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  • ItemOpen AccessAccepted version Peer-reviewed
    Division-independent differentiation mandates proliferative competition among stem cells.
    (National Academy of Sciences, 2018-04-03) Reilein, Amy; Melamed, David; Tavaré, Simon; Kalderon, Daniel
    Cancer-initiating gatekeeper mutations that arise in stem cells would be especially potent if they stabilize and expand an affected stem cell lineage. It is therefore important to understand how different stem cell organization strategies promote or prevent variant stem cell amplification in response to different types of mutation, including those that activate proliferation. Stem cell numbers can be maintained constant while producing differentiated products through individually asymmetrical division outcomes or by population asymmetry strategies in which individual stem cell lineages necessarily compete for niche space. We considered alternative mechanisms underlying population asymmetry and used quantitative modeling to predict starkly different consequences of altering proliferation rate: A variant, faster proliferating mutant stem cell should compete better only when stem cell division and differentiation are independent processes. For most types of stem cells, it has not been possible to ascertain experimentally whether division and differentiation are coupled. However,Drosophilafollicle stem cells (FSCs) provided a favorable system with which to investigate population asymmetry mechanisms and also for measuring the impact of altered proliferation on competition. We found from detailed cell lineage studies that division and differentiation of an individual FSC are not coupled. We also found that FSC representation, reflecting maintenance and amplification, was highly responsive to genetic changes that altered only the rate of FSC proliferation. The FSC paradigm therefore provides definitive experimental evidence for the general principle that relative proliferation rate will always be a major determinant of competition among stem cells specifically when stem cell division and differentiation are independent.
  • ItemOpen AccessAccepted version Peer-reviewed
    Combined Inhibition of mTOR and CDK4/6 Is Required for Optimal Blockade of E2F Function and Long-term Growth Inhibition in Estrogen Receptor-positive Breast Cancer.
    (American Association for Cancer Research, 2018-05) Michaloglou, Chrysiis; Crafter, Claire; Siersbaek, Rasmus; Delpuech, Oona; Curwen, Jon O; Carnevalli, Larissa S; Staniszewska, Anna D; Polanska, Urszula M; Cheraghchi-Bashi, Azadeh; Lawson, Mandy; Chernukhin, Igor; McEwen, Robert; Carroll, Jason S; Cosulich, Sabina C; Carroll, Jason [0000-0003-3643-0080]
    The cyclin dependent kinase (CDK)-retinoblastoma (RB)-E2F pathway plays a critical role in the control of cell cycle in estrogen receptor-positive (ER+) breast cancer. Small-molecule inhibitors of CDK4/6 have shown promise in this tumor type in combination with hormonal therapies, reflecting the particular dependence of this subtype of cancer on cyclin D1 and E2F transcription factors. mTOR inhibitors have also shown potential in clinical trials in this disease setting. Recent data have suggested cooperation between the PI3K/mTOR pathway and CDK4/6 inhibition in preventing early adaptation and eliciting growth arrest, but the mechanisms of the interplay between these pathways have not been fully elucidated. Here we show that profound and durable inhibition of ER+ breast cancer growth is likely to require multiple hits on E2F-mediated transcription. We demonstrate that inhibition of mTORC1/2 does not affect ER function directly, but does cause a decrease in cyclin D1 protein, RB phosphorylation, and E2F-mediated transcription. Combination of an mTORC1/2 inhibitor with a CDK4/6 inhibitor results in more profound effects on E2F-dependent transcription, which translates into more durable growth arrest and a delay in the onset of resistance. Combined inhibition of mTORC1/2, CDK4/6, and ER delivers even more profound and durable regressions in breast cancer cell lines and xenografts. Furthermore, we show that CDK4/6 inhibitor-resistant cell lines reactivate the CDK-RB-E2F pathway, but remain sensitive to mTORC1/2 inhibition, suggesting that mTORC1/2 inhibitors may represent an option for patients that have relapsed on CDK4/6 therapy. Mol Cancer Ther; 17(5); 908-20. ©2018 AACR.
  • ItemOpen AccessPublished version Peer-reviewed
    Protein aggregation of the p63 transcription factor underlies severe skin fragility in AEC syndrome.
