Publications by authors named "Louise van der Weyden"

83 Publications

Combinatorial CRISPR screen identifies fitness effects of gene paralogues.

Nat Commun 2021 02 26;12(1):1302. Epub 2021 Feb 26.

Wellcome Sanger Institute, Wellcome Trust Genome Campus, Cambridge, UK.

Genetic redundancy has evolved as a way for human cells to survive the loss of genes that are single copy and essential in other organisms, but also allows tumours to survive despite having highly rearranged genomes. In this study we CRISPR screen 1191 gene pairs, including paralogues and known and predicted synthetic lethal interactions to identify 105 gene combinations whose co-disruption results in a loss of cellular fitness. 27 pairs influence fitness across multiple cell lines including the paralogues FAM50A/FAM50B, two genes of unknown function. Silencing of FAM50B occurs across a range of tumour types and in this context disruption of FAM50A reduces cellular fitness whilst promoting micronucleus formation and extensive perturbation of transcriptional programmes. Our studies reveal the fitness effects of FAM50A/FAM50B in cancer cells.
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http://dx.doi.org/10.1038/s41467-021-21478-9DOI Listing
February 2021

Histological Characterization of Feline Bladder Urothelial Carcinoma.

J Comp Pathol 2021 Jan 5;182:9-14. Epub 2020 Dec 5.

Synlab's VPG Histology, Bristol, UK. Electronic address:

Urothelial (transitional cell) carcinoma (UC) is the most common type of bladder cancer in humans, dogs and cats, although the incidence in cats is comparatively low. This retrospective study details the histopathological features of UC of the urinary bladder in 38 samples from 35 cats. Of the 38 samples, eight had a papillary architecture and in nine the tumour cells formed tubular or acinar structures. Tumour cell invasion of the bladder wall varied from confinement within the lamina propria or submucosa to transmural or extending to the serosa. The tumour stroma varied from sparse to abundant, with a scirrhous, myxomatous or mucinous appearance in eleven cases, three cases and one case, respectively. The degrees of tumour cell necrosis and inflammation were highly variable. We confirm that the histopathological features of bladder UC in cats have many similarities to the corresponding tumours in dogs and humans.
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http://dx.doi.org/10.1016/j.jcpa.2020.11.001DOI Listing
January 2021

The use of CRISPR/Cas9-based gene editing strategies to explore cancer gene function in mice.

Curr Opin Genet Dev 2021 Jan 7;66:57-62. Epub 2021 Jan 7.

Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, CB10 1SA, United Kingdom. Electronic address:

CRISPR/Cas9 systems have revolutionised the field of gene editing, allowing for precise modifications to be generated in vivo to mimic the genetic events found in human cancer cells. These systems may be used to generate germline or somatic loss-of-function of events, and also chromosomal rearrangements, either constitutively or in a spatiotemporally controlled manner. Forward genetic screens have also been performed using CRISPR/Cas9 systems to identify new driver genes and approaches using catalytically inactive Cas9 fused to base editors have enabled genome editing with single-base precision. Here we discuss the many 'flavours' of the CRISPR/Cas9 system and give examples of their use for the generation of clinically-relevant mouse models of cancer.
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http://dx.doi.org/10.1016/j.gde.2020.12.005DOI Listing
January 2021

Tumors induce de novo steroid biosynthesis in T cells to evade immunity.

Nat Commun 2020 07 17;11(1):3588. Epub 2020 Jul 17.

Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, CB10 1SA, UK.

Tumors subvert immune cell function to evade immune responses, yet the complex mechanisms driving immune evasion remain poorly understood. Here we show that tumors induce de novo steroidogenesis in T lymphocytes to evade anti-tumor immunity. Using a transgenic steroidogenesis-reporter mouse line we identify and characterize de novo steroidogenic immune cells, defining the global gene expression identity of these steroid-producing immune cells and gene regulatory networks by using single-cell transcriptomics. Genetic ablation of T cell steroidogenesis restricts primary tumor growth and metastatic dissemination in mouse models. Steroidogenic T cells dysregulate anti-tumor immunity, and inhibition of the steroidogenesis pathway is sufficient to restore anti-tumor immunity. This study demonstrates T cell de novo steroidogenesis as a mechanism of anti-tumor immunosuppression and a potential druggable target.
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http://dx.doi.org/10.1038/s41467-020-17339-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7368057PMC
July 2020

Spontaneously occurring melanoma in animals and their relevance to human melanoma.

J Pathol 2020 Sep 31;252(1):4-21. Epub 2020 Jul 31.

Wellcome Sanger Institute, Wellcome Genome Campus, Cambridge, UK.

