) had a sensitivity of 100% (95% CI 68.8-100%) and specificity of 89% (95% CI 80.0-95.0%) for prediction of bacteraemia, correctly identifying all eight bacteraemia episodes and classifying 16 FN episodes as high-risk. There was limited additive benefit of incorporating clinical variables to this model. On Day 2, there was an 11-fold increase in PCT in episodes with a bacteraemia which was significantly higher than that observed in the non-bacteraemia episodes.

Conclusion: Elevated PCT and IL-10 accurately identified all bacteraemia episodes in our FN cohort and may enhance the early risk stratification process in this population. Prospective validation and implementation is required to determine the impact on health service utilisation.
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http://dx.doi.org/10.3389/fimmu.2021.641879DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8173204PMC
May 2021

Extracellular Vesicles in Synovial Fluid from Rheumatoid Arthritis Patients Contain miRNAs with Capacity to Modulate Inflammation.

Int J Mol Sci 2021 May 6;22(9). Epub 2021 May 6.

The Walter and Eliza Hall Institute of Medical Research, Parkville 3052, Australia.

In rheumatoid arthritis (RA), extracellular vesicles (EVs) are associated with both the propagation and attenuation of joint inflammation and destruction. However, the specific EV content responsible for these processes is largely unknown. Investigations into identifying EV content are confounded by the challenges in obtaining high-quality EV preparations from synovial fluid. Implementing a size exclusion chromatography-based method of EV isolation, coupled with small RNA sequencing, we accurately characterised EV miRNAs in synovial fluid obtained from RA patients and investigated the differences between joints with high- and low-grade inflammation. Synovial fluid was obtained from the joints of 12 RA patients and, based on leukocyte counts, classified as either high ( = 7)- or low ( = 5)-grade inflammation. Using size exclusion chromatography, EVs were purified and small RNA was extracted and sequenced on a NextSeq 500. Sequencing reads were aligned to miRBase v21, and differences in miRNA profiles between RA patients with high- and low-grade joint inflammation were analysed. In total, 1972 distinct miRNAs were identified from RA synovial fluid EVs. miRNAs with less than five reads in fewer than five patients were filtered out, leaving 318 miRNAs for analysis. Analysis of the most abundant miRNAs suggested that they negatively regulate multiple genes relevant to inflammation, including signal transducer and activator of transcription 3 (STAT3), which lies downstream of IL-6 and has a pro-inflammatory role in RA. Synovial fluid from joints with high-grade inflammation contained 3.5-fold more EV miRNA per mL of synovial fluid ( = 0.0017). Seventy-eight EV miRNAs were differentially expressed between RA joints with high- and low-grade inflammation, and pathway analysis revealed that their target genes were commonly involved a variety of processes, including cellular apoptosis, proliferation and migration. Of the 49 miRNAs that were elevated in joints with high-grade inflammation, pathway analysis revealed that genes involved in cytokine-mediated signalling pathways were significantly enriched targets. In contrast, genes associated with reactive oxygen species signalling were significantly enriched as targets of the 29 miRNAs elevated in joints with low-grade inflammation. Our study identified an abundance of EV miRNAs from the synovial fluid of RA patients with the potential to modulate inflammation. In doing so, we defined potential mechanisms by which synovial fluid EVs may contribute to RA pathophysiology.
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http://dx.doi.org/10.3390/ijms22094910DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8125513PMC
May 2021

Targeting histone acetylation dynamics and oncogenic transcription by catalytic P300/CBP inhibition.

Mol Cell 2021 05;81(10):2183-2200.e13

The Walter and Eliza Hall Institute of Medical Research, Parkville, 3052, Australia; Department of Medical Biology, The University of Melbourne, Parkville, 3010, Australia.

To separate causal effects of histone acetylation on chromatin accessibility and transcriptional output, we used integrated epigenomic and transcriptomic analyses following acute inhibition of major cellular lysine acetyltransferases P300 and CBP in hematological malignancies. We found that catalytic P300/CBP inhibition dynamically perturbs steady-state acetylation kinetics and suppresses oncogenic transcriptional networks in the absence of changes to chromatin accessibility. CRISPR-Cas9 screening identified NCOR1 and HDAC3 transcriptional co-repressors as the principal antagonists of P300/CBP by counteracting acetylation turnover kinetics. Finally, deacetylation of H3K27 provides nucleation sites for reciprocal methylation switching, a feature that can be exploited therapeutically by concomitant KDM6A and P300/CBP inhibition. Overall, this study indicates that the steady-state histone acetylation-methylation equilibrium functions as a molecular rheostat governing cellular transcription that is amenable to therapeutic exploitation as an anti-cancer regimen.
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http://dx.doi.org/10.1016/j.molcel.2021.04.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8183601PMC
May 2021

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

EMBO J 2021 Jun 5;40(11):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
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8167363PMC
June 2021

Type 1 conventional dendritic cell fate and function are controlled by DC-SCRIPT.

