Publications by authors named "Melissa J Davis"

75 Publications

Metastasis-entrained eosinophils enhance lymphocyte-mediated anti-tumor immunity.

Cancer Res 2021 Aug 24. Epub 2021 Aug 24.

Clinical Microbiology and Immunology, Tel Aviv University

The recognition of the immune system as a key component of the tumor microenvironment (TME) led to promising therapeutics. Since such therapies benefit only subsets of patients, understanding the activities of immune cells in the TME is required. Eosinophils are an integral part of the TME especially in mucosal tumors. Nonetheless, their role in the TME and the environmental cues that direct their activities are largely unknown. We report that breast cancer lung metastasis are characterized by resident and recruited eosinophils. Eosinophil recruitment to the metastatic sites in the lung was regulated by G protein-coupled receptor signaling but independent of CCR3. Functionally, eosinophils promoted lymphocyte-mediated anti-tumor immunity. Transcriptome and proteomic analyses identified the TME rather than intrinsic differences between eosinophil subsets as a key instructing factor directing anti-tumorigenic eosinophil activities. Specifically, TNF-alpha/IFN-gamma-activated eosinophils facilitated CD4+ and CD8+ T cell infiltration and promoted anti-tumor immunity. Collectively, we identify a mechanism by which the TME trains eosinophils to adopt anti-tumorigenic properties, which may lead to the development of eosinophil-targeted therapeutics.
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http://dx.doi.org/10.1158/0008-5472.CAN-21-0839DOI Listing
August 2021

SOX9 Defines Distinct Populations of Cells in SHH Medulloblastoma but Is Not Required for Math1-Driven Tumor Formation.

Mol Cancer Res 2021 Jul 30. Epub 2021 Jul 30.

The University of Queensland Diamantina Institute, University of Queensland, Woolloongabba, Queensland, Australia.

Medulloblastoma is the most common malignant pediatric brain tumor and there is an urgent need for molecularly targeted and subgroup-specific therapies. The stem cell factor SOX9, has been proposed as a potential therapeutic target for the treatment of Sonic Hedgehog medulloblastoma (SHH-MB) subgroup tumors, given its role as a downstream target of Hedgehog signaling and in functionally promoting SHH-MB metastasis and treatment resistance. However, the functional requirement for SOX9 in the genesis of medulloblastoma remains to be determined. Here we report a previously undocumented level of SOX9 expression exclusively in proliferating granule cell precursors (GCP) of the postnatal mouse cerebellum, which function as the medulloblastoma-initiating cells of SHH-MBs. Wild-type GCPs express comparatively lower levels of SOX9 than neural stem cells and mature astroglia and SOX9 GCP-like tumor cells constitute the bulk of both infant (Math1Cre: ) and adult ( ) SHH-MB mouse models. Human medulloblastoma single-cell RNA data analyses reveal three distinct populations present in SHH-MB and noticeably absent in other medulloblastoma subgroups: (GCP), (astrocytes) and (potential tumor-derived astrocytes). To functionally address whether SOX9 is required as a downstream effector of Hedgehog signaling in medulloblastoma tumor cells, we ablated using a Math1Cre model system. Surprisingly, targeted ablation of in GCPs (Math1Cre: ) revealed no overt phenotype and loss of in SHH-MB (Math1Cre: ) does not affect tumor formation. IMPLICATIONS: Despite preclinical data indicating SOX9 plays a key role in SHH-MB biology, our data argue against SOX9 as a viable therapeutic target.
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http://dx.doi.org/10.1158/1541-7786.MCR-21-0117DOI Listing
July 2021

The site of breast cancer metastases dictates their clonal composition and reversible transcriptomic profile.

Sci Adv 2021 Jul 7;7(28). Epub 2021 Jul 7.

Olivia Newton-John Cancer Research Institute, Heidelberg, VIC 3084, Australia.

Intratumoral heterogeneity is a driver of breast cancer progression, but the nature of the clonal interactive network involved in this process remains unclear. Here, we optimized the use of optical barcoding to visualize and characterize 31 cancer subclones in vivo. By mapping the clonal composition of thousands of metastases in two clinically relevant sites, the lungs and liver, we found that metastases were highly polyclonal in lungs but not in the liver. Furthermore, the transcriptome of the subclones varied according to their metastatic niche. We also identified a reversible niche-driven signature that was conserved in lung and liver metastases collected during patient autopsies. Among this signature, we found that the tumor necrosis factor-α pathway was up-regulated in lung compared to liver metastases, and inhibition of this pathway affected metastasis diversity. These results highlight that the cellular and molecular heterogeneity observed in metastases is largely dictated by the tumor microenvironment.
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http://dx.doi.org/10.1126/sciadv.abf4408DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8262813PMC
July 2021

Systems pharmacogenomics identifies novel targets and clinically actionable therapeutics for medulloblastoma.

Genome Med 2021 Jun 21;13(1):103. Epub 2021 Jun 21.

The University of Queensland Diamantina Institute, The University of Queensland, Woolloongabba, QLD, 4102, Australia.