    (National Academy of Sciences, 2018-01-30) Russo, Claudia; Osterburg, Christian; Sirico, Anna; Antonini, Dario; Ambrosio, Raffaele; Würz, Julia Maren; Rinnenthal, Jörg; Ferniani, Marco; Kehrloesser, Sebastian; Schäfer, Birgit; Güntert, Peter; Sinha, Satrajit; Dötsch, Volker; Missero, Caterina; Kehrloesser, Sebastian [0000-0002-6791-2421]
    The p63 gene encodes a master regulator of epidermal commitment, development, and differentiation. Heterozygous mutations in the C-terminal domain of the p63 gene can cause ankyloblepharon-ectodermal defects-cleft lip/palate (AEC) syndrome, a life-threatening disorder characterized by skin fragility and severe, long-lasting skin erosions. Despite deep knowledge of p63 functions, little is known about mechanisms underlying disease pathology and possible treatments. Here, we show that multiple AEC-associated p63 mutations, but not those causative of other diseases, lead to thermodynamic protein destabilization, misfolding, and aggregation, similar to the known p53 gain-of-function mutants found in cancer. AEC mutant proteins exhibit impaired DNA binding and transcriptional activity, leading to dominant negative effects due to coaggregation with wild-type p63 and p73. Importantly, p63 aggregation occurs also in a conditional knock-in mouse model for the disorder, in which the misfolded p63 mutant protein leads to severe epidermal defects. Variants of p63 that abolish aggregation of the mutant proteins are able to rescue p63's transcriptional function in reporter assays as well as in a human fibroblast-to-keratinocyte conversion assay. Our studies reveal that AEC syndrome is a protein aggregation disorder and opens avenues for therapeutic intervention.
  • ItemOpen AccessAccepted version Peer-reviewed
    Asparagine bioavailability governs metastasis in a model of breast cancer.
    (Springer nature, 2018-02-15) Knott, Simon RV; Wagenblast, Elvin; Khan, Showkhin; Kim, Sun Y; Soto, Mar; Wagner, Michel; Turgeon, Marc-Olivier; Fish, Lisa; Erard, Nicolas; Gable, Annika L; Maceli, Ashley R; Dickopf, Steffen; Papachristou, Evangelia K; D'Santos, Clive S; Carey, Lisa A; Wilkinson, John E; Harrell, J Chuck; Perou, Charles M; Goodarzi, Hani; Poulogiannis, George; Hannon, Gregory J; Papachristou, Evangelia [0000-0002-5835-2055]; Hannon, Gregory [0000-0003-4021-3898]
    Using a functional model of breast cancer heterogeneity, we previously showed that clonal sub-populations proficient at generating circulating tumour cells were not all equally capable of forming metastases at secondary sites. A combination of differential expression and focused in vitro and in vivo RNA interference screens revealed candidate drivers of metastasis that discriminated metastatic clones. Among these, asparagine synthetase expression in a patient's primary tumour was most strongly correlated with later metastatic relapse. Here we show that asparagine bioavailability strongly influences metastatic potential. Limiting asparagine by knockdown of asparagine synthetase, treatment with l-asparaginase, or dietary asparagine restriction reduces metastasis without affecting growth of the primary tumour, whereas increased dietary asparagine or enforced asparagine synthetase expression promotes metastatic progression. Altering asparagine availability in vitro strongly influences invasive potential, which is correlated with an effect on proteins that promote the epithelial-to-mesenchymal transition. This provides at least one potential mechanism for how the bioavailability of a single amino acid could regulate metastatic progression.
  • ItemOpen AccessAccepted version Peer-reviewed
    Deconstruction of a Metastatic Tumor Microenvironment Reveals a Common Matrix Response in Human Cancers.
    (AACR, 2018-03) Pearce, Oliver MT; Delaine-Smith, Robin M; Maniati, Eleni; Nichols, Sam; Wang, Jun; Böhm, Steffen; Rajeeve, Vinothini; Ullah, Dayem; Chakravarty, Probir; Jones, Roanne R; Montfort, Anne; Dowe, Tom; Gribben, John; Jones, J Louise; Kocher, Hemant M; Serody, Jonathan S; Vincent, Benjamin G; Connelly, John; Brenton, James D; Chelala, Claude; Cutillas, Pedro R; Lockley, Michelle; Bessant, Conrad; Knight, Martin M; Balkwill, Frances R; Brenton, James [0000-0002-5738-6683]
    We have profiled, for the first time, an evolving human metastatic microenvironment by measuring gene expression, matrisome proteomics, cytokine and chemokine levels, cellularity, extracellular matrix organization, and biomechanical properties, all on the same sample. Using biopsies of high-grade serous ovarian cancer metastases that ranged from minimal to extensive disease, we show how nonmalignant cell densities and cytokine networks evolve with disease progression. Multivariate integration of the different components allowed us to define, for the first time, gene and protein profiles that predict extent of disease and tissue stiffness, while also revealing the complexity and dynamic nature of matrisome remodeling during development of metastases. Although we studied a single metastatic site from one human malignancy, a pattern of expression of 22 matrisome genes distinguished patients with a shorter overall survival in ovarian and 12 other primary solid cancers, suggesting that there may be a common matrix response to human cancer.Significance: Conducting multilevel analysis with data integration on biopsies with a range of disease involvement identifies important features of the evolving tumor microenvironment. The data suggest that despite the large spectrum of genomic alterations, some human malignancies may have a common and potentially targetable matrix response that influences the course of disease. Cancer Discov; 8(3); 304-19. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 253.