Sci Immunol 2021 Apr;6(58)

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

The functional diversification of dendritic cells (DCs) is a key step in establishing protective immune responses. Despite the importance of DC lineage diversity, its genetic basis is not fully understood. The transcription factor DC-SCRIPT is expressed in conventional DCs (cDCs) and their committed bone marrow progenitors but not in plasmacytoid DCs (pDCs). We show that mice lacking DC-SCRIPT displayed substantially impaired development of IRF8 (interferon regulatory factor 8)-dependent cDC1, whereas cDC2 numbers increased marginally. The residual DC-SCRIPT-deficient cDC1s had impaired capacity to capture and present cell-associated antigens and to secrete IL-12p40, two functional hallmarks of this population. Genome-wide mapping of DC-SCRIPT binding and gene expression analyses revealed a key role for DC-SCRIPT in maintaining cDC1 identity via the direct regulation of cDC1 signature genes, including Our study reveals DC-SCRIPT to be a critical component of the gene regulatory program shaping the functional attributes of cDC1s.
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http://dx.doi.org/10.1126/sciimmunol.abf4432DOI Listing
April 2021

Chromosomes distribute randomly to, but not within, human neutrophil nuclear lobes.

iScience 2021 Mar 7;24(3):102161. Epub 2021 Feb 7.

The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, 3052, Australia.

The proximity pattern and radial distribution of chromosome territories within spherical nuclei are random and non-random, respectively. Whether this distribution pattern is conserved in the partitioned or lobed nuclei of polymorphonuclear cells is unclear. Here we use chromosome paint technology to examine the chromosome territories of all 46 chromosomes in hundreds of single human neutrophils - an abundant and famously polymorphonuclear immune cell. By comparing the distribution of chromosomes to randomly shuffled controls and validating with orthogonal chromosome conformation capture technology, we show for the first time that human chromosomes randomly distribute to neutrophil nuclear lobes, while maintaining a non-random radial distribution within these lobes. Furthermore, we demonstrate that chromosome length correlates with three-dimensional volume not only in neutrophils but other human immune cells. This work demonstrates that chromosomes are largely passive passengers during the neutrophil lobing process but are able to subsequently maintain their macro-level organization within lobes.
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http://dx.doi.org/10.1016/j.isci.2021.102161DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7905186PMC
March 2021

Pre-mitotic genome re-organisation bookends the B cell differentiation process.

Nat Commun 2021 02 26;12(1):1344. Epub 2021 Feb 26.

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

During cellular differentiation chromosome conformation is intricately remodelled to support the lineage-specific transcriptional programs required for initiating and maintaining lineage identity. When these changes occur in relation to cell cycle, division and time in response to cellular activation and differentiation signals has yet to be explored, although it has been proposed to occur during DNA synthesis or after mitosis. Here, we elucidate the chromosome conformational changes in B lymphocytes as they differentiate and expand from a naive, quiescent state into antibody secreting plasma cells. We find gene-regulatory chromosome reorganization in late G1 phase before the first division, and that this configuration is remarkably stable as the cells massively and rapidly clonally expand. A second wave of conformational change occurs as cells terminally differentiate into plasma cells, coincident with increased time in G1 phase. These results provide further explanation for how lymphocyte fate is imprinted prior to the first division. They also suggest that chromosome reconfiguration occurs prior to DNA replication and mitosis, and is linked to a gene expression program that controls the differentiation process required for the generation of immunity.
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http://dx.doi.org/10.1038/s41467-021-21536-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7910489PMC
February 2021

A guide to creating design matrices for gene expression experiments.

F1000Res 2020 10;9:1444. Epub 2020 Dec 10.

The Walter and Eliza Hall Institute of Medical Research, Parkville, 3052, Australia.