Background: Medulloblastoma (MB) is the most common malignant paediatric brain tumour and a leading cause of cancer-related mortality and morbidity. Existing treatment protocols are aggressive in nature resulting in significant neurological, intellectual and physical disabilities for the children undergoing treatment. Thus, there is an urgent need for improved, targeted therapies that minimize these harmful side effects.

Methods: We identified candidate drugs for MB using a network-based systems-pharmacogenomics approach: based on results from a functional genomics screen, we identified a network of interactions implicated in human MB growth regulation. We then integrated drugs and their known mechanisms of action, along with gene expression data from a large collection of medulloblastoma patients to identify drugs with potential to treat MB.

Results: Our analyses identified drugs targeting CDK4, CDK6 and AURKA as strong candidates for MB; all of these genes are well validated as drug targets in other tumour types. We also identified non-WNT MB as a novel indication for drugs targeting TUBB, CAD, SNRPA, SLC1A5, PTPRS, P4HB and CHEK2. Based upon these analyses, we subsequently demonstrated that one of these drugs, the new microtubule stabilizing agent, ixabepilone, blocked tumour growth in vivo in mice bearing patient-derived xenograft tumours of the Sonic Hedgehog and Group 3 subtype, providing the first demonstration of its efficacy in MB.

Conclusions: Our findings confirm that this data-driven systems pharmacogenomics strategy is a powerful approach for the discovery and validation of novel therapeutic candidates relevant to MB treatment, and along with data validating ixabepilone in PDX models of the two most aggressive subtypes of medulloblastoma, we present the network analysis framework as a resource for the field.
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http://dx.doi.org/10.1186/s13073-021-00920-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8215804PMC
June 2021

Measuring pathway database coverage of the phosphoproteome.

PeerJ 2021 25;9:e11298. Epub 2021 May 25.

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

Protein phosphorylation is one of the best known post-translational mechanisms playing a key role in the regulation of cellular processes. Over 100,000 distinct phosphorylation sites have been discovered through constant improvement of mass spectrometry based phosphoproteomics in the last decade. However, data saturation is occurring and the bottleneck of assigning biologically relevant functionality to phosphosites needs to be addressed. There has been finite success in using data-driven approaches to reveal phosphosite functionality due to a range of limitations. The alternate, more suitable approach is making use of prior knowledge from literature-derived databases. Here, we analysed seven widely used databases to shed light on their suitability to provide functional insights into phosphoproteomics data. We first determined the global coverage of each database at both the protein and phosphosite level. We also determined how consistent each database was in its phosphorylation annotations compared to a global standard. Finally, we looked in detail at the coverage of each database over six experimental datasets. Our analysis highlights the relative strengths and weaknesses of each database, providing a guide in how each can be best used to identify biological mechanisms in phosphoproteomic data.
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http://dx.doi.org/10.7717/peerj.11298DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8162239PMC
May 2021

Blockade of the co-inhibitory molecule PD-1 unleashes ILC2-dependent antitumor immunity in melanoma.

Nat Immunol 2021 07 7;22(7):851-864. Epub 2021 Jun 7.

Innate Pharma Research Labs, Marseille, France.

Group 2 innate lymphoid cells (ILC2s) are essential to maintain tissue homeostasis. In cancer, ILC2s can harbor both pro-tumorigenic and anti-tumorigenic functions, but we know little about their underlying mechanisms or whether they could be clinically relevant or targeted to improve patient outcomes. Here, we found that high ILC2 infiltration in human melanoma was associated with a good clinical prognosis. ILC2s are critical producers of the cytokine granulocyte-macrophage colony-stimulating factor, which coordinates the recruitment and activation of eosinophils to enhance antitumor responses. Tumor-infiltrating ILC2s expressed programmed cell death protein-1, which limited their intratumoral accumulation, proliferation and antitumor effector functions. This inhibition could be overcome in vivo by combining interleukin-33-driven ILC2 activation with programmed cell death protein-1 blockade to significantly increase antitumor responses. Together, our results identified ILC2s as a critical immune cell type involved in melanoma immunity and revealed a potential synergistic approach to harness ILC2 function for antitumor immunotherapies.
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http://dx.doi.org/10.1038/s41590-021-00943-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7611091PMC
July 2021

Elp2 mutations perturb the epitranscriptome and lead to a complex neurodevelopmental phenotype.

Nat Commun 2021 05 11;12(1):2678. Epub 2021 May 11.

Queensland Brain Institute, The University of Queensland, Brisbane, QLD, Australia.

Intellectual disability (ID) and autism spectrum disorder (ASD) are the most common neurodevelopmental disorders and are characterized by substantial impairment in intellectual and adaptive functioning, with their genetic and molecular basis remaining largely unknown. Here, we identify biallelic variants in the gene encoding one of the Elongator complex subunits, ELP2, in patients with ID and ASD. Modelling the variants in mice recapitulates the patient features, with brain imaging and tractography analysis revealing microcephaly, loss of white matter tract integrity and an aberrant functional connectome. We show that the Elp2 mutations negatively impact the activity of the complex and its function in translation via tRNA modification. Further, we elucidate that the mutations perturb protein homeostasis leading to impaired neurogenesis, myelin loss and neurodegeneration. Collectively, our data demonstrate an unexpected role for tRNA modification in the pathogenesis of monogenic ID and ASD and define Elp2 as a key regulator of brain development.
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http://dx.doi.org/10.1038/s41467-021-22888-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8113450PMC
May 2021

Measuring and Modelling the Epithelial- Mesenchymal Hybrid State in Cancer: Clinical Implications.

