Centre for Cancer Research - Research Publications

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    A Matched Molecular and Clinical Analysis of the Epithelioid Haemangioendothelioma Cohort in the Stafford Fox Rare Cancer Program and Contextual Literature Review
    Abdelmogod, A ; Papadopoulos, L ; Riordan, S ; Wong, M ; Weltman, M ; Lim, R ; Mcevoy, C ; Fellowes, A ; Fox, S ; Bedo, J ; Penington, J ; Pham, K ; Hofmann, O ; Vissers, JHA ; Grimmond, S ; Ratnayake, G ; Christie, M ; Mitchell, C ; Murray, WK ; Mcclymont, K ; Luk, P ; Papenfuss, AT ; Kee, D ; Scott, CL ; Goldstein, D ; Barker, HE (MDPI, 2023-09)
    BACKGROUND: Epithelioid haemangioendothelioma (EHE) is an ultra-rare malignant vascular tumour with a prevalence of 1 per 1,000,000. It is typically molecularly characterised by a WWTR1::CAMTA1 gene fusion in approximately 90% of cases, or a YAP1::TFE3 gene fusion in approximately 10% of cases. EHE cases are typically refractory to therapies, and no anticancer agents are reimbursed for EHE in Australia. METHODS: We report a cohort of nine EHE cases with comprehensive histologic and molecular profiling from the Walter and Eliza Hall Institute of Medical Research Stafford Fox Rare Cancer Program (WEHI-SFRCP) collated via nation-wide referral to the Australian Rare Cancer (ARC) Portal. The diagnoses of EHE were confirmed by histopathological and immunohistochemical (IHC) examination. Molecular profiling was performed using the TruSight Oncology 500 assay, the TruSight RNA fusion panel, whole genome sequencing (WGS), or whole exome sequencing (WES). RESULTS: Molecular analysis of RNA, DNA or both was possible in seven of nine cases. The WWTR1::CAMTA1 fusion was identified in five cases. The YAP1::TFE3 fusion was identified in one case, demonstrating unique morphology compared to cases with the more common WWTR1::CAMTA1 fusion. All tumours expressed typical endothelial markers CD31, ERG, and CD34 and were negative for pan-cytokeratin. Cases with a WWTR1::CAMTA1 fusion displayed high expression of CAMTA1 and the single case with a YAP1::TFE3 fusion displayed high expression of TFE3. Survival was highly variable and unrelated to molecular profile. CONCLUSIONS: This cohort of EHE cases provides molecular and histopathological characterisation and matching clinical information that emphasises the molecular patterns and variable clinical outcomes and adds to our knowledge of this ultra-rare cancer. Such information from multiple studies will advance our understanding, potentially improving treatment options.
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    The landscape of cell-free mitochondrial DNA in liquid biopsy for cancer detection
    van der Pol, Y ; Moldovan, N ; Ramaker, J ; Bootsma, S ; Lenos, KJ ; Vermeulen, L ; Sandhu, S ; Bahce, I ; Pegtel, DM ; Wong, SQ ; Dawson, S-J ; Chandrananda, D ; Mouliere, F (BMC, 2023-10-12)
    BACKGROUND: Existing methods to detect tumor signal in liquid biopsy have focused on the analysis of nuclear cell-free DNA (cfDNA). However, non-nuclear cfDNA and in particular mitochondrial DNA (mtDNA) has been understudied. We hypothesize that an increase in mtDNA in plasma could reflect the presence of cancer, and that leveraging cell-free mtDNA could enhance cancer detection. RESULTS: We survey 203 healthy and 664 cancer plasma samples from three collection centers covering 12 cancer types with whole genome sequencing to catalogue the plasma mtDNA fraction. The mtDNA fraction is increased in individuals with cholangiocarcinoma, colorectal, liver, pancreatic, or prostate cancer, in comparison to that in healthy individuals. We detect almost no increase of mtDNA fraction in individuals with other cancer types. The mtDNA fraction in plasma correlates with the cfDNA tumor fraction as determined by somatic mutations and/or copy number aberrations. However, the mtDNA fraction is also elevated in a fraction of patients without an apparent increase in tumor-derived cfDNA. A predictive model integrating mtDNA and copy number analysis increases the area under the curve (AUC) from 0.73 when using copy number alterations alone to an AUC of 0.81. CONCLUSIONS: The mtDNA signal retrieved by whole genome sequencing has the potential to boost the detection of cancer when combined with other tumor-derived signals in liquid biopsies.