Differential expression analysis of genomic data types, such as RNA-sequencing experiments, use linear models to determine the size and direction of the changes in gene expression. For RNA-sequencing, there are several established software packages for this purpose accompanied with analysis pipelines that are well described. However, there are two crucial steps in the analysis process that can be a stumbling block for many -- the set up an appropriate model via design matrices and the set up of comparisons of interest via contrast matrices. These steps are particularly troublesome because an extensive catalogue for design and contrast matrices does not currently exist. One would usually search for example case studies across different platforms and mix and match the advice from those sources to suit the dataset they have at hand. This article guides the reader through the basics of how to set up design and contrast matrices. We take a practical approach by providing code and graphical representation of each case study, starting with simpler examples (e.g. models with a single explanatory variable) and move onto more complex ones (e.g. interaction models, mixed effects models, higher order time series and cyclical models). Although our work has been written specifically with a -style pipeline in mind, most of it is also applicable to other software packages for differential expression analysis, and the ideas covered can be adapted to data analysis of other high-throughput technologies. Where appropriate, we explain the interpretation and differences between models to aid readers in their own model choices. Unnecessary jargon and theory is omitted where possible so that our work is accessible to a wide audience of readers, from beginners to those with experience in genomics data analysis.
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http://dx.doi.org/10.12688/f1000research.27893.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7873980PMC
May 2021

Multi-level remodelling of chromatin underlying activation of human T cells.

Sci Rep 2021 Jan 12;11(1):528. Epub 2021 Jan 12.

The Walter and Eliza Hall Institute of Medical Research, Parkville, 3052, Australia.

Remodelling of chromatin architecture is known to regulate gene expression and has been well characterized in cell lineage development but less so in response to cell perturbation. Activation of T cells, which triggers extensive changes in transcriptional programs, serves as an instructive model to elucidate how changes in chromatin architecture orchestrate gene expression in response to cell perturbation. To characterize coordinate changes at different levels of chromatin architecture, we analyzed chromatin accessibility, chromosome conformation and gene expression in activated human T cells. T cell activation was characterized by widespread changes in chromatin accessibility and interactions that were shared between activated CD4 and CD8 T cells, and with the formation of active regulatory regions associated with transcription factors relevant to T cell biology. Chromatin interactions that increased and decreased were coupled, respectively, with up- and down-regulation of corresponding target genes. Furthermore, activation was associated with disruption of long-range chromatin interactions and with partitioning of topologically associating domains (TADs) and remodelling of their TAD boundaries. Newly formed/strengthened TAD boundaries were associated with higher nucleosome occupancy and lower accessibility, linking changes in lower and higher order chromatin architecture. T cell activation exemplifies coordinate multi-level remodelling of chromatin underlying gene transcription.
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http://dx.doi.org/10.1038/s41598-020-80165-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7804404PMC
January 2021

Identification and characterization of the long noncoding RNA Dreg1 as a novel regulator of Gata3.

Immunol Cell Biol 2021 Mar 17;99(3):323-332. Epub 2020 Oct 17.

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

The eukaryotic genome is three-dimensionally segregated into discrete globules of topologically associating domains (TADs), within which numerous cis-regulatory elements such as enhancers and promoters interact to regulate gene expression. In this study, we identify a T-cell-specific sub-TAD containing the Gata3 locus, and reveal a previously uncharacterized long noncoding RNA (Dreg1) within a distant enhancer lying approximately 280 kb downstream of Gata3. Dreg1 expression is highly correlated with that of Gata3 during early immune system development and T helper type 2 cell differentiation. Inhibition and overexpression of Dreg1 suggest that it may be involved in the establishment, but not in the maintenance of Gata3 expression. Overall, we propose that Dreg1 is a novel regulator of Gata3 and may inform therapeutic strategies in diseases such allergy and lymphoma, where Gata3 has a pathological role.
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http://dx.doi.org/10.1111/imcb.12408DOI Listing
March 2021

An Erg-driven transcriptional program controls B cell lymphopoiesis.

Nat Commun 2020 06 15;11(1):3013. Epub 2020 Jun 15.

Blood Cells and Blood Cancer Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, 3052, Australia.

B lymphoid development is initiated by the differentiation of hematopoietic stem cells into lineage committed progenitors, ultimately generating mature B cells. This highly regulated process generates clonal immunological diversity via recombination of immunoglobulin V, D and J gene segments. While several transcription factors that control B cell development and V(D)J recombination have been defined, how these processes are initiated and coordinated into a precise regulatory network remains poorly understood. Here, we show that the transcription factor ETS Related Gene (Erg) is essential for early B lymphoid differentiation. Erg initiates a transcriptional network involving the B cell lineage defining genes, Ebf1 and Pax5, which directly promotes expression of key genes involved in V(D)J recombination and formation of the B cell receptor. Complementation of Erg deficiency with a productively rearranged immunoglobulin gene rescued B lineage development, demonstrating that Erg is an essential and stage-specific regulator of the gene regulatory network controlling B lymphopoiesis.
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http://dx.doi.org/10.1038/s41467-020-16828-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7296042PMC
June 2020

Circulating Small Noncoding RNA Biomarkers of Response to Triple Disease-modifying Antirheumatic Drug Therapy in White Women With Early Rheumatoid Arthritis.