Cells Tissues Organs 2021 Apr 26:1-24. Epub 2021 Apr 26.

Queensland University of Technology, Institute of Health and Biomedical Innovation and School of Biomedical Sciences, Brisbane, Queensland, Australia.

The epithelial-mesenchymal (E/M) hybrid state has emerged as an important mediator of elements of cancer progression, facilitated by epithelial mesenchymal plasticity (EMP). We review here evidence for the presence, prognostic significance, and therapeutic potential of the E/M hybrid state in carcinoma. We further assess modelling predictions and validation studies to demonstrate stabilised E/M hybrid states along the spectrum of EMP, as well as computational approaches for characterising and quantifying EMP phenotypes, with particular attention to the emerging realm of single-cell approaches through RNA sequencing and protein-based techniques.
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http://dx.doi.org/10.1159/000515289DOI Listing
April 2021

PRMT1-mediated H4R3me2a recruits SMARCA4 to promote colorectal cancer progression by enhancing EGFR signaling.

Genome Med 2021 04 14;13(1):58. Epub 2021 Apr 14.

The State Key Laboratory of Pharmaceutical Biotechnology, Department of Hematology, the Affiliated Drum Tower Hospital of Nanjing University Medical School, China-Australia Institute of Translational Medicine, School of Life Sciences, Nanjing University, 163 Xianlin Avenue, Nanjing, 210023, China.

Background: Aberrant changes in epigenetic mechanisms such as histone modifications play an important role in cancer progression. PRMT1 which triggers asymmetric dimethylation of histone H4 on arginine 3 (H4R3me2a) is upregulated in human colorectal cancer (CRC) and is essential for cell proliferation. However, how this dysregulated modification might contribute to malignant transitions of CRC remains poorly understood.

Methods: In this study, we integrated biochemical assays including protein interaction studies and chromatin immunoprecipitation (ChIP), cellular analysis including cell viability, proliferation, colony formation, and migration assays, clinical sample analysis, microarray experiments, and ChIP-Seq data to investigate the potential genomic recognition pattern of H4R3me2s in CRC cells and its effect on CRC progression.

Results: We show that PRMT1 and SMARCA4, an ATPase subunit of the SWI/SNF chromatin remodeling complex, act cooperatively to promote colorectal cancer (CRC) progression. We find that SMARCA4 is a novel effector molecule of PRMT1-mediated H4R3me2a. Mechanistically, we show that H4R3me2a directly recruited SMARCA4 to promote the proliferative, colony-formative, and migratory abilities of CRC cells by enhancing EGFR signaling. We found that EGFR and TNS4 were major direct downstream transcriptional targets of PRMT1 and SMARCA4 in colon cells, and acted in a PRMT1 methyltransferase activity-dependent manner to promote CRC cell proliferation. In vivo, knockdown or inhibition of PRMT1 profoundly attenuated the growth of CRC cells in the C57BL/6 J-Apc CRC mice model. Importantly, elevated expression of PRMT1 or SMARCA4 in CRC patients were positively correlated with expression of EGFR and TNS4, and CRC patients had shorter overall survival. These findings reveal a critical interplay between epigenetic and transcriptional control during CRC progression, suggesting that SMARCA4 is a novel key epigenetic modulator of CRC. Our findings thus highlight PRMT1/SMARCA4 inhibition as a potential therapeutic intervention strategy for CRC.

Conclusion: PRMT1-mediated H4R3me2a recruits SMARCA4, which promotes colorectal cancer progression by enhancing EGFR signaling.
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http://dx.doi.org/10.1186/s13073-021-00871-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8048298PMC
April 2021

Type 2 Innate Lymphoid Cells Protect against Colorectal Cancer Progression and Predict Improved Patient Survival.

Cancers (Basel) 2021 Feb 1;13(3). Epub 2021 Feb 1.

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

Chronic inflammation of the gastrointestinal (GI) tract contributes to colorectal cancer (CRC) progression. While the role of adaptive T cells in CRC is now well established, the role of innate immune cells, specifically innate lymphoid cells (ILCs), is not well understood. To define the role of ILCs in CRC we employed complementary heterotopic and chemically-induced CRC mouse models. We discovered that ILCs were abundant in CRC tumours and contributed to anti-tumour immunity. We focused on ILC2 and showed that ILC2-deficient mice developed a higher tumour burden compared with littermate wild-type controls. We generated an ILC2 gene signature and using machine learning models revealed that CRC patients with a high intratumor ILC2 gene signature had a favourable clinical prognosis. Collectively, our results highlight a critical role for ILC2 in CRC, suggesting a potential new avenue to improve clinical outcomes through ILC2-agonist based therapeutic approaches.
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http://dx.doi.org/10.3390/cancers13030559DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7867134PMC
February 2021

Hhex Directly Represses BIM-Dependent Apoptosis to Promote NK Cell Development and Maintenance.