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    Venetoclax treatment in patients with cancer has limited impact on circulating T and NK cells
    Teh, CE ; Peng, H ; Luo, M-X ; Tan, T ; Trussart, M ; Howson, LJ ; Chua, CC ; Muttiah, C ; Brown, F ; Ritchie, ME ; Wei, AH ; Roberts, AW ; Bryant, VL ; Anderson, MA ; Lindeman, GJ ; Huang, DCS ; Thijssen, R ; Gray, DHD (ELSEVIER, 2023-06-27)
    Venetoclax is an effective treatment for certain blood cancers, such as chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML). However, most patients relapse while on venetoclax and further treatment options are limited. Combining venetoclax with immunotherapies is an attractive approach; however, a detailed understanding of how venetoclax treatment impacts normal immune cells in patients is lacking. In this study, we performed deep profiling of peripheral blood (PB) cells from patients with CLL and AML before and after short-term treatment with venetoclax using mass cytometry (cytometry by time of flight) and found no impact on the concentrations of key T-cell subsets or their expression of checkpoint molecules. We also analyzed PB from patients with breast cancer receiving venetoclax long-term using a single-cell multiomics approach (cellular indexing of transcriptomes and epitopes by sequencing) and functional assays. We found significant depletion of B-cell populations with low expression of MCL-1 relative to other immune cells, attended by extensive transcriptomic changes. By contrast, there was less impact on circulating T cells and natural killer (NK) cells, with no changes in their subset composition, transcriptome, or function following venetoclax treatment. Our data indicate that venetoclax has minimal impact on circulating T or NK cells, supporting the rationale of combining this BH3 mimetic drug with cancer immunotherapies for more durable antitumor responses.
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    Inhibition of METTL3 Results in a Cell-Intrinsic Interferon Response That Enhances Antitumor Immunity
    Guirguis, AA ; Ofir-Rosenfeld, Y ; Knezevic, K ; Blackaby, W ; Hardick, D ; Chan, Y-C ; Motazedian, A ; Gillespie, A ; Vassiliadis, D ; Tran, K ; Lam, EYN ; Andrews, B ; Harbour, ME ; Vasiliauskaite, L ; Saunders, CJ ; Tsagkogeorga, G ; Azevedo, A ; Obacz, J ; Pilka, ES ; Carkill, M ; Macpherson, L ; Wainwright, EN ; Liddicoat, B ; Blyth, BJ ; Albertella, MR ; Rausch, O ; Dawson, MA (AMER ASSOC CANCER RESEARCH, 2023-10-05)
    UNLABELLED: Therapies that enhance antitumor immunity have altered the natural history of many cancers. Consequently, leveraging nonoverlapping mechanisms to increase immunogenicity of cancer cells remains a priority. Using a novel enzymatic inhibitor of the RNA methyl-transferase METTL3, we demonstrate a global decrease in N6-methyladenosine (m6A) results in double-stranded RNA (dsRNA) formation and a profound cell-intrinsic interferon response. Through unbiased CRISPR screens, we establish dsRNA-sensing and interferon signaling are primary mediators that potentiate T-cell killing of cancer cells following METTL3 inhibition. We show in a range of immunocompetent mouse models that although METTL3 inhibition is equally efficacious to anti-PD-1 therapy, the combination has far greater preclinical activity. Using SPLINTR barcoding, we demonstrate that anti-PD-1 therapy and METTL3 inhibition target distinct malignant clones, and the combination of these therapies overcomes clones insensitive to the single agents. These data provide the mole-cular and preclinical rationale for employing METTL3 inhibitors to promote antitumor immunity in the clinic. SIGNIFICANCE: This work demonstrates that METTL3 inhibition stimulates a cell-intrinsic interferon response through dsRNA formation. This immunomodulatory mechanism is distinct from current immunotherapeutic agents and provides the molecular rationale for combination with anti-PD-1 immune-checkpoint blockade to augment antitumor immunity. This article is featured in Selected Articles from This Issue, p. 2109.
