Publications by authors named "David J Adams"

469 Publications

Ulcerated Melanoma: Systems Biology Evidence of Inflammatory Imbalance Toward Pro-tumorigenicity.

Pigment Cell Melanoma Res 2021 Nov 26. Epub 2021 Nov 26.

Leeds Institute of Medical Research at St James's, University of Leeds, UK, LS9 7TF.

Microscopic ulceration is an independent predictor of melanoma death. Here we used systems biology to query the role of host and tumor specific processes in defining the phenotype. Measures of systemic inflammation were predictive of fewer tumor infiltrating lymphocytes and poorer survival. Ulcerated melanomas were thicker and more mitotically active (transcriptomic upregulated cell-cycle pathways). Sequencing identified tumoral p53 and APC mutations, and TUBB2B amplification as associated with the phenotype. Ulcerated tumors had perturbed expression of cytokine genes, consistent with protumorigenic inflammation and histological and transcriptomic evidence for reduced adaptive immune cell infiltration. Pathway/network analysis of multi-omic data using neural networks highlighted a role for the β-catenin pathway in the ulceration, linking genomic changes in the tumor to immunosuppression and cell proliferation. In summary the data suggest that ulceration is in part associated with genomic changes but that host factors predict melanoma death with evidence of reduced immune responses to the tumor.
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http://dx.doi.org/10.1111/pcmr.13023DOI Listing
November 2021

Variant Library Annotation Tool (VaLiAnT): an oligonucleotide library design and annotation tool for Saturation Genome Editing and other Deep Mutational Scanning experiments.

Bioinformatics 2021 Nov 16. Epub 2021 Nov 16.

Cancer, Ageing and Somatic Mutation Programme.

Motivation: CRISPR/Cas9-based technology allows for the functional analysis of genetic variants at single nucleotide resolution whilst maintaining genomic context (Findlay et al., 2018). This approach, known as saturation genome editing (SGE), a form of deep mutational scanning (DMS), systematically alters each position in a target region to explore its function. SGE experiments require the design and synthesis of oligonucleotide variant libraries which are introduced into the genome. This technology is applicable to diverse fields such as disease variant identification, drug development, structure-function studies, synthetic biology, evolutionary genetics and host-pathogen interactions. Here we present the Variant Library Annotation Tool (VaLiAnT) which can be used to generate variant libraries from user-defined genomic coordinates and standard input files. The software can accommodate user-specified species, reference sequences and transcript annotations.

Results: Coordinates for a genomic range are provided by the user to retrieve a corresponding oligonucleotide reference sequence. A user-specified range within this sequence is then subject to systematic, nucleotide and/or amino acid saturating mutator functions. VaLiAnT provides a novel way to retrieve, mutate and annotate genomic sequences for oligonucleotide library generation. Specific features for SGE library generation can be employed. In addition, VaLiAnT is configurable, allowing for cDNA and prime editing saturation library generation, with other diverse applications possible.

Availability: VaLiAnT is a command line tool written in Python. Source code, testing data, example input and output files, and executables are available (https://github.com/cancerit/VaLiAnT ) in addition to a detailed user manual (https://github.com/cancerit/VaLiAnT/wiki). VaLiAnT is licensed under AGPLv3.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btab776DOI Listing
November 2021

ɑO-Conotoxin GeXIVA isomers modulate N-type calcium (Ca 2.2) channels and inwardly-rectifying potassium (GIRK) channels via GABA receptor activation.

J Neurochem 2021 Nov 4. Epub 2021 Nov 4.

Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong, New South Wales, Australia.

αO-Conotoxin GeXIVA is a 28 amino acid peptide derived from the venom of the marine snail Conus generalis. The presence of four cysteine residues in the structure of GeXIVA allows it to have three different disulfide isomers, that is, the globular, ribbon or bead isomer. All three isomers are active at α9α10 nicotinic acetylcholine receptors, with the bead isomer, GeXIVA[1,2], being the most potent and exhibiting analgesic activity in animal models of neuropathic pain. The original report of GeXIVA activity failed to observe any effect of the isomers on high voltage-activated (HVA) calcium channel currents in rat dorsal root ganglion (DRG) neurons. In this study, we report, for the first time, the activity of globular GeXIVA[1,3] at G protein-coupled GABA receptors (GABA R) inhibiting HVA N-type calcium (Cav2.2) channels and reducing membrane excitability in mouse DRG neurons. The inhibition of HVA Ba currents and neuroexcitability by GeXIVA[1,3] was partially reversed by the selective GABA R antagonist CGP 55845. In transfected HEK293T cells co-expressing human GABA R1 and R2 subunits and Cav2.2 channels, both GeXIVA[1,3] and GeXIVA[1,4] inhibited depolarization-activated Ba currents mediated by Cav2.2 channels, whereas GeXIVA[1,2] had no effect. The effects of three cyclized GeXIVA[1,4] ribbon isomers were also tested, with cGeXIVA GAG being the most potent at human GABA R-coupled Cav2.2 channels. Interestingly, globular GeXIVA[1,3] also reversibly potentiated inwardly-rectifying K currents mediated by human GIRK1/2 channels co-expressed with GABA R in HEK293T cells. This study highlights GABA R as a potentially important receptor target for the activity of αO-conotoxin GeXIVA to mediate analgesia.
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http://dx.doi.org/10.1111/jnc.15535DOI Listing
November 2021

Combined presentation and immunogenicity analysis reveals a recurrent RAS.Q61K neoantigen in melanoma.