Cell Rep 2020 10;33(3):108285

The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, 3052, Australia; Department of Medical Biology, Faculty of Medicine, Dentistry and Health Sciences, The University of Melbourne, Melbourne, Victoria, 3010, Australia; Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria, 3800, Australia; oNKo-Innate Pty Ltd., 27 Norwood Cres, Moonee Ponds, Victoria, 3039, Australia. Electronic address:

Hhex encodes a homeobox transcriptional regulator important for embryonic development and hematopoiesis. Hhex is highly expressed in NK cells, and its germline deletion results in significant defects in lymphoid development, including NK cells. To determine if Hhex is intrinsically required throughout NK cell development or for NK cell function, we generate mice that specifically lack Hhex in NK cells. NK cell frequency is dramatically reduced, while NK cell differentiation, IL-15 responsiveness, and function at the cellular level remain largely normal in the absence of Hhex. Increased IL-15 availability fails to fully reverse NK lymphopenia following conditional Hhex deletion, suggesting that Hhex regulates developmental pathways extrinsic to those dependent on IL-15. Gene expression and functional genetic approaches reveal that Hhex regulates NK cell survival by directly binding Bcl2l11 (Bim) and repressing expression of this key apoptotic mediator. These data implicate Hhex as a transcriptional regulator of NK cell homeostasis and immunity.
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http://dx.doi.org/10.1016/j.celrep.2020.108285DOI Listing
October 2020

Stable gene expression for normalisation and single-sample scoring.

Nucleic Acids Res 2020 11;48(19):e113

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

Gene expression signatures have been critical in defining the molecular phenotypes of cells, tissues, and patient samples. Their most notable and widespread clinical application is stratification of breast cancer patients into molecular (PAM50) subtypes. The cost and relatively large amounts of fresh starting material required for whole-transcriptome sequencing has limited clinical application of thousands of existing gene signatures captured in repositories such as the Molecular Signature Database. We identified genes with stable expression across a range of abundances, and with a preserved relative ordering across thousands of samples, allowing signature scoring and supporting general data normalisation for transcriptomic data. Our new method, stingscore, quantifies and summarises relative expression levels of signature genes from individual samples through the inclusion of these 'stably-expressed genes'. We show that our list of stable genes has better stability across cancer and normal tissue data than previously proposed gene sets. Additionally, we show that signature scores computed from targeted transcript measurements using stingscore can predict docetaxel response in breast cancer patients. This new approach to gene expression signature analysis will facilitate the development of panel-type tests for gene expression signatures, thus supporting clinical translation of the powerful insights gained from cancer transcriptomic studies.
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http://dx.doi.org/10.1093/nar/gkaa802DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7641762PMC
November 2020

Eukaryote-Conserved Methylarginine Is Absent in Diplomonads and Functionally Compensated in Giardia.

Mol Biol Evol 2020 12;37(12):3525-3549

Population Health and Immunity Division, The Walter and Eliza Hall Institute of Medical Research, Melbourne, VIC, Australia.

Methylation is a common posttranslational modification of arginine and lysine in eukaryotic proteins. Methylproteomes are best characterized for higher eukaryotes, where they are functionally expanded and evolved complex regulation. However, this is not the case for protist species evolved from the earliest eukaryotic lineages. Here, we integrated bioinformatic, proteomic, and drug-screening data sets to comprehensively explore the methylproteome of Giardia duodenalis-a deeply branching parasitic protist. We demonstrate that Giardia and related diplomonads lack arginine-methyltransferases and have remodeled conserved RGG/RG motifs targeted by these enzymes. We also provide experimental evidence for methylarginine absence in proteomes of Giardia but readily detect methyllysine. We bioinformatically infer 11 lysine-methyltransferases in Giardia, including highly diverged Su(var)3-9, Enhancer-of-zeste and Trithorax proteins with reduced domain architectures, and novel annotations demonstrating conserved methyllysine regulation of eukaryotic elongation factor 1 alpha. Using mass spectrometry, we identify more than 200 methyllysine sites in Giardia, including in species-specific gene families involved in cytoskeletal regulation, enriched in coiled-coil features. Finally, we use known methylation inhibitors to show that methylation plays key roles in replication and cyst formation in this parasite. This study highlights reduced methylation enzymes, sites, and functions early in eukaryote evolution, including absent methylarginine networks in the Diplomonadida. These results challenge the view that arginine methylation is eukaryote conserved and demonstrate that functional compensation of methylarginine was possible preceding expansion and diversification of these key networks in higher eukaryotes.
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http://dx.doi.org/10.1093/molbev/msaa186DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7743719PMC
December 2020

Cotargeting BCL-2 and MCL-1 in high-risk B-ALL.

Blood Adv 2020 06;4(12):2762-2767

Centre for Cancer Research, University of Melbourne, Parkville, Melbourne, Australia.