In contrast to other cancer types, melanoma incidence has been increasing over the last 50 years, and while it still represents less than 5% of all cutaneous malignancies, melanoma accounts for the majority of skin cancer deaths, due to its propensity to metastasise. Whilst melanoma most commonly affects the skin, it can also arise in mucosal surfaces, the eye, and the brain. For new therapies to be developed, a better understanding of the genetic landscape, signalling pathways, and tumour-microenvironmental interactions is needed. This is where animal models are of critical importance. The mouse is the foremost used model of human melanoma. Arguably this is due to its plethora of benefits as a laboratory animal; however, it is important to note that unlike humans, melanocytes are not present at the dermal-epidermal junction in mice and mice do not develop melanoma without genetic manipulation. In contrast, there are numerous reports of animals that spontaneously develop melanoma, ranging from sharks and parrots to hippos and monkeys. In addition, several domesticated and laboratory-bred animals spontaneously develop melanoma or UV-induced melanoma, specifically, fish, opossums, pigs, horses, cats, and dogs. In this review, we look at spontaneously occurring animal 'models' of melanoma and discuss their relevance to the different types of melanoma found in humans. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland..
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http://dx.doi.org/10.1002/path.5505DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7497193PMC
September 2020

Association of aberrant ASNS imprinting with asparaginase sensitivity and chromosomal abnormality in childhood BCP-ALL.

Blood 2020 Nov;136(20):2319-2333

Department of Pediatrics and.

Karyotype is an important prognostic factor in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL), but the underlying pharmacogenomics remain unknown. Asparaginase is an integral component in current chemotherapy for childhood BCP-ALL. Asparaginase therapy depletes serum asparagine. Normal hematopoietic cells can produce asparagine by asparagine synthetase (ASNS) activity, but ALL cells are unable to synthesize adequate amounts of asparagine. The ASNS gene has a typical CpG island in its promoter. Thus, methylation of the ASNS CpG island could be one of the epigenetic mechanisms for ASNS gene silencing in BCP-ALL. To gain deep insights into the pharmacogenomics of asparaginase therapy, we investigated the association of ASNS methylation status with asparaginase sensitivity. The ASNS CpG island is largely unmethylated in normal hematopoietic cells, but it is allele-specifically methylated in BCP-ALL cells. The ASNS gene is located at 7q21, an evolutionally conserved imprinted gene cluster. ASNS methylation in childhood BCP-ALL is associated with an aberrant methylation of the imprinted gene cluster at 7q21. Aberrant methylation of mouse Asns and a syntenic imprinted gene cluster is also confirmed in leukemic spleen samples from ETV6-RUNX1 knockin mice. In 3 childhood BCP-ALL cohorts, ASNS is highly methylated in BCP-ALL patients with favorable karyotypes but is mostly unmethylated in BCP-ALL patients with poor prognostic karyotypes. Higher ASNS methylation is associated with higher L-asparaginase sensitivity in BCP-ALL through lower ASNS gene and protein expression levels. These observations demonstrate that silencing of the ASNS gene as a result of aberrant imprinting is a pharmacogenetic mechanism for the leukemia-specific activity of asparaginase therapy in BCP-ALL.
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http://dx.doi.org/10.1182/blood.2019004090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7702480PMC
November 2020

A Genome-Wide Screen in Mice To Identify Cell-Extrinsic Regulators of Pulmonary Metastatic Colonisation.

G3 (Bethesda) 2020 06 1;10(6):1869-1877. Epub 2020 Jun 1.

Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, CB10 1SA, United Kingdom.

Metastatic colonization, whereby a disseminated tumor cell is able to survive and proliferate at a secondary site, involves both tumor cell-intrinsic and -extrinsic factors. To identify tumor cell-extrinsic (microenvironmental) factors that regulate the ability of metastatic tumor cells to effectively colonize a tissue, we performed a genome-wide screen utilizing the experimental metastasis assay on mutant mice. Mutant and wildtype (control) mice were tail vein-dosed with murine metastatic melanoma B16-F10 cells and 10 days later the number of pulmonary metastatic colonies were counted. Of the 1,300 genes/genetic locations (1,344 alleles) assessed in the screen 34 genes were determined to significantly regulate pulmonary metastatic colonization (15 increased and 19 decreased; < 0.005 and genotype effect <-55 or >+55). While several of these genes have known roles in immune system regulation (, , , , , , , , , , and ) most are involved in a disparate range of biological processes, ranging from ubiquitination () to diphthamide synthesis () to Rho GTPase-activation ( and ), with no previous reports of a role in the regulation of metastasis. Thus, we have identified numerous novel regulators of pulmonary metastatic colonization, which may represent potential therapeutic targets.
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http://dx.doi.org/10.1534/g3.120.401128DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7263671PMC
June 2020

Companion canines: an under-utilised model to aid in translating anti-metastatics to the clinic.

Clin Exp Metastasis 2020 02 5;37(1):7-12. Epub 2019 Nov 5.

Pathobiology Department, Ontario Veterinary College, University of Guelph, Guelph, ON, N1G 2W1, Canada.

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http://dx.doi.org/10.1007/s10585-019-10002-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7007897PMC
February 2020

Vitamin D-VDR Signaling Inhibits Wnt/β-Catenin-Mediated Melanoma Progression and Promotes Antitumor Immunity.

Cancer Res 2019 12 5;79(23):5986-5998. Epub 2019 Nov 5.

University of Leeds School of Medicine, Leeds, United Kingdom.