J Clin Invest 2021 10;131(20)

Department of Immunology, Weizmann Institute of Science, Rehovot, Israel.

Neoantigens are now recognized drivers of the antitumor immune response. Recurrent neoantigens, shared among groups of patients, have thus become increasingly coveted therapeutic targets. Here, we report on the data-driven identification of a robustly presented, immunogenic neoantigen that is derived from the combination of HLA-A*01:01 and RAS.Q61K. Analysis of large patient cohorts indicated that this combination applies to 3% of patients with melanoma. Using HLA peptidomics, we were able to demonstrate robust endogenous presentation of the neoantigen in 10 tumor samples. We detected specific reactivity to the mutated peptide within tumor-infiltrating lymphocytes (TILs) from 2 unrelated patients, thus confirming its natural immunogenicity. We further investigated the neoantigen-specific clones and their T cell receptors (TCRs) via a combination of TCR sequencing, TCR overexpression, functional assays, and single-cell transcriptomics. Our analysis revealed a diverse repertoire of neoantigen-specific clones with both intra- and interpatient TCR similarities. Moreover, 1 dominant clone proved to cross-react with the highly prevalent RAS.Q61R variant. Transcriptome analysis revealed a high association of TCR clones with specific T cell phenotypes in response to cognate melanoma, with neoantigen-specific cells showing an activated and dysfunctional phenotype. Identification of recurrent neoantigens and their reactive TCRs can promote "off-the-shelf" precision immunotherapies, alleviating limitations of personalized treatments.
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http://dx.doi.org/10.1172/JCI129466DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8516471PMC
October 2021

Analgesic α-conotoxins modulate native and recombinant GIRK1/2 channels via activation of GABA receptors and reduce neuroexcitability.

Br J Pharmacol 2021 Oct 1. Epub 2021 Oct 1.

Illawarra Health and Medical Research Institute (IHMRI), University of Wollongong, Wollongong, NSW, Australia.

Background And Purpose: Activation of GIRK channels via G protein-coupled GABA receptors has been shown to attenuate nociceptive transmission. The analgesic α-conotoxin Vc1.1 activates GABA receptors resulting in inhibition of Ca 2.2 and Ca 2.3 channels in mammalian primary afferent neurons. Here, we investigated the effects of analgesic α-conotoxins on recombinant and native GIRK-mediated K currents and on neuronal excitability.

Experimental Approach: The effects of analgesic α-conotoxins, Vc1.1, RgIA, and PeIA, were investigated on inwardly-rectifying K currents in HEK293T cells recombinantly co-expressing either heteromeric human GIRK1/2 or homomeric GIRK2 subunits, with GABA receptors. The effects of α-conotoxin Vc1.1 and baclofen were studied on GIRK-mediated K currents and the passive and active electrical properties of adult mouse dorsal root ganglion neurons.

Key Results: Analgesic α-conotoxins Vc1.1, RgIA, and PeIA potentiate inwardly-rectifying K currents in HEK293T cells recombinantly expressing human GIRK1/2 channels and GABA receptors. GABA receptor-dependent GIRK channel potentiation by Vc1.1 and baclofen occurs via a pertussis toxin-sensitive G protein and is inhibited by the selective GABA receptor antagonist CGP 55845. In adult mouse dorsal root ganglion neurons, GABA receptor-dependent GIRK channel potentiation by Vc1.1 and baclofen hyperpolarizes the cell membrane potential and reduces excitability.

Conclusions And Implications: This is the first report of GIRK channel potentiation via allosteric α-conotoxin Vc1.1-GABA receptor agonism, leading to decreased neuronal excitability. Such action potentially contributes to the analgesic effects of Vc1.1 and baclofen observed in vivo.
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http://dx.doi.org/10.1111/bph.15690DOI Listing
October 2021

Electrical properties and synaptic transmission in mouse intracardiac ganglion neurons in situ.

Physiol Rep 2021 Sep;9(18):e15056

Illawarra Health and Medical Research Institute (IHMRI), University of Wollongong, Wollongong, New South Wales, Australia.