Improving survival outcomes in adult B-cell acute lymphoblastic leukemia (B-ALL) remains a clinical challenge. Relapsed disease has a poor prognosis despite the use of tyrosine kinase inhibitors (TKIs) for Philadelphia chromosome positive (Ph+ ALL) cases and immunotherapeutic approaches, including blinatumomab and chimeric antigen receptor T cells. Targeting aberrant cell survival pathways with selective small molecule BH3-mimetic inhibitors of BCL-2 (venetoclax, S55746), BCL-XL (A1331852), or MCL1 (S63845) is an emerging therapeutic option. We report that combined targeting of BCL-2 and MCL1 is synergistic in B-ALL in vitro. The combination demonstrated greater efficacy than standard chemotherapeutics and TKIs in primary samples from adult B-ALL with Ph+ ALL, Ph-like ALL, and other B-ALL. Moreover, combined BCL-2 or MCL1 inhibition with dasatinib showed potent killing in primary Ph+ B-ALL cases, but the BH3-mimetic combination appeared superior in vitro in a variety of Ph-like ALL samples. In PDX models, combined BCL-2 and MCL1 targeting eradicated ALL from Ph- and Ph+ B-ALL cases, although fatal tumor lysis was observed in some instances of high tumor burden. We conclude that a dual BH3-mimetic approach is highly effective in diverse models of high-risk human B-ALL and warrants assessment in clinical trials that incorporate tumor lysis precautions.
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http://dx.doi.org/10.1182/bloodadvances.2019001416DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7322969PMC
June 2020

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

Harnessing Natural Killer Immunity in Metastatic SCLC.

J Thorac Oncol 2020 09 26;15(9):1507-1521. Epub 2020 May 26.

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

Introduction: SCLC is the most aggressive subtype of lung cancer, and though most patients initially respond to platinum-based chemotherapy, resistance develops rapidly. Immunotherapy holds promise in the treatment of lung cancer; however, patients with SCLC exhibit poor overall responses highlighting the necessity for alternative approaches. Natural killer (NK) cells are an alternative to T cell-based immunotherapies that do not require sensitization to antigens presented on the surface of tumor cells.

Methods: We investigated the immunophenotype of human SCLC tumors by both flow cytometry on fresh samples and bioinformatic analysis. Cell lines generated from murine SCLC were transplanted into mice lacking key cytotoxic immune cells. Subcutaneous tumor growth, metastatic dissemination, and activation of CD8 T and NK cells were evaluated by histology and flow cytometry.

Results: Transcriptomic analysis of human SCLC tumors revealed heterogeneous immune checkpoint and cytotoxic signature profiles. Using sophisticated, genetically engineered mouse models, we reported that the absence of NK cells, but not CD8 T cells, substantially enhanced metastatic dissemination of SCLC tumor cells in vivo. Moreover, hyperactivation of NK cell activity through augmentation of interleukin-15 or transforming growth factor-β signaling pathways ameliorated SCLC metastases, an effect that was enhanced when combined with antiprogrammed cell death-1 therapy.

Conclusions: These proof-of-principle findings provide a rationale for exploiting the antitumor functions of NK cells in the treatment of patients with SCLC. Moreover, the distinct immune profiles of SCLC subtypes reveal an unappreciated level of heterogeneity that warrants further investigation in the stratification of patients for immunotherapy.
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http://dx.doi.org/10.1016/j.jtho.2020.05.008DOI Listing
September 2020

Snail induces epithelial cell extrusion by regulating RhoA contractile signalling and cell-matrix adhesion.

J Cell Sci 2020 07 6;133(13). Epub 2020 Jul 6.

Division of Cell and Developmental Biology, Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Brisbane, Queensland 4072, Australia

Cell extrusion is a morphogenetic process that is implicated in epithelial homeostasis and elicited by stimuli ranging from apoptosis to oncogenic transformation. To explore whether the morphogenetic transcription factor Snail (SNAI1) induces extrusion, we inducibly expressed a stabilized Snail transgene in confluent MCF-7 monolayers. When expressed in small clusters (less than three cells) within otherwise wild-type confluent monolayers, Snail expression induced apical cell extrusion. In contrast, larger clusters or homogenous cultures of Snail cells did not show enhanced apical extrusion, but eventually displayed sporadic basal delamination. Transcriptomic profiling revealed that Snail did not substantively alter the balance of epithelial and mesenchymal genes. However, we identified a transcriptional network that led to upregulated RhoA signalling and cortical contractility in cells expressing Snail Enhanced contractility was necessary, but not sufficient, to drive extrusion, suggesting that Snail collaborates with other factors. Indeed, we found that the transcriptional downregulation of cell-matrix adhesion cooperates with contractility to mediate basal delamination. This provides a pathway for Snail to influence epithelial morphogenesis independently of classic epithelial-to-mesenchymal transition.
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http://dx.doi.org/10.1242/jcs.235622DOI Listing
July 2020

The EMT modulator SNAI1 contributes to AML pathogenesis via its interaction with LSD1.

Blood 2020 08;136(8):957-973

Australian Centre for Blood Diseases, Monash University, Melbourne, VIC, Australia.

Modulators of epithelial-to-mesenchymal transition (EMT) have recently emerged as novel players in the field of leukemia biology. The mechanisms by which EMT modulators contribute to leukemia pathogenesis, however, remain to be elucidated. Here we show that overexpression of SNAI1, a key modulator of EMT, is a pathologically relevant event in human acute myeloid leukemia (AML) that contributes to impaired differentiation, enhanced self-renewal, and proliferation of immature myeloid cells. We demonstrate that ectopic expression of Snai1 in hematopoietic cells predisposes mice to AML development. This effect is mediated by interaction with the histone demethylase KDM1A/LSD1. Our data shed new light on the role of SNAI1 in leukemia development and identify a novel mechanism of LSD1 corruption in cancer. This is particularly pertinent given the current interest surrounding the use of LSD1 inhibitors in the treatment of multiple different malignancies, including AML.
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http://dx.doi.org/10.1182/blood.2019002548DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7441169PMC
August 2020

NK cell-derived GM-CSF potentiates inflammatory arthritis and is negatively regulated by CIS.