  • ItemOpen AccessPublished version Peer-reviewed
    Using single-cell genomics to understand developmental processes and cell fate decisions.
    (Wiley, 2018-04-16) Griffiths, Jonathan A; Scialdone, Antonio; Marioni, John C; Griffiths, Jonathan [0000-0002-2010-2296]; Marioni, John [0000-0001-9092-0852]
    High-throughput -omics techniques have revolutionised biology, allowing for thorough and unbiased characterisation of the molecular states of biological systems. However, cellular decision-making is inherently a unicellular process to which "bulk" -omics techniques are poorly suited, as they capture ensemble averages of cell states. Recently developed single-cell methods bridge this gap, allowing high-throughput molecular surveys of individual cells. In this review, we cover core concepts of analysis of single-cell gene expression data and highlight areas of developmental biology where single-cell techniques have made important contributions. These include understanding of cell-to-cell heterogeneity, the tracing of differentiation pathways, quantification of gene expression from specific alleles, and the future directions of cell lineage tracing and spatial gene expression analysis.
  • ItemOpen AccessPublished version Peer-reviewed
    Disease-Associated Mutations in CEP120 Destabilize the Protein and Impair Ciliogenesis.
    (Elsevier BV, 2018-05-29) Joseph, Nimesh; Al-Jassar, Caezar; Johnson, Christopher M; Andreeva, Antonina; Barnabas, Deepak D; Freund, Stefan MV; Gergely, Fanni; van Breugel, Mark; Gergely, Fanni [0000-0002-2441-8095]
    Ciliopathies are a group of genetic disorders caused by a failure to form functional cilia. Due to a lack of structural information, it is currently poorly understood how ciliopathic mutations affect protein functionality to give rise to the underlying disease. Using X-ray crystallography, we show that the ciliopathy-associated centriolar protein CEP120 contains three C2 domains. The point mutations V194A and A199P, which cause Joubert syndrome (JS) and Jeune asphyxiating thoracic dystrophy (JATD), respectively, both reduce the thermostability of the second C2 domain by targeting residues that point toward its hydrophobic core. Genome-engineered cells homozygous for these mutations have largely normal centriole numbers but show reduced CEP120 levels, compromised recruitment of distal centriole markers, and deficient cilia formation. Our results provide insight into the disease mechanism of two ciliopathic mutations in CEP120, identify putative binding partners of CEP120 C2B, and suggest a complex genotype-phenotype relation of the CEP120 ciliopathy alleles.
  • ItemOpen AccessPublished version Peer-reviewed
    Dynamics of multiple resistance mechanisms in plasma DNA during EGFR-targeted therapies in non-small cell lung cancer.
    (Springer Science and Business Media LLC, 2018-06) Tsui, Dana Wai Yi; Murtaza, Muhammed; Wong, Alvin Seng Cheong; Rueda, Oscar M; Smith, Christopher G; Chandrananda, Dineika; Soo, Ross A; Lim, Hong Liang; Goh, Boon Cher; Caldas, Carlos; Forshew, Tim; Gale, Davina; Liu, Wei; Morris, James; Marass, Francesco; Eisen, Tim; Chin, Tan Min; Rosenfeld, Nitzan; Tsui, Dana Wai Yi [0000-0002-0595-6664]; Chin, Tan Min [0000-0002-3289-8498]; Rosenfeld, Nitzan [0000-0002-2825-4788]
    Tumour heterogeneity leads to the development of multiple resistance mechanisms during targeted therapies. Identifying the dominant driver(s) is critical for treatment decision. We studied the relative dynamics of multiple oncogenic drivers in longitudinal plasma of 50 EGFR-mutant non-small-cell lung cancer patients receiving gefitinib and hydroxychloroquine. We performed digital PCR and targeted sequencing on samples from all patients and shallow whole-genome sequencing on samples from three patients who underwent histological transformation to small-cell lung cancer. In 43 patients with known EGFR mutations from tumour, we identified them accurately in plasma of 41 patients (95%, 41/43). We also found additional mutations, including EGFR T790M (31/50, 62%), TP53 (23/50, 46%), PIK3CA (7/50, 14%) and PTEN (4/50, 8%). Patients with both TP53 and EGFR mutations before treatment had worse overall survival than those with only EGFR Patients who progressed without T790M had worse PFS during TKI continuation and developed alternative alterations, including small-cell lung cancer-associated copy number changes and TP53 mutations, that tracked subsequent treatment responses. Longitudinal plasma analysis can help identify dominant resistance mechanisms, including non-druggable genetic information that may guide clinical management.
  • ItemOpen AccessAccepted version Peer-reviewed
    Structural basis of G-quadruplex unfolding by the DEAH/RHA helicase DHX36.