The intrinsic cardiac nervous system represents the final site of signal integration for neurotransmission to the myocardium to enable local control of cardiac performance. The electrophysiological characteristics and ganglionic transmission of adult mouse intrinsic cardiac ganglion (ICG) neurons were investigated using a whole-mount ganglion preparation of the excised right atrial ganglion plexus and intracellular microelectrode recording techniques. The passive and active electrical properties of ICG neurons and synaptic transmission including synaptic response strength and efficacy as a function of stimulation frequency were examined. The resting membrane potential and input resistance of ICG neurons were -47.9 ± 4.0 mV and 197.2 ± 81.5 MΩ, respectively. All neurons had somatic action potentials with overshoots of >+15 mV and after-hyperpolarizations having an average of 10 mV amplitude and ~45 ms half duration. Phasic discharge activities were recorded from the majority of neurons studied and several types of excitatory synaptic responses were recorded following inputs from the vagus or interganglionic nerve trunk(s). Most postganglionic neurons (>75%) received a strong, suprathreshold synaptic input and reliably followed high-frequency repetitive nerve stimulation up to at least 50 Hz. Nerve-evoked synaptic transmission was blocked by extracellular Cd , ω-conotoxin CVIE, or α-conotoxin RegIIA, a selective α3-containing nicotinic acetylcholine receptor antagonist. Synaptic transmission and the electrical properties of murine ICG neurons contribute to the pattern of discharge which regulates chronotropic, dromotropic, and inotropic elements of cardiac function.
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http://dx.doi.org/10.14814/phy2.15056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8477906PMC
September 2021

Ethanol-induced formation of colorectal tumours and precursors in a mouse model of Lynch syndrome.

J Pathol 2021 Dec 13;255(4):464-474. Epub 2021 Oct 13.

Division of Pathology, Centre for Comparative Pathology, CRUK Edinburgh Centre, Institute of Genetics and Cancer, Western General Hospital, University of Edinburgh, Edinburgh, UK.

Lynch syndrome (LS) confers inherited cancer predisposition due to germline mutations in a DNA mismatch repair (MMR) gene, e.g. MSH2. MMR is a repair pathway for removal of base mismatches and insertion/deletion loops caused by endogenous and exogenous factors. Loss of MMR through somatic alteration of the wild-type allele in LS results in defective MMR (dMMR). Lifestyle/environmental factors can modify colorectal cancer risk in sporadic and LS patients. Ethanol and its metabolite acetaldehyde are classified as group one carcinogens, and acetaldehyde causes a range of DNA lesions. However, DNA repair pathways responsible for correcting most of such DNA lesions remain uncharacterised. We hypothesised that MMR plays a role in protecting colorectal epithelium from ethanol/acetaldehyde-induced DNA damage. Here, an LS mouse model (intestinal epithelial conditional-knockout for Msh2) was used to determine if there is a gene-environment interaction between dMMR and ethanol/acetaldehyde that accelerates colorectal tumourigenesis in LS. Mice underwent either long-term ethanol treatment or water treatment. Most ethanol-treated mice demonstrated colonic hyperproliferation and adenoma formation (with some invasive adenocarcinomas) within 6 months (15/23, 65%), compared with one colonic tumour after 15 months in water-treated mice (1/23, 4%) (p < 0.0001, Fisher's exact test). A significantly greater number of dMMR colonic crypt foci precursors were observed in ethanol-treated compared with water-treated mice (p = 0.0029, Student's t-test). Moreover, increased plasma acetaldehyde levels were detected in ethanol-treated compared with water-treated mice (p = 0.0019, Mann-Whitney U-test), along with significantly increased DNA damage response in the colonic epithelium. Long-term ethanol treatment was associated with significantly increased colonic epithelial proliferation and markedly reduced apoptosis in dMMR adenomas, consistent with enhanced survival of aberrant dMMR relative to MMR-proficient colonic epithelium. In conclusion, there is strong evidence for a gene-environment interaction between dMMR and acetaldehyde, causing acceleration of dMMR-driven colonic tumour formation in this LS model, indicating that advice to limit alcohol consumption should be considered for LS patients. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.
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http://dx.doi.org/10.1002/path.5796DOI Listing
December 2021

Cooperation between melanoma cell states promotes metastasis through heterotypic cluster formation.

Dev Cell 2021 Oct 15;56(20):2808-2825.e10. Epub 2021 Sep 15.

Cancer Biology and Genetics, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA. Electronic address:

Melanomas can have multiple coexisting cell states, including proliferative (PRO) versus invasive (INV) subpopulations that represent a "go or grow" trade-off; however, how these populations interact is poorly understood. Using a combination of zebrafish modeling and analysis of patient samples, we show that INV and PRO cells form spatially structured heterotypic clusters and cooperate in the seeding of metastasis, maintaining cell state heterogeneity. INV cells adhere tightly to each other and form clusters with a rim of PRO cells. Intravital imaging demonstrated cooperation in which INV cells facilitate dissemination of less metastatic PRO cells. We identified the TFAP2 neural crest transcription factor as a master regulator of clustering and PRO/INV states. Isolation of clusters from patients with metastatic melanoma revealed a subset with heterotypic PRO-INV clusters. Our data suggest a framework for the co-existence of these two divergent cell populations, in which heterotypic clusters promote metastasis via cell-cell cooperation.
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http://dx.doi.org/10.1016/j.devcel.2021.08.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8551056PMC
October 2021

Comprehensive prediction of robust synthetic lethality between paralog pairs in cancer cell lines.

Cell Syst 2021 Sep 10. Epub 2021 Sep 10.