1α,25-Dihydroxyvitamin D3 signals via the vitamin D receptor (VDR). Higher serum vitamin D is associated with thinner primary melanoma and better outcome, although a causal mechanism has not been established. As patients with melanoma commonly avoid sun exposure, and consequent vitamin D deficiency might worsen outcomes, we interrogated 703 primary melanoma transcriptomes to understand the role of vitamin D-VDR signaling and replicated the findings in The Cancer Genome Atlas metastases. expression was independently protective for melanoma-related death in both primary and metastatic disease. High tumor expression was associated with upregulation of pathways mediating antitumor immunity and corresponding with higher imputed immune cell scores and histologically detected tumor-infiltrating lymphocytes. High -expressing tumors had downregulation of proliferative pathways, notably Wnt/β-catenin signaling. Deleterious low levels resulted from promoter methylation and gene deletion in metastases. Vitamin D deficiency (<25 nmol/L ∼ 10 ng/mL) shortened survival in primary melanoma in a -dependent manner. functional validation studies showed that elevated vitamin D-VDR signaling inhibited Wnt/β-catenin signaling genes. Murine melanoma cells overexpressing produced fewer pulmonary metastases than controls in tail-vein metastasis assays. In summary, vitamin D-VDR signaling contributes to controlling pro-proliferative/immunosuppressive Wnt/β-catenin signaling in melanoma and this is associated with less metastatic disease and stronger host immune responses. This is evidence of a causal relationship between vitamin D-VDR signaling and melanoma survival, which should be explored as a therapeutic target in primary resistance to checkpoint blockade. SIGNIFICANCE: VDR expression could potentially be used as a biomarker to stratify patients with melanoma that may respond better to immunotherapy.
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http://dx.doi.org/10.1158/0008-5472.CAN-18-3927DOI Listing
December 2019

Whole-genome landscape of mucosal melanoma reveals diverse drivers and therapeutic targets.

Nat Commun 2019 07 18;10(1):3163. Epub 2019 Jul 18.

Melanoma Institute Australia, The University of Sydney, Sydney, NSW, 2065, Australia.

Knowledge of key drivers and therapeutic targets in mucosal melanoma is limited due to the paucity of comprehensive mutation data on this rare tumor type. To better understand the genomic landscape of mucosal melanoma, here we describe whole genome sequencing analysis of 67 tumors and validation of driver gene mutations by exome sequencing of 45 tumors. Tumors have a low point mutation burden and high numbers of structural variants, including recurrent structural rearrangements targeting TERT, CDK4 and MDM2. Significantly mutated genes are NRAS, BRAF, NF1, KIT, SF3B1, TP53, SPRED1, ATRX, HLA-A and CHD8. SF3B1 mutations occur more commonly in female genital and anorectal melanomas and CTNNB1 mutations implicate a role for WNT signaling defects in the genesis of some mucosal melanomas. TERT aberrations and ATRX mutations are associated with alterations in telomere length. Mutation profiles of the majority of mucosal melanomas suggest potential susceptibility to CDK4/6 and/or MEK inhibitors.
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http://dx.doi.org/10.1038/s41467-019-11107-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6639323PMC
July 2019

ALPK1 hotspot mutation as a driver of human spiradenoma and spiradenocarcinoma.

Nat Commun 2019 05 17;10(1):2213. Epub 2019 May 17.

Experimental Cancer Genetics, Wellcome Trust Sanger Institute, Cambridge, CB10 1SA, UK.

Spiradenoma and cylindroma are distinctive skin adnexal tumors with sweat gland differentiation and potential for malignant transformation and aggressive behaviour. We present the genomic analysis of 75 samples from 57 representative patients including 15 cylindromas, 17 spiradenomas, 2 cylindroma-spiradenoma hybrid tumors, and 24 low- and high-grade spiradenocarcinoma cases, together with morphologically benign precursor regions of these cancers. We reveal somatic or germline alterations of the CYLD gene in 15/15 cylindromas and 5/17 spiradenomas, yet only 2/24 spiradenocarcinomas. Notably, we find a recurrent missense mutation in the kinase domain of the ALPK1 gene in spiradenomas and spiradenocarcinomas, which is mutually exclusive from mutation of CYLD and can activate the NF-κB pathway in reporter assays. In addition, we show that high-grade spiradenocarcinomas carry loss-of-function TP53 mutations, while cylindromas may have disruptive mutations in DNMT3A. Thus, we reveal the genomic landscape of adnexal tumors and therapeutic targets.
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http://dx.doi.org/10.1038/s41467-019-09979-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6525246PMC
May 2019

FBXO7 sensitivity of phenotypic traits elucidated by a hypomorphic allele.

PLoS One 2019 6;14(3):e0212481. Epub 2019 Mar 6.

Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridgeshire, United Kingdom.

FBXO7 encodes an F box containing protein that interacts with multiple partners to facilitate numerous cellular processes and has a canonical role as part of an SCF E3 ubiquitin ligase complex. Mutation of FBXO7 is responsible for an early onset Parkinsonian pyramidal syndrome and genome-wide association studies have linked variants in FBXO7 to erythroid traits. A putative orthologue in Drosophila, nutcracker, has been shown to regulate the proteasome, and deficiency of nutcracker results in male infertility. Therefore, we reasoned that modulating Fbxo7 levels in a murine model could provide insights into the role of this protein in mammals. We used a targeted gene trap model which retained 4-16% residual gene expression and assessed the sensitivity of phenotypic traits to gene dosage. Fbxo7 hypomorphs showed regenerative anaemia associated with a shorter erythrocyte half-life, and male mice were infertile. Alterations to T cell phenotypes were also observed, which intriguingly were both T cell intrinsic and extrinsic. Hypomorphic mice were also sensitive to infection with Salmonella, succumbing to a normally sublethal challenge. Despite these phenotypes, Fbxo7 hypomorphs were produced at a normal Mendelian ratio with a normal lifespan and no evidence of neurological symptoms. These data suggest that erythrocyte survival, T cell development and spermatogenesis are particularly sensitive to Fbxo7 gene dosage.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0212481PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6402633PMC
November 2019

Metastasis in the wild: investigating metastasis in non-laboratory animals.