J Exp Med 2020 05;217(5)

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

Despite increasing recognition of the importance of GM-CSF in autoimmune disease, it remains unclear how GM-CSF is regulated at sites of tissue inflammation. Using GM-CSF fate reporter mice, we show that synovial NK cells produce GM-CSF in autoantibody-mediated inflammatory arthritis. Synovial NK cells promote a neutrophilic inflammatory cell infiltrate, and persistent arthritis, via GM-CSF production, as deletion of NK cells, or specific ablation of GM-CSF production in NK cells, abrogated disease. Synovial NK cell production of GM-CSF is IL-18-dependent. Furthermore, we show that cytokine-inducible SH2-containing protein (CIS) is crucial in limiting GM-CSF signaling not only during inflammatory arthritis but also in experimental allergic encephalomyelitis (EAE), a murine model of multiple sclerosis. Thus, a cellular cascade of synovial macrophages, NK cells, and neutrophils mediates persistent joint inflammation via production of IL-18 and GM-CSF. Endogenous CIS provides a key brake on signaling through the GM-CSF receptor. These findings shed new light on GM-CSF biology in sterile tissue inflammation and identify several potential therapeutic targets.
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http://dx.doi.org/10.1084/jem.20191421DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7201918PMC
May 2020

NK Cell Priming From Endogenous Homeostatic Signals Is Modulated by CIS.

Front Immunol 2020 31;11:75. Epub 2020 Jan 31.

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

Natural killer (NK) cell activation is controlled by a balance of activating and inhibitory signals and cytokines such as IL-15. We previously identified cytokine-inducible SH2-containing protein (CIS) as a negative regulator of IL-15 signaling in NK cells under inflammatory conditions. While the functional effect of -deficiency in NK cells was obvious by their increased anti-tumor immunity and hyper-proliferative response to IL-15, it remained unclear how CIS regulates NK cell biology in steady-state. Here, we investigated the role of CIS in the homeostatic maintenance of NK cells and found CIS-ablation promoted terminal differentiation of NK cells and increased turnover, suggesting that under steady-state conditions, CIS plays a role in maintaining IL-15 driven regulation of NK cells . However, hyper-responsiveness to IL-15 did not manifest in NK cell accumulation, even when the essential NK cell apoptosis mediator, (BIM) was deleted in addition to . Instead, loss of CIS conferred a lower activation threshold, evidenced by augmented functionality on a per cell basis both and without prior priming. We conclude that regulates IL-15 signaling in NK cells , and through the rewiring of several activation pathways leads to a reduction in activation threshold, decreasing the requirement for priming and improving NK cell anti-tumor function. Furthermore, this study highlights the tight regulation of NK cell homeostasis by several pathways which prevent NK cell accumulation when IL-15 signaling and intrinsic apoptosis are dysregulated.
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http://dx.doi.org/10.3389/fimmu.2020.00075DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7005222PMC
April 2021

Using singscore to predict mutation status in acute myeloid leukemia from transcriptomic signatures.

F1000Res 2019 3;8:776. Epub 2019 Jun 3.

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

Advances in RNA sequencing (RNA-seq) technologies that measure the transcriptome of biological samples have revolutionised our ability to understand transcriptional regulatory programs that underpin diseases such as cancer. We recently published singscore - a single sample, rank-based gene set scoring method which quantifies how concordant the transcriptional profile of individual samples are relative to specific gene sets of interest. Here we demonstrate the application of singscore to investigate transcriptional profiles associated with specific mutations or genetic lesions in acute myeloid leukemia. Using matched genomic and transcriptomic data available through the TCGA we show that scoring of appropriate signatures can distinguish samples with corresponding mutations, reflecting the ability of these mutations to drive aberrant transcriptional programs involved in leukemogenesis. We believe the singscore method is particularly useful for studying heterogeneity within a specific subsets of cancers, and as demonstrated, we show the ability of singscore to identify where alternative mutations appear to drive similar transcriptional programs.
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http://dx.doi.org/10.12688/f1000research.19236.3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6844140PMC
June 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

Therapeutic blockade of activin-A improves NK cell function and antitumor immunity.

Sci Signal 2019 08 27;12(596). Epub 2019 Aug 27.

Division of Molecular Immunology, The Walter and Eliza Hall Institute of Medical Research and Department of Medical Biology, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Parkville, Victoria 3052, Australia.