    (Springer Science and Business Media LLC, 2018-06) Chen, Michael C; Tippana, Ramreddy; Demeshkina, Natalia A; Murat, Pierre; Balasubramanian, Shankar; Myong, Sua; Ferré-D'Amaré, Adrian R; Balasubramanian, Shankar [0000-0002-0281-5815]
    Guanine-rich nucleic acid sequences challenge the replication, transcription, and translation machinery by spontaneously folding into G-quadruplexes, the unfolding of which requires forces greater than most polymerases can exert1,2. Eukaryotic cells contain numerous helicases that can unfold G-quadruplexes 3 . The molecular basis of the recognition and unfolding of G-quadruplexes by helicases remains poorly understood. DHX36 (also known as RHAU and G4R1), a member of the DEAH/RHA family of helicases, binds both DNA and RNA G-quadruplexes with extremely high affinity4-6, is consistently found bound to G-quadruplexes in cells7,8, and is a major source of G-quadruplex unfolding activity in HeLa cell lysates 6 . DHX36 is a multi-functional helicase that has been implicated in G-quadruplex-mediated transcriptional and post-transcriptional regulation, and is essential for heart development, haematopoiesis, and embryogenesis in mice9-12. Here we report the co-crystal structure of bovine DHX36 bound to a DNA with a G-quadruplex and a 3' single-stranded DNA segment. We show that the N-terminal DHX36-specific motif folds into a DNA-binding-induced α-helix that, together with the OB-fold-like subdomain, selectively binds parallel G-quadruplexes. Comparison with unliganded and ATP-analogue-bound DHX36 structures, together with single-molecule fluorescence resonance energy transfer (FRET) analysis, suggests that G-quadruplex binding alone induces rearrangements of the helicase core; by pulling on the single-stranded DNA tail, these rearrangements drive G-quadruplex unfolding one residue at a time.
  • ItemOpen AccessPublished version Peer-reviewed
    beachmat: A Bioconductor C++ API for accessing high-throughput biological data from a variety of R matrix types.
    (Public Library of Science (PLoS), 2018-05) Lun, Aaron TL; Pagès, Hervé; Smith, Mike L; Lun, Aaron TL [0000-0002-3564-4813]; Smith, Mike L [0000-0002-7800-3848]
    Biological experiments involving genomics or other high-throughput assays typically yield a data matrix that can be explored and analyzed using the R programming language with packages from the Bioconductor project. Improvements in the throughput of these assays have resulted in an explosion of data even from routine experiments, which poses a challenge to the existing computational infrastructure for statistical data analysis. For example, single-cell RNA sequencing (scRNA-seq) experiments frequently generate large matrices containing expression values for each gene in each cell, requiring sparse or file-backed representations for memory-efficient manipulation in R. These alternative representations are not easily compatible with high-performance C++ code used for computationally intensive tasks in existing R/Bioconductor packages. Here, we describe a C++ interface named beachmat, which enables agnostic data access from various matrix representations. This allows package developers to write efficient C++ code that is interoperable with dense, sparse and file-backed matrices, amongst others. We evaluated the performance of beachmat for accessing data from each matrix representation using both simulated and real scRNA-seq data, and defined a clear memory/speed trade-off to motivate the choice of an appropriate representation. We also demonstrate how beachmat can be incorporated into the code of other packages to drive analyses of a very large scRNA-seq data set.
  • ItemOpen AccessPublished version Peer-reviewed
    Analytical validation of a next generation sequencing liquid biopsy assay for high sensitivity broad molecular profiling.
    (Public Library of Science (PLoS), 2018) Plagnol, Vincent; Woodhouse, Samuel; Howarth, Karen; Lensing, Stefanie; Smith, Matt; Epstein, Michael; Madi, Mikidache; Smalley, Sarah; Leroy, Catherine; Hinton, Jonathan; de Kievit, Frank; Musgrave-Brown, Esther; Herd, Colin; Baker-Neblett, Katherine; Brennan, Will; Dimitrov, Peter; Campbell, Nathan; Morris, Clive; Rosenfeld, Nitzan; Clark, James; Gale, Davina; Platt, Jamie; Calaway, John; Jones, Greg; Forshew, Tim; Forshew, Tim [0000-0003-2093-7020]
    Circulating tumor DNA (ctDNA) analysis is being incorporated into cancer care; notably in profiling patients to guide treatment decisions. Responses to targeted therapies have been observed in patients with actionable mutations detected in plasma DNA at variant allele fractions (VAFs) below 0.5%. Highly sensitive methods are therefore required for optimal clinical use. To enable objective assessment of assay performance, detailed analytical validation is required. We developed the InVisionFirst™ assay, an assay based on enhanced tagged amplicon sequencing (eTAm-Seq™) technology to profile 36 genes commonly mutated in non-small cell lung cancer (NSCLC) and other cancer types for actionable genomic alterations in cell-free DNA. The assay has been developed to detect point mutations, indels, amplifications and gene fusions that commonly occur in NSCLC. For analytical validation, two 10mL blood tubes were collected from NSCLC patients and healthy volunteer donors. In addition, contrived samples were used to represent a wide spectrum of genetic aberrations and VAFs. Samples were analyzed by multiple operators, at different times and using different reagent Lots. Results were compared with digital PCR (dPCR). The InVisionFirst assay demonstrated an excellent limit of detection, with 99.48% sensitivity for SNVs present at VAF range 0.25%-0.33%, 92.46% sensitivity for indels at 0.25% VAF and a high rate of detection at lower frequencies while retaining high specificity (99.9997% per base). The assay also detected ALK and ROS1 gene fusions, and DNA amplifications in ERBB2, FGFR1, MET and EGFR with high sensitivity and specificity. Comparison between the InVisionFirst assay and dPCR in a series of cancer patients showed high concordance. This analytical validation demonstrated that the InVisionFirst assay is highly sensitive, specific and robust, and meets analytical requirements for clinical applications.