J Rheumatol 2020 12 15;47(12):1746-1751. Epub 2020 Jun 15.

I.P. Wicks, MB BS, PhD, The Walter and Eliza Hall Institute of Medical Research, and Department of Rheumatology, Royal Melbourne Hospital, Parkville, Australia.

Objective: To identify small noncoding RNA (sncRNA) serum biomarkers that predict response to triple disease-modifying antirheumatic drug (DMARD) therapy in patients with early rheumatoid arthritis (RA).

Methods: Early RA patients entered into a treat-to-target management algorithm, with triple DMARD therapy (methotrexate, sulfasalazine, hydroxychloroquine). Patients were assessed following 6 months of therapy and classified as European League Against Rheumatism responders or nonresponders. RNA was isolated from 42 archived serum samples, collected prior to commencement of triple DMARD therapy. Small RNA sequencing was performed and the reads mapped to annotations in a database of human sncRNA. Differential expression analysis was performed, comparing responders (n = 24) and nonresponders (n = 18).

Results: Pretreatment levels of 4 sncRNA were significantly increased in nonresponders: chr1. tRNA131-GlyCCC (4.1-fold, adjusted = 0.01), chr2.tRNA13-AlaCGC (2.2-fold, adjusted 0.02), U2-L166 (6.6-fold, adjusted 0.02), and piR-35982 (2.4-fold, adjusted 0.03). 5S-L612 was the only sncRNA significantly increased in responders (3.3-fold; adjusted 0.01). Reads for chr1. tRNA131-GlyCCC and chr2.tRNA13-AlaCGC mapped to the 5' end of each tRNA gene and were truncated at the anticodon loop, consistent with these sncRNA having roles as 5' translation interfering tRNA halves (tiRNA).

Conclusion: Pretreatment levels of specific serum sncRNA might facilitate identification of patients more likely to respond to triple DMARD therapy.
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http://dx.doi.org/10.3899/jrheum.191012DOI Listing
December 2020

Tissue-resident ductal macrophages survey the mammary epithelium and facilitate tissue remodelling.

Nat Cell Biol 2020 05 27;22(5):546-558. Epub 2020 Apr 27.

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

Macrophages are diverse immune cells that reside in all tissues. Although macrophages have been implicated in mammary-gland function, their diversity has not been fully addressed. By exploiting high-resolution three-dimensional imaging and flow cytometry, we identified a unique population of tissue-resident ductal macrophages that form a contiguous network between the luminal and basal layers of the epithelial tree throughout postnatal development. Ductal macrophages are long lived and constantly survey the epithelium through dendrite movement, revealed via advanced intravital imaging. Although initially originating from embryonic precursors, ductal macrophages derive from circulating monocytes as they expand during puberty. Moreover, they undergo proliferation in pregnancy to maintain complete coverage of the epithelium in lactation, when they are poised to phagocytose milk-producing cells post-lactation and facilitate remodelling. Interestingly, ductal macrophages strongly resemble mammary tumour macrophages and form a network that pervades the tumour. Thus, the mammary epithelium programs specialized resident macrophages in both physiological and tumorigenic contexts.
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http://dx.doi.org/10.1038/s41556-020-0505-0DOI Listing
May 2020

Targeting triple-negative breast cancers with the Smac-mimetic birinapant.

Cell Death Differ 2020 10 27;27(10):2768-2780. Epub 2020 Apr 27.

The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, 3052, Australia.

Smac mimetics target inhibitor of apoptosis (IAP) proteins, thereby suppressing their function to facilitate tumor cell death. Here we have evaluated the efficacy of the preclinical Smac-mimetic compound A and the clinical lead birinapant on breast cancer cells. Both exhibited potent in vitro activity in triple-negative breast cancer (TNBC) cells, including those from patient-derived xenograft (PDX) models. Birinapant was further studied using in vivo PDX models of TNBC and estrogen receptor-positive (ER) breast cancer. Birinapant exhibited single agent activity in all TNBC PDX models and augmented response to docetaxel, the latter through induction of TNF. Transcriptomic analysis of TCGA datasets revealed that genes encoding mediators of Smac-mimetic-induced cell death were expressed at higher levels in TNBC compared with ER breast cancer, resulting in a molecular signature associated with responsiveness to Smac mimetics. In addition, the cell death complex was preferentially formed in TNBCs versus ER cells in response to Smac mimetics. Taken together, our findings provide a rationale for prospectively selecting patients whose breast tumors contain a competent death receptor signaling pathway for the further evaluation of birinapant in the clinic.
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http://dx.doi.org/10.1038/s41418-020-0541-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7492458PMC
October 2020

Dual Targeting of CDK4/6 and BCL2 Pathways Augments Tumor Response in Estrogen Receptor-Positive Breast Cancer.