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    Benchmarking single-cell hashtag oligo demultiplexing methods
    Howitt, G ; Feng, Y ; Tobar, L ; Vassiliadis, D ; Hickey, P ; Dawson, MA ; Ranganathan, S ; Shanthikumar, S ; Neeland, M ; Maksimovic, J ; Oshlack, A (OXFORD UNIV PRESS, 2023-10-11)
    Sample multiplexing is often used to reduce cost and limit batch effects in single-cell RNA sequencing (scRNA-seq) experiments. A commonly used multiplexing technique involves tagging cells prior to pooling with a hashtag oligo (HTO) that can be sequenced along with the cells' RNA to determine their sample of origin. Several tools have been developed to demultiplex HTO sequencing data and assign cells to samples. In this study, we critically assess the performance of seven HTO demultiplexing tools: hashedDrops, HTODemux, GMM-Demux, demuxmix, deMULTIplex, BFF (bimodal flexible fitting) and HashSolo. The comparison uses data sets where each sample has also been demultiplexed using genetic variants from the RNA, enabling comparison of HTO demultiplexing techniques against complementary data from the genetic 'ground truth'. We find that all methods perform similarly where HTO labelling is of high quality, but methods that assume a bimodal count distribution perform poorly on lower quality data. We also suggest heuristic approaches for assessing the quality of HTO counts in an scRNA-seq experiment.
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    CRISPR-ChIP reveals selective regulation of H3K79me2 by Menin in MLL leukemia
    Gilan, O ; Talarmain, L ; Bell, CC ; Neville, D ; Knezevic, K ; Ferguson, DT ; Boudes, M ; Chan, Y-C ; Davidovich, C ; Lam, EYN ; Dawson, MA (NATURE PORTFOLIO, 2023-10)
    Chromatin regulation involves the selective recruitment of chromatin factors to facilitate DNA repair, replication and transcription. Here we demonstrate the utility of coupling unbiased functional genomics with chromatin immunoprecipitation (CRISPR-ChIP) to identify the factors associated with active chromatin modifications in mammalian cells. Specifically, an integrated reporter containing a cis-regulatory element of interest and a single guide RNA provide a chromatinized template for a direct readout for regulators of histone modifications associated with actively transcribed genes such as H3K4me3 and H3K79me2. With CRISPR-ChIP, we identify all the nonredundant COMPASS complex members required for H3K4me3 and demonstrate that RNA polymerase II is dispensable for the maintenance of H3K4me3. As H3K79me2 has a putative oncogenic function in leukemia cells driven by MLL translocations, using CRISPR-ChIP we reveal a functional partitioning of H3K79 methylation into two distinct regulatory units: an oncogenic DOT1L complex directed by the MLL fusion protein in a Menin-dependent manner and a separate endogenous DOT1L complex, where catalytic activity is directed by MLLT10. Overall, CRISPR-ChIP provides a powerful tool for the unbiased interrogation of the mechanisms underpinning chromatin regulation.