  • ItemOpen AccessPublished version Peer-reviewed
    Development of a highly sensitive liquid biopsy platform to detect clinically-relevant cancer mutations at low allele fractions in cell-free DNA.
    (Public Library of Science (PLoS), 2018) Gale, Davina; Lawson, Andrew RJ; Howarth, Karen; Madi, Mikidache; Durham, Bradley; Smalley, Sarah; Calaway, John; Blais, Shannon; Jones, Greg; Clark, James; Dimitrov, Peter; Pugh, Michelle; Woodhouse, Samuel; Epstein, Michael; Fernandez-Gonzalez, Ana; Whale, Alexandra S; Huggett, Jim F; Foy, Carole A; Jones, Gerwyn M; Raveh-Amit, Hadas; Schmitt, Karin; Devonshire, Alison; Green, Emma; Forshew, Tim; Plagnol, Vincent; Rosenfeld, Nitzan; Gale, Davina [0000-0002-4521-8199]
    INTRODUCTION: Detection and monitoring of circulating tumor DNA (ctDNA) is rapidly becoming a diagnostic, prognostic and predictive tool in cancer patient care. A growing number of gene targets have been identified as diagnostic or actionable, requiring the development of reliable technology that provides analysis of multiple genes in parallel. We have developed the InVision™ liquid biopsy platform which utilizes enhanced TAm-Seq™ (eTAm-Seq™) technology, an amplicon-based next generation sequencing method for the identification of clinically-relevant somatic alterations at low frequency in ctDNA across a panel of 35 cancer-related genes. MATERIALS AND METHODS: We present analytical validation of the eTAm-Seq technology across two laboratories to determine the reproducibility of mutation identification. We assess the quantitative performance of eTAm-Seq technology for analysis of single nucleotide variants in clinically-relevant genes as compared to digital PCR (dPCR), using both established DNA standards and novel full-process control material. RESULTS: The assay detected mutant alleles down to 0.02% AF, with high per-base specificity of 99.9997%. Across two laboratories, analysis of samples with optimal amount of DNA detected 94% mutations at 0.25%-0.33% allele fraction (AF), with 90% of mutations detected for samples with lower amounts of input DNA. CONCLUSIONS: These studies demonstrate that eTAm-Seq technology is a robust and reproducible technology for the identification and quantification of somatic mutations in circulating tumor DNA, and support its use in clinical applications for precision medicine.
  • ItemOpen AccessPublished version Peer-reviewed
    RAD51 foci as a functional biomarker of homologous recombination repair and PARP inhibitor resistance in germline BRCA-mutated breast cancer.