School of Computer Science, University College Dublin, Dublin, Ireland; Systems Biology Ireland, University College Dublin, Dublin, Ireland. Electronic address:

Pairs of paralogs may share common functionality and, hence, display synthetic lethal interactions. As the majority of human genes have an identifiable paralog, exploiting synthetic lethality between paralogs may be a broadly applicable approach for targeting gene loss in cancer. However, only a biased subset of human paralog pairs has been tested for synthetic lethality to date. Here, by analyzing genome-wide CRISPR screens and molecular profiles of over 700 cancer cell lines, we identify features predictive of synthetic lethality between paralogs, including shared protein-protein interactions and evolutionary conservation. We develop a machine-learning classifier based on these features to predict which paralog pairs are most likely to be synthetic lethal and to explain why. We show that our classifier accurately predicts the results of combinatorial CRISPR screens in cancer cell lines and furthermore can distinguish pairs that are synthetic lethal in multiple cell lines from those that are cell-line specific. A record of this paper's transparent peer review process is included in the supplemental information.
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http://dx.doi.org/10.1016/j.cels.2021.08.006DOI Listing
September 2021

Comparison of the oncogenomic landscape of canine and feline hemangiosarcoma shows novel parallels with human angiosarcoma.

Dis Model Mech 2021 Jul 23;14(7). Epub 2021 Jul 23.

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

Angiosarcoma (AS) is a highly aggressive tumor of blood and lymphatic vessels in humans that shares many similarities with spontaneously occurring hemangiosarcoma (HSA) in dogs and cats. To investigate the genetic suitability of HSA as a model for AS, we sequenced ∼1000 cancer genes in 41 cases of HSA and matched germline tissue: 15 canine visceral HSAs, 13 canine skin HSAs and 13 feline skin HSAs. Analysis of visceral HSAs from dogs presenting with concurrent splenic and cardiac neoplasms showed that the tumors were not independent primaries, consistent with the highly metastatic nature of HSA. Comparison of HSA to AS revealed that several driver genes were recurrently mutated in both species, such as TP53, PIK3CA, ATRX, GRIN2A and LRP1B. Similar to AS, a UV mutational signature was found in a subset of canine cutaneous HSAs and both species show differing mutational profiles between tissue sites. Our characterization of canine and feline HSA demonstrates many important parallels to AS and provides hope that future studies on these cancers will benefit of all three species.
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http://dx.doi.org/10.1242/dmm.049044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8319545PMC
July 2021

Predictive Immune-Checkpoint Blockade Classifiers Identify Tumors Responding to Inhibition of PD-1 and/or CTLA-4.

Clin Cancer Res 2021 Jul 6. Epub 2021 Jul 6.

Department of Molecular Oncology and Immunology, Oncode Institute, The Netherlands Cancer Institute, Amsterdam, the Netherlands.

Purpose: Combining anti-PD-1 + anti-CTLA-4 immune-checkpoint blockade (ICB) shows improved patient benefit, but it is associated with severe immune-related adverse events and exceedingly high cost. Therefore, there is a dire need to predict which patients respond to monotherapy and which require combination ICB treatment.

Experimental Design: In patient-derived melanoma xenografts (PDX), human tumor microenvironment (TME) cells were swiftly replaced by murine cells upon transplantation. Using our XenofilteR deconvolution algorithm we curated human tumor cell RNA reads, which were subsequently subtracted from bulk (tumor cell + TME) patients' melanoma RNA. This produced a purely tumor cell-intrinsic signature ("InTumor") and a signature comprising tumor cell-extrinsic RNA reads ("ExTumor").

Results: We show that whereas the InTumor signature predicts response to anti-PD-1, the ExTumor predicts anti-CTLA-4 benefit. In PDX, InTumor, but not InTumor, tumors are effectively eliminated by cytotoxic T cells. When used in conjunction, the InTumor and ExTumor signatures identify not only patients who have a substantially higher chance of responding to combination treatment than to either monotherapy, but also those who are likely to benefit little from anti-CTLA-4 on top of anti-PD-1.

Conclusions: These signatures may be exploited to distinguish melanoma patients who need combination ICB blockade from those who likely benefit from either monotherapy.
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http://dx.doi.org/10.1158/1078-0432.CCR-20-4218DOI Listing
July 2021

More than just acral melanoma: the controversies of defining the disease.

J Pathol Clin Res 2021 Nov 2;7(6):531-541. Epub 2021 Jul 2.

Program of Immunology and Tumour Biology, Brazilian National Cancer Institute, Rio de Janeiro, Brazil.

Acral melanoma (AM) is a malignant cutaneous melanocytic tumour specifically located on the palms, soles, and nail apparatus, which are areas of glabrous (hairless) skin. Acral lentiginous melanoma, a subtype of AM, represents a histopathological subtype diagnosis of cutaneous melanoma with unique morphological and structural features. Despite clear definitions, the misuse of these terms and the inconsistency in reporting the histopathological features of AM cases have become a major obstacle to the study of the disease. In this review, we discuss the epidemiology, histopathological features, prognosis, and genetic profile of AM, highlighting the differences observed when histopathological subtypes are considered. The increasing global effort to characterise AM cases from ethnically diverse populations would benefit greatly from a more consistent classification of the disease.
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http://dx.doi.org/10.1002/cjp2.233DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8503895PMC
November 2021

Molecular pathology of skin adnexal tumors.

Histopathology 2021 Jul 1. Epub 2021 Jul 1.