Natural killer (NK) cells are innate lymphocytes that play a major role in immunosurveillance against tumor initiation and metastatic spread. The signals and checkpoints that regulate NK cell fitness and function in the tumor microenvironment are not well defined. Transforming growth factor-β (TGF-β) is a suppressor of NK cells that inhibits interleukin-15 (IL-15)-dependent signaling events and increases the abundance of receptors that promote tissue residency. Here, we showed that NK cells express the type I activin receptor ALK4, which, upon binding to its ligand activin-A, phosphorylated SMAD2/3 to suppress IL-15-mediated NK cell metabolism. Activin-A impaired human and mouse NK cell proliferation and reduced the production of granzyme B to impair tumor killing. Similar to TGF-β, activin-A also induced SMAD2/3 phosphorylation and stimulated NK cells to increase their cell surface expression of several markers of ILC1 cells. Activin-A also induced these changes in TGF-β receptor-deficient NK cells, suggesting that activin-A and TGF-β stimulate independent pathways that drive SMAD2/3-mediated NK cell suppression. Last, inhibition of activin-A by follistatin substantially slowed orthotopic melanoma growth in mice. These data highlight the relevance of examining TGF-β-independent SMAD2/3 signaling mechanisms as a therapeutic axis to relieve NK cell suppression and promote antitumor immunity.
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http://dx.doi.org/10.1126/scisignal.aat7527DOI Listing
August 2019

Extensive transcriptional responses are co-ordinated by microRNAs as revealed by Exon-Intron Split Analysis (EISA).

Nucleic Acids Res 2019 09;47(16):8606-8619

Centre for Cancer Biology, an alliance of SA Pathology and University of South Australia, Adelaide, SA, Australia.

Epithelial-mesenchymal transition (EMT) has been a subject of intense scrutiny as it facilitates metastasis and alters drug sensitivity. Although EMT-regulatory roles for numerous miRNAs and transcription factors are known, their functions can be difficult to disentangle, in part due to the difficulty in identifying direct miRNA targets from complex datasets and in deciding how to incorporate 'indirect' miRNA effects that may, or may not, represent biologically relevant information. To better understand how miRNAs exert effects throughout the transcriptome during EMT, we employed Exon-Intron Split Analysis (EISA), a bioinformatic technique that separates transcriptional and post-transcriptional effects through the separate analysis of RNA-Seq reads mapping to exons and introns. We find that in response to the manipulation of miRNAs, a major effect on gene expression is transcriptional. We also find extensive co-ordination of transcriptional and post-transcriptional regulatory mechanisms during both EMT and mesenchymal to epithelial transition (MET) in response to TGF-β or miR-200c respectively. The prominent transcriptional influence of miRNAs was also observed in other datasets where miRNA levels were perturbed. This work cautions against a narrow approach that is limited to the analysis of direct targets, and demonstrates the utility of EISA to examine complex regulatory networks involving both transcriptional and post-transcriptional mechanisms.
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http://dx.doi.org/10.1093/nar/gkz664DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6895270PMC
September 2019

Integrative Analysis of Somatic Mutations in Non-coding Regions Altering RNA Secondary Structures in Cancer Genomes.

Sci Rep 2019 06 3;9(1):8205. Epub 2019 Jun 3.

Ministry of Education Key Laboratory of Contemporary Anthropology, School of Life Sciences, Fudan University, Shanghai, 200433, China.

RNA secondary structure may influence many cellular processes, including RNA processing, stability, localization, and translation. Single-nucleotide variations (SNVs) that alter RNA secondary structure, referred to as riboSNitches, are potentially causative of human diseases, especially in untranslated regions (UTRs) and noncoding RNAs (ncRNAs). The functions of somatic mutations that act as riboSNitches in cancer development remain poorly understood. In this study, we developed a computational pipeline called SNIPER (riboSNitch-enriched or depleted elements in cancer genomes), which employs MeanDiff and EucDiff to detect riboSNitches and then identifies riboSNitch-enriched or riboSNitch-depleted non-coding elements across tumors. SNIPER is available at github: https://github.com/suzhixi/SNIPER/ . We found that riboSNitches were more likely to be pathogenic. Moreover, we predicted several UTRs and lncRNAs (long non-coding RNA) that significantly enriched or depleted riboSNitches in cancer genomes, indicative of potential cancer driver or essential noncoding elements. Our study highlights the possibly neglected importance of RNA secondary structure in cancer genomes and provides a new strategy to identify new cancer-associated genes.
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http://dx.doi.org/10.1038/s41598-019-44489-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6546760PMC
June 2019

TCF-1 limits the formation of Tc17 cells via repression of the MAF-RORγt axis.

J Exp Med 2019 07 29;216(7):1682-1699. Epub 2019 May 29.

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

Interleukin (IL)-17-producing CD8 T (Tc17) cells have emerged as key players in host-microbiota interactions, infection, and cancer. The factors that drive their development, in contrast to interferon (IFN)-γ-producing effector CD8 T cells, are not clear. Here we demonstrate that the transcription factor TCF-1 () regulates CD8 T cell fate decisions in double-positive (DP) thymocytes through the sequential suppression of MAF and RORγt, in parallel with TCF-1-driven modulation of chromatin state. Ablation of TCF-1 resulted in enhanced Tc17 cell development and exposed a gene set signature to drive tissue repair and lipid metabolism, which was distinct from other CD8 T cell subsets. IL-17-producing CD8 T cells isolated from healthy humans were also distinct from CD8IL-17 T cells and enriched in pathways driven by and Overall, our study reveals how TCF-1 exerts central control of T cell differentiation in the thymus by normally repressing Tc17 differentiation and promoting an effector fate outcome.
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http://dx.doi.org/10.1084/jem.20181778DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6605755PMC
July 2019

A Gene Signature Predicting Natural Killer Cell Infiltration and Improved Survival in Melanoma Patients.