    (Elsevier BV, 2018-05-01) Cruz, C; Castroviejo-Bermejo, M; Gutiérrez-Enríquez, S; Llop-Guevara, A; Ibrahim, YH; Gris-Oliver, A; Bonache, S; Morancho, B; Bruna, A; Rueda, OM; Lai, Z; Polanska, UM; Jones, GN; Kristel, P; de Bustos, L; Guzman, M; Rodríguez, O; Grueso, J; Montalban, G; Caratú, G; Mancuso, F; Fasani, R; Jiménez, J; Howat, WJ; Dougherty, B; Vivancos, A; Nuciforo, P; Serres-Créixams, X; Rubio, IT; Oaknin, A; Cadogan, E; Barrett, JC; Caldas, C; Baselga, J; Saura, C; Cortés, J; Arribas, J; Jonkers, J; Díez, O; O'Connor, MJ; Balmaña, J; Serra, V; Bruna, Alejandra [0000-0003-1214-9665]; Caldas, Carlos [0000-0003-3547-1489]
    BACKGROUND: BRCA1 and BRCA2 (BRCA1/2)-deficient tumors display impaired homologous recombination repair (HRR) and enhanced sensitivity to DNA damaging agents or to poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi). Their efficacy in germline BRCA1/2 (gBRCA1/2)-mutated metastatic breast cancers has been recently confirmed in clinical trials. Numerous mechanisms of PARPi resistance have been described, whose clinical relevance in gBRCA-mutated breast cancer is unknown. This highlights the need to identify functional biomarkers to better predict PARPi sensitivity. PATIENTS AND METHODS: We investigated the in vivo mechanisms of PARPi resistance in gBRCA1 patient-derived tumor xenografts (PDXs) exhibiting differential response to PARPi. Analysis included exome sequencing and immunostaining of DNA damage response proteins to functionally evaluate HRR. Findings were validated in a retrospective sample set from gBRCA1/2-cancer patients treated with PARPi. RESULTS: RAD51 nuclear foci, a surrogate marker of HRR functionality, were the only common feature in PDX and patient samples with primary or acquired PARPi resistance. Consistently, low RAD51 was associated with objective response to PARPi. Evaluation of the RAD51 biomarker in untreated tumors was feasible due to endogenous DNA damage. In PARPi-resistant gBRCA1 PDXs, genetic analysis found no in-frame secondary mutations, but BRCA1 hypomorphic proteins in 60% of the models, TP53BP1-loss in 20% and RAD51-amplification in one sample, none mutually exclusive. Conversely, one of three PARPi-resistant gBRCA2 tumors displayed BRCA2 restoration by exome sequencing. In PDXs, PARPi resistance could be reverted upon combination of a PARPi with an ataxia-telangiectasia mutated (ATM) inhibitor. CONCLUSION: Detection of RAD51 foci in gBRCA tumors correlates with PARPi resistance regardless of the underlying mechanism restoring HRR function. This is a promising biomarker to be used in the clinic to better select patients for PARPi therapy. Our study also supports the clinical development of PARPi combinations such as those with ATM inhibitors.
  • ItemOpen AccessPublished version Peer-reviewed
    Mitotic spindle association of TACC3 requires Aurora-A-dependent stabilization of a cryptic α-helix.
    (Springer Science and Business Media LLC, 2018-04-13) Burgess, Selena G; Mukherjee, Manjeet; Sabir, Sarah; Joseph, Nimesh; Gutiérrez-Caballero, Cristina; Richards, Mark W; Huguenin-Dezot, Nicolas; Chin, Jason W; Kennedy, Eileen J; Pfuhl, Mark; Royle, Stephen J; Gergely, Fanni; Bayliss, Richard; Pfuhl, Mark [0000-0001-9592-6639]; Royle, Stephen J [0000-0001-8927-6967]; Gergely, Fanni [0000-0002-2441-8095]; Bayliss, Richard [0000-0003-0604-2773]
    Aurora-A regulates the recruitment of TACC3 to the mitotic spindle through a phospho-dependent interaction with clathrin heavy chain (CHC). Here, we describe the structural basis of these interactions, mediated by three motifs in a disordered region of TACC3. A hydrophobic docking motif binds to a previously uncharacterized pocket on Aurora-A that is blocked in most kinases. Abrogation of the docking motif causes a delay in late mitosis, consistent with the cellular distribution of Aurora-A complexes. Phosphorylation of Ser558 engages a conformational switch in a second motif from a disordered state, needed to bind the kinase active site, into a helical conformation. The helix extends into a third, adjacent motif that is recognized by a helical-repeat region of CHC, not a recognized phospho-reader domain. This potentially widespread mechanism of phospho-recognition provides greater flexibility to tune the molecular details of the interaction than canonical recognition motifs that are dominated by phosphate binding.
  • ItemOpen AccessPublished version Peer-reviewed
    BRCA1-like signature in triple negative breast cancer: Molecular and clinical characterization reveals subgroups with therapeutic potential.