Clin Exp Metastasis 2019 02 9;36(1):15-28. Epub 2019 Feb 9.

Experimental Cancer Genetics (T113), Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, CB10 1SA, UK.

Humans are not the only species to spontaneously develop metastatic cancer as cases of metastasis have been reported in a wide range of animals, including dinosaurs. Mouse models have been an invaluable tool in experimental and clinical metastasis research, with the use of genetically-engineered mouse models that spontaneously develop metastasis or ectopic/orthotopic transplantation of tumour cells to wildtype or immunodeficient mice being responsible for many key advances in our understanding of metastasis. However, are there other species that can also be relevant models? Similarities to humans in terms of environmental exposures, life-span, genetics, histopathology and available therapeutics are all factors that can be considered when looking at species other than the laboratory mouse. This review will explore the occurrence of metastasis in multiple species from a variety of domestic, captive and free-living veterinary cases to assist in identifying potential alternative experimental and clinical research models relevant to humans.
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http://dx.doi.org/10.1007/s10585-019-09956-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6394581PMC
February 2019

Cross-species genomic landscape comparison of human mucosal melanoma with canine oral and equine melanoma.

Nat Commun 2019 01 21;10(1):353. Epub 2019 Jan 21.

Wellcome Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK.

Mucosal melanoma is a rare and poorly characterized subtype of human melanoma. Here we perform a cross-species analysis by sequencing tumor-germline pairs from 46 primary human muscosal, 65 primary canine oral and 28 primary equine melanoma cases from mucosal sites. Analysis of these data reveals recurrently mutated driver genes shared between species such as NRAS, FAT4, PTPRJ, TP53 and PTEN, and pathogenic germline alleles of BRCA1, BRCA2 and TP53. We identify a UV mutation signature in a small number of samples, including human cases from the lip and nasal mucosa. A cross-species comparative analysis of recurrent copy number alterations identifies several candidate drivers including MDM2, B2M, KNSTRN and BUB1B. Comparison of somatic mutations in recurrences and metastases to those in the primary tumor suggests pervasive intra-tumor heterogeneity. Collectively, these studies suggest a convergence of some genetic changes in mucosal melanomas between species but also distinctly different paths to tumorigenesis.
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http://dx.doi.org/10.1038/s41467-018-08081-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6341101PMC
January 2019

Association of the POT1 Germline Missense Variant p.I78T With Familial Melanoma.

JAMA Dermatol 2019 05;155(5):604-609

Experimental Cancer Genetics, The Wellcome Trust Sanger Institute, Hinxton, England.

Importance: The protection of telomeres 1 protein (POT1) is a critical component of the shelterin complex, a multiple-protein machine that regulates telomere length and protects telomere ends. Germline variants in POT1 have been linked to familial melanoma, and somatic mutations are associated with a range of cancers including cutaneous T-cell lymphoma (CTCL).

Objective: To characterize pathogenic variation in POT1 in families with melanoma to inform clinical management.

Design, Setting, And Participants: In this case study and pedigree evaluation, analysis of the pedigree of 1 patient with melanoma revealed a novel germline POT1 variant (p.I78T, c.233T>C, chromosome 7, g.124870933A>G, GRCh38) that was subsequently found in 2 other pedigrees obtained from the GenoMEL Consortium.

Main Outcomes And Measures: (1) Identification of the POT1 p.I78T variant; (2) evaluation of the clinical features and characteristics of patients with this variant; (3) analysis of 3 pedigrees; (4) genomewide single-nucleotide polymorphism genotyping of germline DNA; and (5) a somatic genetic analysis of available nevi and 1 melanoma lesion.

Results: The POT1 p.I78T variant was found in 3 melanoma pedigrees, all of persons who self-reported as being of Jewish descent, and was shown to disrupt POT1-telomere binding. A UV mutation signature was associated with nevus and melanoma formation in POT1 variant carriers, and somatic mutations in driver genes such as BRAF, NRAS, and KIT were associated with lesion development in these patients.

Conclusions And Relevance: POT1 p.I78T is a newly identified, likely pathogenic, variant meriting screening for in families with melanoma after more common predisposition genes such as CDKN2A have been excluded. It could also be included as part of gene panel testing.
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http://dx.doi.org/10.1001/jamadermatol.2018.3662DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6506889PMC
May 2019

Pulmonary metastatic colonisation and granulomas in NOX2-deficient mice.

J Pathol 2018 11 19;246(3):300-310. Epub 2018 Sep 19.

Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, UK.