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    CRISPR-Cas9 screening identifies an IRF1-SOCS1-mediated negative feedback loop that limits CXCL9 expression and antitumor immunity
    House, IG ; Derrick, EB ; Sek, K ; Chen, AXY ; Li, J ; Lai, J ; Todd, KL ; Munoz, I ; Michie, J ; Chan, CW ; Huang, Y-K ; Chan, JD ; Petley, E ; Tong, J ; Nguyen, D ; Engel, S ; Savas, P ; Hogg, SJ ; Vervoort, SJ ; Kearney, CJ ; Burr, ML ; Lam, EYN ; Gilan, O ; Bedoui, S ; Johnstone, RW ; Dawson, MA ; Loi, S ; Darcy, PK ; Beavis, PA (CELL PRESS, 2023-08-29)
    CXCL9 expression is a strong predictor of response to immune checkpoint blockade therapy. Accordingly, we sought to develop therapeutic strategies to enhance the expression of CXCL9 and augment antitumor immunity. To perform whole-genome CRISPR-Cas9 screening for regulators of CXCL9 expression, a CXCL9-GFP reporter line is generated using a CRISPR knockin strategy. This approach finds that IRF1 limits CXCL9 expression in both tumor cells and primary myeloid cells through induction of SOCS1, which subsequently limits STAT1 signaling. Thus, we identify a subset of STAT1-dependent genes that do not require IRF1 for their transcription, including CXCL9. Targeting of either IRF1 or SOCS1 potently enhances CXCL9 expression by intratumoral macrophages, which is further enhanced in the context of immune checkpoint blockade therapy. We hence show a non-canonical role for IRF1 in limiting the expression of a subset of STAT1-dependent genes through induction of SOCS1.
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    Methyl-CpG binding domain 4, DNA glycosylase (MBD4)-associated neoplasia syndrome associated with a homozygous missense variant in MBD4: Expansion of an emerging phenotype
    Blombery, P ; Ryland, GL ; Fox, LC ; Stark, Z ; Wall, M ; Jarmolowicz, A ; Roesley, A ; Thompson, ER ; Grimmond, SM ; Panicker, S ; Kwok, F (WILEY, 2022-07)
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    Denosumab and invasive cervical root resorption: a case report
    Beaumont, S ; Angel, CM ; Dawson, S-J (WILEY, 2022-06)
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    Single-nuclei and bulk-tissue gene-expression analysis of pheochromocytoma and paraganglioma links disease subtypes with tumor microenvironment
    Zethoven, M ; Martelotto, L ; Pattison, A ; Bowen, B ; Balachander, S ; Flynn, A ; Rossello, FJ ; Hogg, A ; Miller, JA ; Frysak, Z ; Grimmond, S ; Fishbein, L ; Tischler, AS ; Gill, AJ ; Hicks, RJ ; Dahia, PLM ; Clifton-Bligh, R ; Pacak, K ; Tothill, RW (NATURE PORTFOLIO, 2022-10-21)
    Pheochromocytomas (PC) and paragangliomas (PG) are rare neuroendocrine tumors associated with autonomic nerves. Here we use single-nuclei RNA-seq and bulk-tissue gene-expression data to characterize the cellular composition of PCPG and normal adrenal tissues, refine tumor gene-expression subtypes and make clinical and genotypic associations. We confirm seven PCPG gene-expression subtypes with significant genotype and clinical associations. Tumors with mutations in VHL, SDH-encoding genes (SDHx) or MAML3-fusions are characterized by hypoxia-inducible factor signaling and neoangiogenesis. PCPG have few infiltrating lymphocytes but abundant macrophages. While neoplastic cells transcriptionally resemble mature chromaffin cells, early chromaffin and neuroblast markers are also features of some PCPG subtypes. The gene-expression profile of metastatic SDHx-related PCPG indicates these tumors have elevated cellular proliferation and a lower number of non-neoplastic Schwann-cell-like cells, while GPR139 is a potential theranostic target. Our findings therefore clarify the diverse transcriptional programs and cellular composition of PCPG and identify biomarkers of potential clinical significance.