    (Wiley, 2015-10) Severson, Tesa M; Peeters, Justine; Majewski, Ian; Michaut, Magali; Bosma, Astrid; Schouten, Philip C; Chin, Suet-Feung; Pereira, Bernard; Goldgraben, Mae A; Bismeijer, Tycho; Kluin, Roelof JC; Muris, Jettie JF; Jirström, Karin; Kerkhoven, Ron M; Wessels, Lodewyk; Caldas, Carlos; Bernards, René; Simon, Iris M; Linn, Sabine; Chin, Suet-Feung [0000-0001-5697-1082]; Goldgraben, Mae [0000-0002-1111-2804]; Caldas, Carlos [0000-0003-3547-1489]
    Triple negative (TN) breast cancers make up some 15% of all breast cancers. Approximately 10-15% are mutant for the tumor suppressor, BRCA1. BRCA1 is required for homologous recombination-mediated DNA repair and deficiency results in genomic instability. BRCA1-mutated tumors have a specific pattern of genomic copy number aberrations that can be used to classify tumors as BRCA1-like or non-BRCA1-like. BRCA1 mutation, promoter methylation, BRCA1-like status and genome-wide expression data was determined for 112 TN breast cancer samples with long-term follow-up. Mutation status for 21 known DNA repair genes and PIK3CA was assessed. Gene expression and mutation frequency in BRCA1-like and non-BRCA1-like tumors were compared. Multivariate survival analysis was performed using the Cox proportional hazards model. BRCA1 germline mutation was identified in 10% of patients and 15% of tumors were BRCA1 promoter methylated. Fifty-five percent of tumors classified as BRCA1-like. The functions of genes significantly up-regulated in BRCA1-like tumors included cell cycle and DNA recombination and repair. TP53 was found to be frequently mutated in BRCA1-like (P < 0.05), while PIK3CA was frequently mutated in non-BRCA1-like tumors (P < 0.05). A significant association with worse prognosis was evident for patients with BRCA1-like tumors (adjusted HR = 3.32, 95% CI = 1.30-8.48, P = 0.01). TN tumors can be further divided into two major subgroups, BRCA1-like and non-BRCA1-like with different mutation and expression patterns and prognoses. Based on these molecular patterns, subgroups may be more sensitive to specific targeted agents such as PI3K or PARP inhibitors.
  • ItemOpen AccessAccepted version Peer-reviewed
    Batch effects in single-cell RNA-sequencing data are corrected by matching mutual nearest neighbors.
    (Springer Science and Business Media LLC, 2018-06) Haghverdi, Laleh; Lun, Aaron TL; Morgan, Michael D; Marioni, John C; Lun, Aaron TL [0000-0002-3564-4813]; Morgan, Michael D [0000-0003-0757-0711]
    Large-scale single-cell RNA sequencing (scRNA-seq) data sets that are produced in different laboratories and at different times contain batch effects that may compromise the integration and interpretation of the data. Existing scRNA-seq analysis methods incorrectly assume that the composition of cell populations is either known or identical across batches. We present a strategy for batch correction based on the detection of mutual nearest neighbors (MNNs) in the high-dimensional expression space. Our approach does not rely on predefined or equal population compositions across batches; instead, it requires only that a subset of the population be shared between batches. We demonstrate the superiority of our approach compared with existing methods by using both simulated and real scRNA-seq data sets. Using multiple droplet-based scRNA-seq data sets, we demonstrate that our MNN batch-effect-correction method can be scaled to large numbers of cells.
  • ItemOpen Access
    Shallow whole genome sequencing for robust copy number profiling of formalin-fixed paraffin-embedded breast cancers.
    (Elsevier BV, 2018-06) Chin, Suet-Feung; Santonja, Angela; Grzelak, Marta; Ahn, Soomin; Sammut, Stephen-John; Clifford, Harry; Rueda, Oscar M; Pugh, Michelle; Goldgraben, Mae A; Bardwell, Helen A; Cho, Eun Yoon; Provenzano, Elena; Rojo, Federico; Alba, Emilio; Caldas, Carlos; Chin, Suet-Feung [0000-0001-5697-1082]; Grzelak, Marta [0000-0002-2653-2895]; Sammut, Stephen [0000-0003-4472-904X]; Rueda Palacio, Oscar [0000-0003-0008-4884]; Goldgraben, Mae [0000-0002-1111-2804]; Caldas, Carlos [0000-0003-3547-1489]
    Pathology archives with linked clinical data are an invaluable resource for translational research, with the limitation that most cancer samples are formalin-fixed paraffin-embedded (FFPE) tissues. Therefore, FFPE tissues are an important resource for genomic profiling studies but are under-utilised due to the low amount and quality of extracted nucleic acids. We profiled the copy number landscape of 356 breast cancer patients using DNA extracted FFPE tissues by shallow whole genome sequencing. We generated a total of 491 sequencing libraries from 2 kits and obtained data from 98.4% of libraries with 86.4% being of good quality. We generated libraries from as low as 3.8 ng of input DNA and found that the success was independent of input DNA amount and quality, processing site and age of the fixed tissues. Since copy number alterations (CNA) play a major role in breast cancer, it is imperative that we are able to use FFPE archives and we have shown in this study that sWGS is a robust method to do such profiling.
  • ItemOpen AccessAccepted version Peer-reviewed
    Mechanistic Distinctions between CHK1 and WEE1 Inhibition Guide the Scheduling of Triple Therapy with Gemcitabine.