Department of Pathology, University of Michigan, Ann Arbor, 48109, MI, USA.

Aims: Tumors of the cutaneous adnexa arise from, or differentiate toward, structures in normal skin such as hair follicles, sweat ducts/glands, sebaceous glands, or a combination of these elements. This class of neoplasms includes benign tumors and highly aggressive carcinomas. Adnexal tumors often present as solitary sporadic lesions, but can herald the presence of an inherited tumor syndrome such as Muir-Torre Syndrome, Cowden Syndrome, or CYLD Cutaneous Syndrome. In contrast to squamous cell carcinoma and basal cell carcinoma, molecular changes in adnexal neoplasia have been poorly characterized, and there are few published reviews on the current state of knowledge.

Methods And Results: We reviewed findings in peer-reviewed literature on molecular investigations of cutaneous adnexal tumors published through June 2021.

Conclusions: Recent discoveries have revealed diverse oncogenic drivers and tumor suppressor alterations in this class of tumors, implicating pathways including Ras/MAPK, PI3K, YAP/TAZ, beta-catenin, and NF-kappaB. These observations have identified novel markers, such as NUT for poroma and porocarcinoma, and PLAG1 for mixed tumors. Here, we provide a comprehensive overview and update of the molecular findings associated with adnexal tumors of the skin.
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http://dx.doi.org/10.1111/his.14441DOI Listing
July 2021

The clinicopathologic spectrum and genomic landscape of de-/trans-differentiated melanoma.

Mod Pathol 2021 Nov 21;34(11):2009-2019. Epub 2021 Jun 21.

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

Dedifferentiation and transdifferentiation are rare and only poorly understood phenomena in cutaneous melanoma. To study this disease more comprehensively we have retrieved 11 primary cutaneous melanomas from our pathology archives showing biphasic features characterized by a conventional melanoma and additional areas of de-/trans-differentiation as defined by a lack of immunohistochemical expression of all conventional melanocytic markers (S-100 protein, SOX10, Melan-A, and HMB-45). The clinical, histologic, and immunohistochemical findings were recorded and follow-up was obtained. The patients were mostly elderly (median: 81 years; range: 42-86 years) without significant gender predilection, and the sun-exposed skin of the head and neck area was most commonly affected. The tumors were deeply invasive with a mean depth of 7 mm (range: 4-80 mm). The dedifferentiated component showed atypical fibroxanthoma-like features in the majority of cases (7), while additional rhabdomyosarcomatous and epithelial transdifferentiation was noted histologically and/or immunohistochemically in two tumors each. The background conventional melanoma component was of desmoplastic (4), superficial spreading (3), nodular (2), lentigo maligna (1), or spindle cell (1) types. For the seven patients with available follow-up data (median follow-up period of 25 months; range: 8-36 months), two died from their disease, and three developed metastases. Next-generation sequencing of the cohort revealed somatic mutations of established melanoma drivers including mainly NF1 mutations (5) in the conventional component, which was also detected in the corresponding de-/trans-differentiated component. In summary, the diagnosis of primary cutaneous de-/trans-differentiated melanoma is challenging and depends on the morphologic identification of conventional melanoma. Molecular analysis is diagnostically helpful as the mutated gene profile is shared between the conventional and de-/trans-differentiated components. Importantly, de-/trans-differentiation does not appear to confer a more aggressive behavior.
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http://dx.doi.org/10.1038/s41379-021-00857-zDOI Listing
November 2021

Hi-C scaffolded short- and long-read genome assemblies of the California sea lion are broadly consistent for syntenic inference across 45 million years of evolution.

Mol Ecol Resour 2021 Oct 27;21(7):2455-2470. Epub 2021 Jun 27.

Division of Evolutionary Biology, Faculty of Biology, LMU Munich, München, Germany.

With the advent of chromatin-interaction maps, chromosome-level genome assemblies have become a reality for a wide range of organisms. Scaffolding quality is, however, difficult to judge. To explore this gap, we generated multiple chromosome-scale genome assemblies of an emerging wild animal model for carcinogenesis, the California sea lion (Zalophus californianus). Short-read assemblies were scaffolded with two independent chromatin interaction mapping data sets (Hi-C and Chicago), and long-read assemblies with three data types (Hi-C, optical maps and 10X linked reads) following the "Vertebrate Genomes Project (VGP)" pipeline. In both approaches, 18 major scaffolds recovered the karyotype (2n = 36), with scaffold N50s of 138 and 147 Mb, respectively. Synteny relationships at the chromosome level with other pinniped genomes (2n = 32-36), ferret (2n = 34), red panda (2n = 36) and domestic dog (2n = 78) were consistent across approaches and recovered known fissions and fusions. Comparative chromosome painting and multicolour chromosome tiling with a panel of 264 genome-integrated single-locus canine bacterial artificial chromosome probes provided independent evaluation of genome organization. Broad-scale discrepancies between the approaches were observed within chromosomes, most commonly in translocations centred around centromeres and telomeres, which were better resolved in the VGP assembly. Genomic and cytological approaches agreed on near-perfect synteny of the X chromosome, and in combination allowed detailed investigation of autosomal rearrangements between dog and sea lion. This study presents high-quality genomes of an emerging cancer model and highlights that even highly fragmented short-read assemblies scaffolded with Hi-C can yield reliable chromosome-level scaffolds suitable for comparative genomic analyses.
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http://dx.doi.org/10.1111/1755-0998.13443DOI Listing
October 2021

Globular and ribbon isomers of Conus geographus α-conotoxins antagonize human nicotinic acetylcholine receptors.