Clin Cancer Res 2020 08 3;26(15):4120-4134. Epub 2020 Apr 3.

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

Purpose: Although cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors significantly extend tumor response in patients with metastatic estrogen receptor-positive (ER) breast cancer, relapse is almost inevitable. This may, in part, reflect the failure of CDK4/6 inhibitors to induce apoptotic cell death. We therefore evaluated combination therapy with ABT-199 (venetoclax), a potent and selective BCL2 inhibitor.

Experimental Design: BCL2 family member expression was assessed following treatment with endocrine therapy and the CDK4/6 inhibitor palbociclib. Functional assays were used to determine the impact of adding ABT-199 to fulvestrant and palbociclib in ER breast cancer cell lines, patient-derived organoid (PDO), and patient-derived xenograft (PDX) models. A syngeneic ER mouse mammary tumor model was used to study the effect of combination therapy on the immune system.

Results: Triple therapy was well tolerated and produced a superior and more durable tumor response compared with single or doublet therapy. This was associated with marked apoptosis, including of senescent cells, indicative of senolysis. Unexpectedly, ABT-199 resulted in Rb dephosphorylation and reduced G-S cyclins, most notably at high doses, thereby intensifying the fulvestrant/palbociclib-induced cell-cycle arrest. Interestingly, a CRISPR/Cas9 screen suggested that ABT-199 could mitigate loss of Rb (and potentially other mechanisms of acquired resistance) to palbociclib. ABT-199 did not abrogate the favorable immunomodulatory effects of palbociclib in a syngeneic ER mammary tumor model and extended tumor response when combined with anti-PD1 therapy.

Conclusions: This study illustrates the potential for targeting BCL2 in combination with CDK4/6 inhibitors and supports investigation of combination therapy in ER breast cancer.
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http://dx.doi.org/10.1158/1078-0432.CCR-19-1872DOI Listing
August 2020

Germline heterozygous mutations in Nxf1 perturb RNA metabolism and trigger thrombocytopenia and lymphopenia in mice.

Blood Adv 2020 04;4(7):1270-1283

Anatomy and Developmental Biology, Monash Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia.

In eukaryotic cells, messenger RNA (mRNA) molecules are exported from the nucleus to the cytoplasm, where they are translated. The highly conserved protein nuclear RNA export factor1 (Nxf1) is an important mediator of this process. Although studies in yeast and in human cell lines have shed light on the biochemical mechanisms of Nxf1 function, its contribution to mammalian physiology is less clear. Several groups have identified recurrent NXF1 mutations in chronic lymphocytic leukemia (CLL), placing it alongside several RNA-metabolism factors (including SF3B1, XPO, RPS15) whose dysregulation is thought to contribute to CLL pathogenesis. We report here an allelic series of germline point mutations in murine Nxf1. Mice heterozygous for these loss-of-function Nxf1 mutations exhibit thrombocytopenia and lymphopenia, together with milder hematological defects. This is primarily caused by cell-intrinsic defects in the survival of platelets and peripheral lymphocytes, which are sensitized to intrinsic apoptosis. In contrast, Nxf1 mutations have almost no effect on red blood cell homeostasis. Comparative transcriptome analysis of platelets, lymphocytes, and erythrocytes from Nxf1-mutant mice shows that, in response to impaired Nxf1 function, the cytoplasmic representation of transcripts encoding regulators of RNA metabolism is altered in a unique, lineage-specific way. Thus, blood cell lineages exhibit differential requirements for Nxf1-mediated global mRNA export.
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http://dx.doi.org/10.1182/bloodadvances.2019001323DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7160253PMC
April 2020

Author Correction: The neuropeptide VIP confers anticipatory mucosal immunity by regulating ILC3 activity.

Nat Immunol 2020 Mar;21(3):354

Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria, Australia.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41590-020-0606-8DOI Listing
March 2020

qtQDA: quantile transformed quadratic discriminant analysis for high-dimensional RNA-seq data.

PeerJ 2019 18;7:e8260. Epub 2019 Dec 18.