Metastasis is the leading cause of death in cancer patients, and successful colonisation of a secondary organ by circulating tumour cells (CTCs) is the rate-limiting step of this process. We used tail-vein injection of B16-F10 melanoma cells into mice to mimic the presence of CTCs and to allow for the assessment of host (microenvironmental) factors that regulate pulmonary metastatic colonisation. We found that mice deficient for the individual subunits of the NADPH oxidase of myeloid cells, NOX2 (encoded by Cyba, Cybb, Ncf1, Ncf2, and Ncf4), all showed decreased pulmonary metastatic colonisation. To understand the role of NOX2 in controlling tumour cell survival in the pulmonary microenvironment, we focused on Cyba-deficient (Cyba ) mice, which showed the most significant decrease in metastatic colonisation. Interestingly, histological assessment of pulmonary metastatic colonisation was not possible in Cyba mice, owing to the presence of large granulomas composed of galectin-3 (Mac-2)-positive macrophages and eosinophilic deposits; granulomas of variable penetrance and severity were also found in Cyba mice that were not injected with melanoma cells, and these contributed to their decreased survival. The decreased pulmonary metastatic colonisation of Cyba mice was not due to any overt defects in vascular permeability, and bone marrow chimaeras confirmed a role for the haematological system in the reduced metastatic colonisation phenotype. Examination of the lymphocyte populations, which are known key regulators of metastatic colonisation, revealed an enhanced proportion of activated T and natural killer cells in the lungs of Cyba mice, relative to controls. The reduced metastatic colonisation, presence of granulomas and altered immune cell populations observed in Cyba lungs were mirrored in Ncf2-deficient (Ncf2 ) mice. Thus, we show that NOX2 deficiency results in both granulomas and the accumulation of antitumoural immune cells in the lungs that probably mediate the decreased pulmonary metastatic colonisation. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
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http://dx.doi.org/10.1002/path.5140DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6221033PMC
November 2018

CADM1 is essential for KSHV-encoded vGPCR-and vFLIP-mediated chronic NF-κB activation.

PLoS Pathog 2018 04 26;14(4):e1006968. Epub 2018 Apr 26.

Department of Microbiology and Immunology, Viral Oncology Program, Sylvester Comprehensive Cancer Center, Miller School of Medicine, The University of Miami, Miami, FL, United States of America.

Approximately 12% of all human cancers worldwide are caused by infections with oncogenic viruses. Kaposi's sarcoma herpesvirus/human herpesvirus 8 (KSHV/HHV8) is one of the oncogenic viruses responsible for human cancers, including Kaposi's sarcoma (KS), Primary Effusion Lymphoma (PEL), and the lymphoproliferative disorder multicentric Castleman's disease (MCD). Chronic inflammation mediated by KSHV infection plays a decisive role in the development and survival of these cancers. NF-κB, a family of transcription factors regulating inflammation, cell survival, and proliferation, is persistently activated in KSHV-infected cells. The KSHV latent and lytic expressing oncogenes involved in NF-κB activation are vFLIP/K13 and vGPCR, respectively. However, the mechanisms by which NF-κB is activated by vFLIP and vGPCR are poorly understood. In this study, we have found that a host molecule, Cell Adhesion Molecule 1 (CADM1), is robustly upregulated in KSHV-infected PBMCs and KSHV-associated PEL cells. Further investigation determined that both vFLIP and vGPCR interacted with CADM1. The PDZ binding motif localized at the carboxyl terminus of CADM1 is essential for both vGPCR and vFLIP to maintain chronic NF-κB activation. Membrane lipid raft associated CADM1 interaction with vFLIP is critical for the initiation of IKK kinase complex and NF-κB activation in the PEL cells. In addition, CADM1 played essential roles in the survival of KSHV-associated PEL cells. These data indicate that CADM1 plays key roles in the activation of NF-κB pathways during latent and lytic phases of the KSHV life cycle and the survival of KSHV-infected cells.
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http://dx.doi.org/10.1371/journal.ppat.1006968DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5919438PMC
April 2018

Widespread spontaneous hyperproliferation, melanosis and melanoma in Hgf-Cdk4R24C mice.

Melanoma Res 2018 02;28(1):76-78

Experimental Cancer Genetics, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge.

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http://dx.doi.org/10.1097/CMR.0000000000000414DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5739287PMC
February 2018

Comparative genomics reveals that loss of lunatic fringe (LFNG) promotes melanoma metastasis.

Mol Oncol 2018 02 7;12(2):239-255. Epub 2018 Jan 7.

Experimental Cancer Genetics, Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK.

Metastasis is the leading cause of death in patients with advanced melanoma, yet the somatic alterations that aid tumour cell dissemination and colonisation are poorly understood. Here, we deploy comparative genomics to identify and validate clinically relevant drivers of melanoma metastasis. To do this, we identified a set of 976 genes whose expression level was associated with a poor outcome in patients from two large melanoma cohorts. Next, we characterised the genomes and transcriptomes of mouse melanoma cell lines defined as weakly metastatic, and their highly metastatic derivatives. By comparing expression data between species, we identified lunatic fringe (LFNG), among 28 genes whose expression level is predictive of poor prognosis and whose altered expression is associated with a prometastatic phenotype in mouse melanoma cells. CRISPR/Cas9-mediated knockout of Lfng dramatically enhanced the capability of weakly metastatic melanoma cells to metastasise in vivo, a phenotype that could be rescued with the Lfng cDNA. Notably, genomic alterations disrupting LFNG are found exclusively in human metastatic melanomas sequenced as part of The Cancer Genome Atlas. Using comparative genomics, we show that LFNG expression plays a functional role in regulating melanoma metastasis.
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http://dx.doi.org/10.1002/1878-0261.12161DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5792739PMC
February 2018

A high-throughput in vivo screening method in the mouse for identifying regulators of metastatic colonization.