Biochem Pharmacol 2021 08 29;190:114638. Epub 2021 May 29.

Illawarra Health and Medical Research Institute (IHMRI), University of Wollongong, Wollongong, NSW 2522, Australia. Electronic address:

The short disulfide-rich α-conotoxins derived from the venom of Conus snails comprise a conserved CC(m)C(n)C cysteine framework (m and n, number of amino acids) and the majority antagonize nicotinic acetylcholine receptors (nAChRs). Depending on disulfide connectivity, α-conotoxins can exist as either globular (C-C, C-C), ribbon (C-C, C-C) or bead (C-C, C-C) isomers. In the present study, C. geographus α-conotoxins GI, GIB, G1.5 and G1.9 were chemically synthesized as globular and ribbon isomers and their activity investigated at human nAChRs expressed in Xenopus oocytes using the two-electrode voltage clamp recording technique. Both the globular and ribbon isomers of the 3/5 (m/n) α-conotoxins GI and GIB selectively inhibit heterologous human muscle-type α1β1δε nAChRs, whereas G1.5, a 4/7 α-conotoxin, selectively antagonizes neuronal (non-muscle) nAChR subtypes particularly human α3β2, α7 and α9α10 nAChRs. In contrast, globular and ribbon isomers of G1.9, a novel C-terminal elongated 4/8 α-conotoxin exhibited no activity at the human nAChR subtypes studied. This study reinforces earlier observations that 3/5 α-conotoxins selectively target the muscle nAChR subtypes, although interestingly, GIB is also active at α7 and α9 α10 nAChRs. The 4/7 α-conotoxins target human neuronal nAChR subtypes whereas the pharmacology of the 4/8 α-conotoxin remains unknown.
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http://dx.doi.org/10.1016/j.bcp.2021.114638DOI Listing
August 2021

α-Conotoxin Bt1.8 from Conus betulinus selectively inhibits α6/α3β2β3 and α3β2 nicotinic acetylcholine receptor subtypes.

J Neurochem 2021 10 22;159(1):90-100. Epub 2021 Jun 22.

Beijing Institute of Biotechnology, Beijing, China.

α-Conotoxins are small disulfide-rich peptides found in the venom of marine cone snails and are potent antagonists of nicotinic acetylcholine receptors (nAChRs). They are valuable pharmacological tools and have potential therapeutic applications for the treatment of chronic pain or neurological diseases and disorders. In the present study, we synthesized and functionally characterized a novel α-conotoxin Bt1.8, which was cloned from Conus betulinus. Bt1.8 selectively inhibited ACh-evoked currents in Xenopus oocytes expressing rat(r) α6/α3β2β3 and rα3β2 nAChRs with an IC of 2.1 nM and 9.4 nM, respectively, and similar potency for human (h) α6/α3β2β3 and hα3β2 nAChRs. Additionally, Bt1.8 had higher binding affinity with a slower dissociation rate for the rα6/α3β2β3 subtype compared to rα3β2. The amino acid sequence of Bt1.8 is significantly different from other reported α-conotoxins targeting the two nAChR subtypes. Further Alanine scanning analyses demonstrated that residues Ile9, Leu10, Asn11, Asn12 and Asn14 are critical for its inhibitory activity at the α6/α3β2β3 and α3β2 subtypes. Moreover, the NMR structure of Bt1.8 indicated the presence of a relatively larger hydrophobic zone than other α4/7-conotoxins which may explain its potent inhibition at α6/α3β2β3 nAChRs.
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http://dx.doi.org/10.1111/jnc.15434DOI Listing
October 2021

Membrane protein regulators of melanoma pulmonary colonisation identified using a CRISPRa screen and spontaneous metastasis assay in mice.

G3 (Bethesda) 2021 May 7. Epub 2021 May 7.

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

Metastasis is the spread of cancer cells to a secondary site within the body, and is the leading cause of death for cancer patients. The lung is a common site of metastasis for many cancer types, including melanoma. Identifying the genes involved in aiding metastasis of melanoma cells to the lungs is critical for the development of better treatments. As the accessibility of cell surface proteins make them attractive therapeutic targets, we performed a CRISPR activation screen using a library of guide RNAs (gRNAs) targeting the transcription start sites of 2,195 membrane protein-encoding genes, to identify genes whose upregulated expression aided pulmonary metastasis. Immunodeficient mice were subcutaneously injected in the flank with murine B16-F0 melanoma cells expressing dCas9 and the membrane protein library gRNAs, and their lungs collected after 14-21 days. Analysis was performed to identify the gRNAs that were enriched in the lungs relative to those present in the cells at the time of administration (day 0). We identified six genes whose increased expression promotes lung metastasis. These genes included several with well-characterised pro-metastatic roles (Fut7, Mgat5 and Pcdh7) that have not previously been linked to melanoma progression, genes linked to tumor progression but that have not previously been described as involved in metastasis (Olfr322 and Olfr441), as well as novel genes (Tmem116). Thus, we have identified genes that, when upregulated in melanoma cells, can aid successful metastasis and colonisation of the lung, and therefore may represent novel therapeutic targets to inhibit pulmonary metastasis.
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http://dx.doi.org/10.1093/g3journal/jkab157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8495943PMC
May 2021

Osteocyte transcriptome mapping identifies a molecular landscape controlling skeletal homeostasis and susceptibility to skeletal disease.