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

Classification on the basis of gene expression data derived from RNA-seq promises to become an important part of modern medicine. We propose a new classification method based on a model where the data is marginally negative binomial but dependent, thereby incorporating the dependence known to be present between measurements from different genes. The method, called qtQDA, works by first performing a quantile transformation (qt) then applying Gaussian quadratic discriminant analysis (QDA) using regularized covariance matrix estimates. We show that qtQDA has excellent performance when applied to real data sets and has advantages over some existing approaches. An R package implementing the method is also available on https://github.com/goknurginer/qtQDA.
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http://dx.doi.org/10.7717/peerj.8260DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6967023PMC
December 2019

Attenuation of TCR-induced transcription by Bach2 controls regulatory T cell differentiation and homeostasis.

Nat Commun 2020 01 14;11(1):252. Epub 2020 Jan 14.

Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, The University of Melbourne, Melbourne, VIC, 3010, Australia.

Differentiation and homeostasis of Foxp3 regulatory T (Treg) cells are strictly controlled by T-cell receptor (TCR) signals; however, molecular mechanisms that govern these processes are incompletely understood. Here we show that Bach2 is an important regulator of Treg cell differentiation and homeostasis downstream of TCR signaling. Bach2 prevents premature differentiation of fully suppressive effector Treg (eTreg) cells, limits IL-10 production and is required for the development of peripherally induced Treg (pTreg) cells in the gastrointestinal tract. Bach2 attenuates TCR signaling-induced IRF4-dependent Treg cell differentiation. Deletion of IRF4 promotes inducible Treg cell differentiation and rescues pTreg cell differentiation in the absence of Bach2. In turn, loss of Bach2 normalizes eTreg cell differentiation of IRF4-deficient Treg cells. Mechanistically, Bach2 counteracts the DNA-binding activity of IRF4 and limits chromatin accessibility, thereby attenuating IRF4-dependent transcription. Thus, Bach2 balances TCR signaling induced transcriptional activity of IRF4 to maintain homeostasis of thymically-derived and peripherally-derived Treg cells.
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http://dx.doi.org/10.1038/s41467-019-14112-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6959360PMC
January 2020

The neuropeptide VIP confers anticipatory mucosal immunity by regulating ILC3 activity.

Nat Immunol 2020 02 23;21(2):168-177. Epub 2019 Dec 23.

Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria, Australia.

Group 3 innate lymphoid cell (ILC3)-mediated production of the cytokine interleukin-22 (IL-22) is critical for the maintenance of immune homeostasis in the gastrointestinal tract. Here, we find that the function of ILC3s is not constant across the day, but instead oscillates between active phases and resting phases. Coordinate responsiveness of ILC3s in the intestine depended on the food-induced expression of the neuropeptide vasoactive intestinal peptide (VIP). Intestinal ILC3s had high expression of the G protein-coupled receptor vasoactive intestinal peptide receptor 2 (VIPR2), and activation by VIP markedly enhanced the production of IL-22 and the barrier function of the epithelium. Conversely, deficiency in signaling through VIPR2 led to impaired production of IL-22 by ILC3s and increased susceptibility to inflammation-induced gut injury. Thus, intrinsic cellular rhythms acted in synergy with the cyclic patterns of food intake to drive the production of IL-22 and synchronize protection of the intestinal epithelium through a VIP-VIPR2 pathway in ILC3s.
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http://dx.doi.org/10.1038/s41590-019-0567-yDOI Listing
February 2020

HBO1 (KAT7) Does Not Have an Essential Role in Cell Proliferation, DNA Replication, or Histone 4 Acetylation in Human Cells.

Mol Cell Biol 2020 01 30;40(4). Epub 2020 Jan 30.

Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia

HBO1 (MYST2/KAT7) is essential for histone 3 lysine 14 acetylation (H3K14ac) but is dispensable for H4 acetylation and DNA replication in mouse tissues. In contrast, previous studies using small interfering RNA (siRNA) knockdown in human cell lines have suggested that HBO1 is essential for DNA replication. To determine if HBO1 has distinctly different roles in immortalized human cell lines and normal mouse cells, we performed siRNA knockdown of HBO1. In addition, we used CRISPR/Cas9 to generate 293T, MCF7, and HeLa cell lines lacking HBO1. Using both techniques, we show that HBO1 is essential for all H3K14ac in human cells and is unlikely to have a direct effect on H4 acetylation and only has minor effects on cell proliferation. Surprisingly, the loss of HBO1 and H3K14ac in HeLa cells led to the secondary loss of almost all H4 acetylation after 4 weeks. Thus, HBO1 is dispensable for DNA replication and cell proliferation in immortalized human cells. However, while cell proliferation proceeded without HBO1 and H3K14ac, gene deletion led to profound changes in cell adhesion, particularly in 293T cells. Consistent with this phenotype, the loss of HBO1 in both 293T and HeLa principally affected genes mediating cell adhesion, with comparatively minor effects on other cellular processes.
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http://dx.doi.org/10.1128/MCB.00506-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6996278PMC
January 2020

Differential co-expression-based detection of conditional relationships in transcriptional data: comparative analysis and application to breast cancer.