Nat Protoc 2017 Dec 2;12(12):2465-2477. Epub 2017 Nov 2.

Experimental Cancer Genetics Group, Wellcome Trust Sanger Institute, Hinxton, UK.

We describe a sensitive, robust, high-throughput method for quantifying the ability of metastatic tumor cells to colonize a secondary organ. Metastasis is the leading cause of death in cancer patients, and successful colonization of the secondary organ is the rate-limiting step in the metastatic process; thus, experimental methods that can be used to interrogate the key factors required for this critical step are of great importance. The experimental metastasis assay we detail here includes tail-vein injection of cancer cells into the mouse and determination of the resulting secondary organ colonization, primarily in the lung, 10 d post dosing. This assay can be used to investigate factors that regulate metastatic colonization both at the tumor-cell-intrinsic level (via manipulation of the tumor cells before injection) and at the tumor-cell-extrinsic level (such as the tissue microenvironment, via the use of genetically modified (GM) mice or agents such as antibodies or drugs). Using this method, we have robustly screened more than 950 GM mouse lines to identify novel microenvironmental regulators of metastatic colonization. The experimental details discussed here include choosing of appropriate cell numbers, handling of the cells, selection of recipient animals and injection techniques. Furthermore, we discuss key experimental design considerations, including the choice of the method used to determine metastatic burden and statistical analysis of the results, as well as provide troubleshooting tips and identification of factors that contribute to experimental variability.
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http://dx.doi.org/10.1038/nprot.2017.118DOI Listing
December 2017

Genome wide in vivo mouse screen data from studies to assess host regulation of metastatic colonisation.

Sci Data 2017 09 12;4:170129. Epub 2017 Sep 12.

Wellcome Trust Sanger Institute, Wellcome Genome Campus, Hinxton CB10 1SA, UK.

The process of metastasis is a multi-stage cascade with prior studies suggesting that the colonisation of the secondary site is the rate limiting step. This process involves contributions from the tumour cells and also non-tumour intrinsic factors such as the stroma and the haematopoietic system. In this study, we present data from screening 810 genetically-modified mouse lines with the experimental metastasis assay where intravenous delivery of murine metastatic melanoma B16-F10 cells was used to assess the formation of pulmonary metastasic foci. To date, these data have been studied with a two-step process cumulating in an integrative data analysis to identify genes controlling metastatic colonisation. We present the raw data, and a description to support fresh analyses where researchers can look both within and across gene sets to further elucidate process that regulate metastatic colonisation.
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http://dx.doi.org/10.1038/sdata.2017.129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5827107PMC
September 2017

Regulation of body weight and energy homeostasis by neuronal cell adhesion molecule 1.

Nat Neurosci 2017 Aug 19;20(8):1096-1103. Epub 2017 Jun 19.

Max Delbrück Center for Molecular Medicine, Berlin, Germany.

Susceptibility to obesity is linked to genes regulating neurotransmission, pancreatic beta-cell function and energy homeostasis. Genome-wide association studies have identified associations between body mass index and two loci near cell adhesion molecule 1 (CADM1) and cell adhesion molecule 2 (CADM2), which encode membrane proteins that mediate synaptic assembly. We found that these respective risk variants associate with increased CADM1 and CADM2 expression in the hypothalamus of human subjects. Expression of both genes was elevated in obese mice, and induction of Cadm1 in excitatory neurons facilitated weight gain while exacerbating energy expenditure. Loss of Cadm1 protected mice from obesity, and tract-tracing analysis revealed Cadm1-positive innervation of POMC neurons via afferent projections originating from beyond the arcuate nucleus. Reducing Cadm1 expression in the hypothalamus and hippocampus promoted a negative energy balance and weight loss. These data identify essential roles for Cadm1-mediated neuronal input in weight regulation and provide insight into the central pathways contributing to human obesity.
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http://dx.doi.org/10.1038/nn.4590DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5533218PMC
August 2017

Eros is a novel transmembrane protein that controls the phagocyte respiratory burst and is essential for innate immunity.

J Exp Med 2017 04 28;214(4):1111-1128. Epub 2017 Mar 28.

Department of Medicine, University of Cambridge, University of Cambridge School of Clinical Medicine, Cambridge CB2 0QQ, England, UK.