Nat Commun 2021 05 5;12(1):2444. Epub 2021 May 5.

St Vincent's Clinical School, Faculty of Medicine, UNSW Sydney, Sydney, NSW, Australia.

Osteocytes are master regulators of the skeleton. We mapped the transcriptome of osteocytes from different skeletal sites, across age and sexes in mice to reveal genes and molecular programs that control this complex cellular-network. We define an osteocyte transcriptome signature of 1239 genes that distinguishes osteocytes from other cells. 77% have no previously known role in the skeleton and are enriched for genes regulating neuronal network formation, suggesting this programme is important in osteocyte communication. We evaluated 19 skeletal parameters in 733 knockout mouse lines and reveal 26 osteocyte transcriptome signature genes that control bone structure and function. We showed osteocyte transcriptome signature genes are enriched for human orthologs that cause monogenic skeletal disorders (P = 2.4 × 10) and are associated with the polygenic diseases osteoporosis (P = 1.8 × 10) and osteoarthritis (P = 1.6 × 10). Thus, we reveal the molecular landscape that regulates osteocyte network formation and function and establish the importance of osteocytes in human skeletal disease.
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http://dx.doi.org/10.1038/s41467-021-22517-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8100170PMC
May 2021

Cut-like homeobox 1 (CUX1) tumor suppressor gene haploinsufficiency induces apoptosis evasion to sustain myeloid leukemia.

Nat Commun 2021 04 30;12(1):2482. Epub 2021 Apr 30.

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

While oncogenes promote tumorigenesis, they also induce deleterious cellular stresses, such as apoptosis, that cancer cells must combat by coopting adaptive responses. Whether tumor suppressor gene haploinsufficiency leads to such phenomena and their mechanistic basis is unclear. Here, we demonstrate that elevated levels of the anti-apoptotic factor, CASP8 and FADD-like apoptosis regulator (CFLAR), promotes apoptosis evasion in acute myeloid leukemia (AML) cells haploinsufficient for the cut-like homeobox 1 (CUX1) transcription factor, whose loss is associated with dismal clinical prognosis. Genome-wide CRISPR/Cas9 screening identifies CFLAR as a selective, acquired vulnerability in CUX1-deficient AML, which can be mimicked therapeutically using inhibitor of apoptosis (IAP) antagonists in murine and human AML cells. Mechanistically, CUX1 deficiency directly alleviates CUX1 repression of the CFLAR promoter to drive CFLAR expression and leukemia survival. These data establish how haploinsufficiency of a tumor suppressor is sufficient to induce advantageous anti-apoptosis cell survival pathways and concurrently nominate CFLAR as potential therapeutic target in these poor-prognosis leukemias.
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http://dx.doi.org/10.1038/s41467-021-22750-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8087769PMC
April 2021

Characterizing genetic intra-tumor heterogeneity across 2,658 human cancer genomes.

Cell 2021 04 7;184(8):2239-2254.e39. Epub 2021 Apr 7.

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

Intra-tumor heterogeneity (ITH) is a mechanism of therapeutic resistance and therefore an important clinical challenge. However, the extent, origin, and drivers of ITH across cancer types are poorly understood. To address this, we extensively characterize ITH across whole-genome sequences of 2,658 cancer samples spanning 38 cancer types. Nearly all informative samples (95.1%) contain evidence of distinct subclonal expansions with frequent branching relationships between subclones. We observe positive selection of subclonal driver mutations across most cancer types and identify cancer type-specific subclonal patterns of driver gene mutations, fusions, structural variants, and copy number alterations as well as dynamic changes in mutational processes between subclonal expansions. Our results underline the importance of ITH and its drivers in tumor evolution and provide a pan-cancer resource of comprehensively annotated subclonal events from whole-genome sequencing data.
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http://dx.doi.org/10.1016/j.cell.2021.03.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8054914PMC
April 2021

Mutagenic mechanisms of cancer-associated DNA polymerase ϵ alleles.

Nucleic Acids Res 2021 04;49(7):3919-3931

The Wellcome/Cancer Research UK Gurdon Institute and Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK.