Genome Biol 2019 11 14;20(1):236. Epub 2019 Nov 14.

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

Background: Elucidation of regulatory networks, including identification of regulatory mechanisms specific to a given biological context, is a key aim in systems biology. This has motivated the move from co-expression to differential co-expression analysis and numerous methods have been developed subsequently to address this task; however, evaluation of methods and interpretation of the resulting networks has been hindered by the lack of known context-specific regulatory interactions.

Results: In this study, we develop a simulator based on dynamical systems modelling capable of simulating differential co-expression patterns. With the simulator and an evaluation framework, we benchmark and characterise the performance of inference methods. Defining three different levels of "true" networks for each simulation, we show that accurate inference of causation is difficult for all methods, compared to inference of associations. We show that a z-score-based method has the best general performance. Further, analysis of simulation parameters reveals five network and simulation properties that explained the performance of methods. The evaluation framework and inference methods used in this study are available in the dcanr R/Bioconductor package.

Conclusions: Our analysis of networks inferred from simulated data show that hub nodes are more likely to be differentially regulated targets than transcription factors. Based on this observation, we propose an interpretation of the inferred differential network that can reconstruct a putative causal network.
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http://dx.doi.org/10.1186/s13059-019-1851-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6857226PMC
November 2019

Author Correction: Increased autophagy in EphrinB2-deficient osteocytes is associated with elevated secondary mineralization and brittle bone.

Nat Commun 2019 Nov 4;10(1):5073. Epub 2019 Nov 4.

Bone Biology and Disease Unit, St. Vincent's Institute of Medical Research, 9 Princes Street, Fitzroy, Melbourne, VIC, 3065, Australia.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41467-019-13040-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6828656PMC
November 2019

Increased autophagy in EphrinB2-deficient osteocytes is associated with elevated secondary mineralization and brittle bone.

Nat Commun 2019 07 31;10(1):3436. Epub 2019 Jul 31.

Bone Biology and Disease Unit, St. Vincent's Institute of Medical Research, 9 Princes Street, Fitzroy, Melbourne, VIC, 3065, Australia.

Mineralized bone forms when collagen-containing osteoid accrues mineral crystals. This is initiated rapidly (primary mineralization), and continues slowly (secondary mineralization) until bone is remodeled. The interconnected osteocyte network within the bone matrix differentiates from bone-forming osteoblasts; although osteoblast differentiation requires EphrinB2, osteocytes retain its expression. Here we report brittle bones in mice with osteocyte-targeted EphrinB2 deletion. This is not caused by low bone mass, but by defective bone material. While osteoid mineralization is initiated at normal rate, mineral accrual is accelerated, indicating that EphrinB2 in osteocytes limits mineral accumulation. No known regulators of mineralization are modified in the brittle cortical bone but a cluster of autophagy-associated genes are dysregulated. EphrinB2-deficient osteocytes displayed more autophagosomes in vivo and in vitro, and EphrinB2-Fc treatment suppresses autophagy in a RhoA-ROCK dependent manner. We conclude that secondary mineralization involves EphrinB2-RhoA-limited autophagy in osteocytes, and disruption leads to a bone fragility independent of bone mass.
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http://dx.doi.org/10.1038/s41467-019-11373-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6668467PMC
July 2019

MOZ directs the distal-less homeobox gene expression program during craniofacial development.

Development 2019 07 24;146(14). Epub 2019 Jul 24.

Walter and Eliza Hall Institute of Medical Research, Melbourne, Parkville, VIC 3052, Australia

Oral clefts are common birth defects. Individuals with oral clefts who have identical genetic mutations regularly present with variable penetrance and severity. Epigenetic or chromatin-mediated mechanisms are commonly invoked to explain variable penetrance. However, specific examples of these are rare. Two functional copies of the (, ) gene, encoding a MYST family lysine acetyltransferase chromatin regulator, are essential for human craniofacial development, but the molecular role of MOZ in this context is unclear. Using genetic interaction and genomic studies, we have investigated the effects of loss of MOZ on the gene expression program during mouse development. Among the more than 500 genes differentially expressed after loss of MOZ, 19 genes had previously been associated with cleft palates. These included four distal-less homeobox (DLX) transcription factor-encoding genes, , , and and DLX target genes (including , , and ). MOZ occupied the locus and was required for normal levels of histone H3 lysine 9 acetylation. MOZ affected Dlx gene expression cell-autonomously within neural crest cells. Our study identifies a specific program by which the chromatin modifier MOZ regulates craniofacial development.
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http://dx.doi.org/10.1242/dev.175042DOI Listing
July 2019

The Selective Expansion and Targeted Accumulation of Bone Marrow-Derived Macrophages Drive Cardiac Vasculitis.