The phagocyte respiratory burst is crucial for innate immunity. The transfer of electrons to oxygen is mediated by a membrane-bound heterodimer, comprising gp91 and p22 subunits. Deficiency of either subunit leads to severe immunodeficiency. We describe Eros (essential for reactive oxygen species), a protein encoded by the previously undefined mouse gene , and show that it is essential for host defense via the phagocyte NAPDH oxidase. Eros is required for expression of the NADPH oxidase components, gp91 and p22 Consequently, -deficient mice quickly succumb to infection. also contributes to the formation of neutrophil extracellular traps (NETS) and impacts on the immune response to melanoma metastases. is an ortholog of the plant protein Ycf4, which is necessary for expression of proteins of the photosynthetic photosystem 1 complex, itself also an NADPH oxio-reductase. We thus describe the key role of the previously uncharacterized protein Eros in host defense.
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http://dx.doi.org/10.1084/jem.20161382DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5379978PMC
April 2017

Genome-wide in vivo screen identifies novel host regulators of metastatic colonization.

Nature 2017 01 4;541(7636):233-236. Epub 2017 Jan 4.

Wellcome Trust Sanger Institute, Wellcome Genome Campus, Cambridge CB10 1SA, UK.

Metastasis is the leading cause of death for cancer patients. This multi-stage process requires tumour cells to survive in the circulation, extravasate at distant sites, then proliferate; it involves contributions from both the tumour cell and tumour microenvironment ('host', which includes stromal cells and the immune system). Studies suggest the early steps of the metastatic process are relatively efficient, with the post-extravasation regulation of tumour growth ('colonization') being critical in determining metastatic outcome. Here we show the results of screening 810 mutant mouse lines using an in vivo assay to identify microenvironmental regulators of metastatic colonization. We identify 23 genes that, when disrupted in mouse, modify the ability of tumour cells to establish metastatic foci, with 19 of these genes not previously demonstrated to play a role in host control of metastasis. The largest reduction in pulmonary metastasis was observed in sphingosine-1-phosphate (S1P) transporter spinster homologue 2 (Spns2)-deficient mice. We demonstrate a novel outcome of S1P-mediated regulation of lymphocyte trafficking, whereby deletion of Spns2, either globally or in a lymphatic endothelial-specific manner, creates a circulating lymphopenia and a higher percentage of effector T cells and natural killer (NK) cells present in the lung. This allows for potent tumour cell killing, and an overall decreased metastatic burden.
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http://dx.doi.org/10.1038/nature20792DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5603286PMC
January 2017

Analyzing tumor heterogeneity and driver genes in single myeloid leukemia cells with SBCapSeq.

Nat Biotechnol 2016 09 1;34(9):962-72. Epub 2016 Aug 1.

Cancer Research Program, Houston Methodist Research Institute, Houston, Texas, USA.

A central challenge in oncology is how to kill tumors containing heterogeneous cell populations defined by different combinations of mutated genes. Identifying these mutated genes and understanding how they cooperate requires single-cell analysis, but current single-cell analytic methods, such as PCR-based strategies or whole-exome sequencing, are biased, lack sequencing depth or are cost prohibitive. Transposon-based mutagenesis allows the identification of early cancer drivers, but current sequencing methods have limitations that prevent single-cell analysis. We report a liquid-phase, capture-based sequencing and bioinformatics pipeline, Sleeping Beauty (SB) capture hybridization sequencing (SBCapSeq), that facilitates sequencing of transposon insertion sites from single tumor cells in a SB mouse model of myeloid leukemia (ML). SBCapSeq analysis of just 26 cells from one tumor revealed the tumor's major clonal subpopulations, enabled detection of clonal insertion events not detected by other sequencing methods and led to the identification of dominant subclones, each containing a unique pair of interacting gene drivers along with three to six cooperating cancer genes with SB-driven expression changes.
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http://dx.doi.org/10.1038/nbt.3637DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6124494PMC
September 2016

Sleeping Beauty screen reveals Pparg activation in metastatic prostate cancer.

Proc Natl Acad Sci U S A 2016 07 29;113(29):8290-5. Epub 2016 Jun 29.

Cancer Research UK Beatson Institute, Bearsden, Glasgow G61 1BD, United Kingdom; Institute of Cancer Sciences, University of Glasgow, Glasgow G61 1QH, United Kingdom;

Prostate cancer (CaP) is the most common adult male cancer in the developed world. The paucity of biomarkers to predict prostate tumor biology makes it important to identify key pathways that confer poor prognosis and guide potential targeted therapy. Using a murine forward mutagenesis screen in a Pten-null background, we identified peroxisome proliferator-activated receptor gamma (Pparg), encoding a ligand-activated transcription factor, as a promoter of metastatic CaP through activation of lipid signaling pathways, including up-regulation of lipid synthesis enzymes [fatty acid synthase (FASN), acetyl-CoA carboxylase (ACC), ATP citrate lyase (ACLY)]. Importantly, inhibition of PPARG suppressed tumor growth in vivo, with down-regulation of the lipid synthesis program. We show that elevated levels of PPARG strongly correlate with elevation of FASN in human CaP and that high levels of PPARG/FASN and PI3K/pAKT pathway activation confer a poor prognosis. These data suggest that CaP patients could be stratified in terms of PPARG/FASN and PTEN levels to identify patients with aggressive CaP who may respond favorably to PPARG/FASN inhibition.
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http://dx.doi.org/10.1073/pnas.1601571113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4961202PMC
July 2016

Alkaline ceramidase 1 is essential for mammalian skin homeostasis and regulating whole-body energy expenditure.

J Pathol 2016 07 30;239(3):374-83. Epub 2016 May 30.

Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK.