    (American Association for Cancer Research (AACR), 2018-06-01) Koh, Siang-Boon; Wallez, Yann; Dunlop, Charles R; Bernaldo de Quirós Fernández, Sandra; Bapiro, Tashinga E; Richards, Frances M; Jodrell, Duncan I; Koh, Siang-Boon [0000-0002-5494-3800]; Dunlop, Charles R [0000-0002-8194-2598]; Richards, Frances M [0000-0001-7947-7853]
    Combination of cytotoxic therapy with emerging DNA damage response inhibitors (DDRi) has been limited by tolerability issues. However, the goal of most combination trials has been to administer DDRi with standard-of-care doses of chemotherapy. We hypothesized that mechanism-guided treatment scheduling could reduce the incidence of dose-limiting toxicities and enable tolerable multitherapeutic regimens. Integrative analyses of mathematical modeling and single-cell assays distinguished the synergy kinetics of WEE1 inhibitor (WEE1i) from CHEK1 inhibitor (CHK1i) by potency, spatiotemporal perturbation, and mitotic effects when combined with gemcitabine. These divergent properties collectively supported a triple-agent strategy, whereby a pulse of gemcitabine and CHK1i followed by WEE1i durably suppressed tumor cell growth. In xenografts, CHK1i exaggerated replication stress without mitotic CDK hyperactivation, enriching a geminin-positive subpopulation and intratumoral gemcitabine metabolite. Without overt toxicity, addition of WEE1i to low-dose gemcitabine and CHK1i was most effective in tumor control compared with single and double agents. Overall, our work provides quantitative insights into the mechanisms of DDRi chemosensitization, leading to the rational development of a tolerable multitherapeutic regimen.Significance: Multiple lines of mechanistic insight regarding DNA damage response inhibitors rationally guide the preclinical development of a tolerable multitherapeutic regimen.Graphical Abstract: Cancer Res; 78(11); 3054-66. ©2018 AACR.
  • ItemOpen AccessPublished version Peer-reviewed
    Whole-body single-cell sequencing reveals transcriptional domains in the annelid larval body.
    (Oxford University Press, 2018-05-01) Achim, Kaia; Eling, Nils; Vergara, Hernando Martinez; Bertucci, Paola Yanina; Musser, Jacob; Vopalensky, Pavel; Brunet, Thibaut; Collier, Paul; Benes, Vladimir; Marioni, John C; Arendt, Detlev; Marioni, John [0000-0001-9092-0852]
    Animal bodies comprise diverse arrays of cells. To characterise cellular identities across an entire body, we have compared the transcriptomes of single cells randomly picked from dissociated whole larvae of the marine annelid Platynereis dumerilii. We identify five transcriptionally distinct groups of differentiated cells, each expressing a unique set of transcription factors and effector genes that implement cellular phenotypes. Spatial mapping of cells into a cellular expression atlas, and wholemount in situ hybridisation of group-specific genes reveals spatially coherent transcriptional domains in the larval body, comprising e.g. apical sensory-neurosecretory cells vs. neural/epidermal surface cells. These domains represent new, basic subdivisions of the annelid body based entirely on differential gene expression, and are composed of multiple, transcriptionally similar cell types. They do not represent clonal domains, as revealed by developmental lineage analysis. We propose that the transcriptional domains that subdivide the annelid larval body represent families of related cell types that have arisen by evolutionary diversification. Their possible evolutionary conservation makes them a promising tool for evo-devo research. (167/250).
  • ItemRestrictedAccepted version Peer-reviewed
    Cleavage of TL1A Differentially Regulates Its Effects on Innate and Adaptive Immune Cells.
    (American Association of Immunologists, 2018-02-15) Ferdinand, John R; Richard, Arianne C; Meylan, Françoise; Al-Shamkhani, Aymen; Siegel, Richard M; Ferdinand, John [0000-0003-0936-0128]; Richard, Arianne [0000-0002-8708-9997]
    TNF superfamily cytokines play major roles in the regulation of adaptive and innate immunity. The TNF superfamily cytokine TL1A (TNFSF15), through its cognate receptor DR3 (TNFRSF25), promotes T cell immunity to pathogens and directly costimulates group 2 and 3 innate lymphoid cells. Polymorphisms in theTNFSF15gene are associated with the risk for various human diseases, including inflammatory bowel disease. Like other cytokines in the TNF superfamily, TL1A is synthesized as a type II transmembrane protein and cleaved from the plasma membrane by metalloproteinases. Membrane cleavage has been shown to alter or abrogate certain activities of other TNF family cytokines; however, the functional capabilities of membrane-bound and soluble forms TL1A are not known. Constitutive expression of TL1A in transgenic mice results in expansion of activated T cells and promotes intestinal hyperplasia and inflammation through stimulation of group 2 innate lymphoid cells. Through the generation of membrane-restricted TL1A-transgenic mice, we demonstrate that membrane TL1A promotes expression of inflammatory cytokines in the lung, dependent upon DR3 expression on T cells. Soluble TL1A alone was unable to produce this phenotype but was still able to induce intestinal type 2 inflammation independently of T cells. These data suggest differential roles for membrane and soluble TL1A on adaptive and innate immune cells and have implications for the consequences of blocking these two forms of TL1A.