J Immunol 2019 06 19;202(11):3282-3296. Epub 2019 Apr 19.

The Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria 3052, Australia;

The adult heart contains macrophages derived from both embryonic and adult bone marrow (BM)-derived precursors. This population diversity prompted us to explore how distinct macrophage subsets localize within the heart, and their relative contributions in cardiac disease. In this study, using the reciprocal expression of Lyve-1 and Ccr2 to distinguish macrophages with distinct origins, we show that, in the steady state, both embryonic (Lyve) and BM-derived (Ccr2) macrophages populate the major vessels of the heart in mice and humans. However, cardiac macrophage populations are markedly perturbed by inflammation. In a mouse model of Kawasaki disease, BM-derived macrophages preferentially increase during acute cardiac inflammation and selectively accumulate around major cardiac vessels. The accumulation of BM-derived macrophages coincides with the loss of their embryonic counterparts and is an initiating, essential step in the emergence of subsequent cardiac vasculitis in this experimental model. Finally, we demonstrate that the accumulation of Ccr2 macrophages (and the development of vasculitis) occurs in close proximity to a population of Ccr2 chemokine ligand-producing epicardial cells, suggesting that the epicardium may be involved in localizing inflammation to cardiac vessels. Collectively, our findings identify the perivascular accumulation of BM-derived macrophages as pivotal in the pathogenesis of cardiac vasculitis and provide evidence about the mechanisms governing their recruitment to the heart.
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http://dx.doi.org/10.4049/jimmunol.1900071DOI Listing
June 2019

Loss of p53 Causes Stochastic Aberrant X-Chromosome Inactivation and Female-Specific Neural Tube Defects.

Cell Rep 2019 04;27(2):442-454.e5

Walter and Eliza Hall Institute of Medical Research, Melbourne, VIC 3052, Australia; Department of Medical Biology, The University of Melbourne, Melbourne, VIC, Australia. Electronic address:

Neural tube defects (NTDs) are common birth defects in humans and show an unexplained female bias. Female mice lacking the tumor suppressor p53 display NTDs with incomplete penetrance. We found that the combined loss of pro-apoptotic BIM and p53 caused 100% penetrant, female-exclusive NTDs, which allowed us to investigate the female-specific functions of p53. We report that female p53 embryonic neural tube samples show fewer cells with inactive X chromosome markers Xist and H3K27me3 and a concomitant increase in biallelic expression of the X-linked genes, Huwe1 and Usp9x. Decreased Xist and increased X-linked gene expression was confirmed by RNA sequencing. Moreover, we found that p53 directly bound response elements in the X chromosome inactivation center (XIC). Together, these findings suggest p53 directly activates XIC genes, without which there is stochastic failure in X chromosome inactivation, and that X chromosome inactivation failure may underlie the female bias in neural tube closure defects.
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http://dx.doi.org/10.1016/j.celrep.2019.03.048DOI Listing
April 2019

Intraclonal Plasticity in Mammary Tumors Revealed through Large-Scale Single-Cell Resolution 3D Imaging.

Cancer Cell 2019 04 28;35(4):618-632.e6. Epub 2019 Mar 28.

Stem Cells and Cancer Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia; Department of Medical Biology, The University of Melbourne, Parkville, VIC 3010, Australia. Electronic address:

Breast tumors are inherently heterogeneous, but the evolving cellular organization through neoplastic progression is poorly understood. Here we report a rapid, large-scale single-cell resolution 3D imaging protocol based on a one-step clearing agent that allows visualization of normal tissue architecture and entire tumors at cellular resolution. Imaging of multicolor lineage-tracing models of breast cancer targeted to either basal or luminal progenitor cells revealed profound clonal restriction during progression. Expression profiling of clones arising in Pten/Trp53-deficient tumors identified distinct molecular signatures. Strikingly, most clones harbored cells that had undergone an epithelial-to-mesenchymal transition, indicating widespread, inherent plasticity. Hence, an integrative pipeline that combines lineage tracing, 3D imaging, and clonal RNA sequencing technologies offers a comprehensive path for studying mechanisms underlying heterogeneity in whole tumors.
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http://dx.doi.org/10.1016/j.ccell.2019.02.010DOI Listing
April 2019