Cancer Immunol Res 2019 07 14;7(7):1162-1174. Epub 2019 May 14.

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

Natural killer (NK) cell activity is essential for initiating antitumor responses and may be linked to immunotherapy success. NK cells and other innate immune components could be exploitable for cancer treatment, which drives the need for tools and methods that identify therapeutic avenues. Here, we extend our gene-set scoring method to investigate NK cell infiltration by applying RNA-seq analysis to samples from bulk tumors. Computational methods have been developed for the deconvolution of immune cell types within solid tumors. We have taken the NK cell gene signatures from several such tools, then curated the gene list using a comparative analysis of tumors and immune cell types. Using a gene-set scoring method to investigate RNA-seq data from The Cancer Genome Atlas (TCGA), we show that patients with metastatic cutaneous melanoma have an improved survival rate if their tumor shows evidence of NK cell infiltration. Furthermore, these survival effects are enhanced in tumors that show higher expression of genes that encode NK cell stimuli such as the cytokine Using this signature, we then examine transcriptomic data to identify tumor and stromal components that may influence the penetrance of NK cells into solid tumors. Our results provide evidence that NK cells play a role in the regulation of human tumors and highlight potential survival effects associated with increased NK cell activity. Our computational analysis identifies putative gene targets that may be of therapeutic value for boosting NK cell antitumor immunity.
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http://dx.doi.org/10.1158/2326-6066.CIR-18-0500DOI Listing
July 2019

Single sample scoring of molecular phenotypes.

BMC Bioinformatics 2018 Nov 6;19(1):404. Epub 2018 Nov 6.

Division of Bioinformatics, Walter and Eliza Hall Institute of Medical Research, Melbourne, VIC, 3051, Australia.

Background: Gene set scoring provides a useful approach for quantifying concordance between sample transcriptomes and selected molecular signatures. Most methods use information from all samples to score an individual sample, leading to unstable scores in small data sets and introducing biases from sample composition (e.g. varying numbers of samples for different cancer subtypes). To address these issues, we have developed a truly single sample scoring method, and associated R/Bioconductor package singscore ( https://bioconductor.org/packages/singscore ).

Results: We use multiple cancer data sets to compare singscore against widely-used methods, including GSVA, z-score, PLAGE, and ssGSEA. Our approach does not depend upon background samples and scores are thus stable regardless of the composition and number of samples being scored. In contrast, scores obtained by GSVA, z-score, PLAGE and ssGSEA can be unstable when less data are available (N < 25). The singscore method performs as well as the best performing methods in terms of power, recall, false positive rate and computational time, and provides consistently high and balanced performance across all these criteria. To enhance the impact and utility of our method, we have also included a set of functions implementing visual analysis and diagnostics to support the exploration of molecular phenotypes in single samples and across populations of data.

Conclusions: The singscore method described here functions independent of sample composition in gene expression data and thus it provides stable scores, which are particularly useful for small data sets or data integration. Singscore performs well across all performance criteria, and includes a suite of powerful visualization functions to assist in the interpretation of results. This method performs as well as or better than other scoring approaches in terms of its power to distinguish samples with distinct biology and its ability to call true differential gene sets between two conditions. These scores can be used for dimensional reduction of transcriptomic data and the phenotypic landscapes obtained by scoring samples against multiple molecular signatures may provide insights for sample stratification.
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http://dx.doi.org/10.1186/s12859-018-2435-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6219008PMC
November 2018

Combinatorial Targeting by MicroRNAs Co-ordinates Post-transcriptional Control of EMT.

Cell Syst 2018 07 11;7(1):77-91.e7. Epub 2018 Jul 11.

Bioinformatics Division, Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC 3052, Australia; Department of Medical Biology, University of Melbourne, Parkville, VIC 3010, Australia; Department of Biochemistry, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Parkville, VIC 3010, Australia. Electronic address:

MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression, functioning in part by facilitating the degradation of target mRNAs. They have an established role in controlling epithelial-mesenchymal transition (EMT), a reversible phenotypic program underlying normal and pathological processes. Many studies demonstrate the role of individual miRNAs using overexpression at levels greatly exceeding physiological abundance. This can influence transcripts with relatively poor targeting and may in part explain why over 130 different miRNAs are directly implicated as EMT regulators. Analyzing a human mammary cell model of EMT we found evidence that a set of miRNAs, including the miR-200 and miR-182/183 family members, co-operate in post-transcriptional regulation, both reinforcing and buffering transcriptional output. Investigating this, we demonstrate that combinatorial treatment altered cellular phenotype with miRNA concentrations much closer to endogenous levels and with less off-target effects. This suggests that co-operative targeting by miRNAs is important for their physiological function and future work classifying miRNAs should consider such combinatorial effects.
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http://dx.doi.org/10.1016/j.cels.2018.05.019DOI Listing
July 2018
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