The epidermis is the outermost layer of skin that acts as a barrier to protect the body from the external environment and to control water and heat loss. This barrier function is established through the multistage differentiation of keratinocytes and the presence of bioactive sphingolipids such as ceramides, the levels of which are tightly regulated by a balance of ceramide synthase and ceramidase activities. Here we reveal the essential role of alkaline ceramidase 1 (Acer1) in the skin. Acer1-deficient (Acer1(-/-) ) mice showed elevated levels of ceramide in the skin, aberrant hair shaft cuticle formation and cyclic alopecia. We demonstrate that Acer1 is specifically expressed in differentiated interfollicular epidermis, infundibulum and sebaceous glands and consequently Acer1(-/-) mice have significant alterations in infundibulum and sebaceous gland architecture. Acer1(-/-) skin also shows perturbed hair follicle stem cell compartments. These alterations result in Acer1(-/-) mice showing increased transepidermal water loss and a hypermetabolism phenotype with associated reduction of fat content with age. We conclude that Acer1 is indispensable for mammalian skin homeostasis and whole-body energy homeostasis. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
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http://dx.doi.org/10.1002/path.4737DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4924601PMC
July 2016

Cross-species models of human melanoma.

J Pathol 2016 Jan 9;238(2):152-65. Epub 2015 Oct 9.

Experimental Cancer Genetics, The Wellcome Trust Sanger Institute, Hinxton, Cambridgeshire, CB10 1SA, UK.

Although transformation of melanocytes to melanoma is rare, the rapid growth, systemic spread, as well as the chemoresistance of melanoma present significant challenges for patient care. Here we review animal models of melanoma, including murine, canine, equine, and zebrafish models, and detail the immense contribution these models have made to our knowledge of human melanoma development, and to melanocyte biology. We also highlight the opportunities for cross-species comparative genomic studies of melanoma to identify the key molecular events that drive this complex disease.
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http://dx.doi.org/10.1002/path.4632DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4832391PMC
January 2016

A novel mouse model identifies cooperating mutations and therapeutic targets critical for chronic myeloid leukemia progression.

J Exp Med 2015 Sep 24;212(10):1551-69. Epub 2015 Aug 24.

Department of Haematology, Cambridge Institute for Medical Research and Addenbrooke's Hospital, University of Cambridge, Cambridge CB2 0XY, England, UK Wellcome Trust - Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Cambridge CB2 1TN, England, UK

The introduction of highly selective ABL-tyrosine kinase inhibitors (TKIs) has revolutionized therapy for chronic myeloid leukemia (CML). However, TKIs are only efficacious in the chronic phase of the disease and effective therapies for TKI-refractory CML, or after progression to blast crisis (BC), are lacking. Whereas the chronic phase of CML is dependent on BCR-ABL, additional mutations are required for progression to BC. However, the identity of these mutations and the pathways they affect are poorly understood, hampering our ability to identify therapeutic targets and improve outcomes. Here, we describe a novel mouse model that allows identification of mechanisms of BC progression in an unbiased and tractable manner, using transposon-based insertional mutagenesis on the background of chronic phase CML. Our BC model is the first to faithfully recapitulate the phenotype, cellular and molecular biology of human CML progression. We report a heterogeneous and unique pattern of insertions identifying known and novel candidate genes and demonstrate that these pathways drive disease progression and provide potential targets for novel therapeutic strategies. Our model greatly informs the biology of CML progression and provides a potent resource for the development of candidate therapies to improve the dismal outcomes in this highly aggressive disease.
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http://dx.doi.org/10.1084/jem.20141661DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4577832PMC
September 2015

Somatic drivers of B-ALL in a model of ETV6-RUNX1; Pax5(+/-) leukemia.

BMC Cancer 2015 Aug 13;15:585. Epub 2015 Aug 13.

Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1HH, UK.

Background: B-cell precursor acute lymphoblastic leukemia (B-ALL) is amongst the leading causes of childhood cancer-related mortality. Its most common chromosomal aberration is the ETV6-RUNX1 fusion gene, with ~25% of ETV6-RUNX1 patients also carrying PAX5 alterations.

Methods: We have recreated this mutation background by inter-crossing Etv6-RUNX1 (Etv6 (RUNX1-SB)) and Pax5(+/-) mice and performed an in vivo analysis to find driver genes using Sleeping Beauty transposon-mediated mutagenesis and also exome sequencing.

Results: Combination of Etv6-RUNX1 and Pax5(+/-) alleles generated a transplantable B220 + CD19+ B-ALL with a significant disease incidence. RNA-seq analysis showed a gene expression pattern consistent with arrest at the pre-B stage. Analysis of the transposon common insertion sites identified genes involved in B-cell development (Zfp423) and the JAK/STAT signaling pathway (Jak1, Stat5 and Il2rb), while exome sequencing revealed somatic hotspot mutations in Jak1 and Jak3 at residues analogous to those mutated in human leukemias, and also mutation of Trp53.

Conclusions: Powerful synergies exists in our model suggesting STAT pathway activation and mutation of Trp53 are potent drivers of B-ALL in the context of Etv6-RUNX1;Pax5(+/-).
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http://dx.doi.org/10.1186/s12885-015-1586-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4542115PMC
August 2015