A single amino acid residue change in the exonuclease domain of human DNA polymerase ϵ, P286R, is associated with the development of colorectal cancers, and has been shown to impart a mutator phenotype. The corresponding Pol ϵ allele in the yeast Saccharomyces cerevisiae (pol2-P301R), was found to drive greater mutagenesis than an entirely exonuclease-deficient Pol ϵ (pol2-4), an unexpected phenotype of ultra-mutagenesis. By studying the impact on mutation frequency, type, replication-strand bias, and sequence context, we show that ultra-mutagenesis is commonly observed in yeast cells carrying a range of cancer-associated Pol ϵ exonuclease domain alleles. Similarities between mutations generated by these alleles and those generated in pol2-4 cells indicate a shared mechanism of mutagenesis that yields a mutation pattern similar to cancer Signature 14. Comparison of POL2 ultra-mutator with pol2-M644G, a mutant in the polymerase domain decreasing Pol ϵ fidelity, revealed unexpected analogies in the sequence context and strand bias of mutations. Analysis of mutational patterns unique to exonuclease domain mutant cells suggests that backtracking of the polymerase, when the mismatched primer end cannot be accommodated in the proofreading domain, results in the observed insertions and T>A mutations in specific sequence contexts.
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http://dx.doi.org/10.1093/nar/gkab160DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8053093PMC
April 2021

Unraveling the cartography of the cancer ecosystem.

Genome Biol 2021 03 24;22(1):87. Epub 2021 Mar 24.

Experimental Cancer Genetics, The Wellcome Sanger Institute, Hinxton, Cambridge, Cambridgeshire, UK.

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http://dx.doi.org/10.1186/s13059-021-02310-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7988951PMC
March 2021

CRISPR activation screen in mice identifies novel membrane proteins enhancing pulmonary metastatic colonisation.

Commun Biol 2021 03 23;4(1):395. Epub 2021 Mar 23.

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

Melanoma represents ~5% of all cutaneous malignancies, yet accounts for the majority of skin cancer deaths due to its propensity to metastasise. To develop new therapies, novel target molecules must to be identified and the accessibility of cell surface proteins makes them attractive targets. Using CRISPR activation technology, we screened a library of guide RNAs targeting membrane protein-encoding genes to identify cell surface molecules whose upregulation enhances the metastatic pulmonary colonisation capabilities of tumour cells in vivo. We show that upregulated expression of the cell surface protein LRRN4CL led to increased pulmonary metastases in mice. Critically, LRRN4CL expression was elevated in melanoma patient samples, with high expression levels correlating with decreased survival. Collectively, our findings uncover an unappreciated role for LRRN4CL in the outcome of melanoma patients and identifies a potential therapeutic target and biomarker.
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http://dx.doi.org/10.1038/s42003-021-01912-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7987976PMC
March 2021

Identification of bacteria-derived HLA-bound peptides in melanoma.

Nature 2021 04 17;592(7852):138-143. Epub 2021 Mar 17.

Department of Melanoma Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.

A variety of species of bacteria are known to colonize human tumours, proliferate within them and modulate immune function, which ultimately affects the survival of patients with cancer and their responses to treatment. However, it is not known whether antigens derived from intracellular bacteria are presented by the human leukocyte antigen class I and II (HLA-I and HLA-II, respectively) molecules of tumour cells, or whether such antigens elicit a tumour-infiltrating T cell immune response. Here we used 16S rRNA gene sequencing and HLA peptidomics to identify a peptide repertoire derived from intracellular bacteria that was presented on HLA-I and HLA-II molecules in melanoma tumours. Our analysis of 17 melanoma metastases (derived from 9 patients) revealed 248 and 35 unique HLA-I and HLA-II peptides, respectively, that were derived from 41 species of bacteria. We identified recurrent bacterial peptides in tumours from different patients, as well as in different tumours from the same patient. Our study reveals that peptides derived from intracellular bacteria can be presented by tumour cells and elicit immune reactivity, and thus provides insight into a mechanism by which bacteria influence activation of the immune system and responses to therapy.
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http://dx.doi.org/10.1038/s41586-021-03368-8DOI Listing
April 2021

Alkyne-Bridged α-Conotoxin Vc1.1 Potently Reverses Mechanical Allodynia in Neuropathic Pain Models.

J Med Chem 2021 03 16;64(6):3222-3233. Epub 2021 Mar 16.

School of Chemistry, Monash University, Clayton, Victoria 3800, Australia.

Several -derived venom peptides are promising lead compounds for the management of neuropathic pain, with α-conotoxins being of particular interest. Modification of the interlocked disulfide framework of α-conotoxin Vc1.1 has been achieved using on-resin alkyne metathesis. Although introduction of a metabolically stable alkyne motif significantly disrupts backbone topography, the structural modification generates a potent and selective GABA receptor agonist that inhibits Ca2.2 channels and exhibits dose-dependent reversal of mechanical allodynia in a behavioral rat model of neuropathic pain. The findings herein support the hypothesis that analgesia can be achieved via activation of GABARs expressed in dorsal root ganglion (DRG) sensory neurons.
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http://dx.doi.org/10.1021/acs.jmedchem.0c02151DOI Listing
March 2021

Editorial overview: The most difficult of years in cancer research.

Curr Opin Genet Dev 2021 02;66:iii-iv

International Laboratory for Human Genome Research, National Autonomous University of Mexico, Mexico; Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK.

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http://dx.doi.org/10.1016/j.gde.2021.02.001DOI Listing
February 2021

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
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7910459PMC
February 2021
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