Loss of Integrin-linked kinase sensitizes breast cancer to SRC inhibitors

SRC is a nonreceptor tyrosine kinase with key roles in breast cancer development and progression. Despite this, SRC tyrosine kinase inhibitors have so far failed to live up to their promise in clinical trials, with poor overall response rates. We aimed to identify possible synergistic gene–drug interactions to discover new rational combination therapies for SRC inhibitors. An unbiased genome-wide CRISPR-Cas9 knockout screen in a model of triple-negative breast cancer revealed that loss of integrin-linked kinase (ILK) and its binding partners α-Parvin and PINCH-1 sensitizes cells to bosutinib, a clinically approved SRC/ABL kinase inhibitor. Sensitivity to bosutinib did not correlate with ABL dependency; instead, bosutinib likely induces these effects by acting as a SRC tyrosine kinase inhibitor. Furthermore, in vitro and in vivo models showed that loss of ILK enhanced sensitivity to eCF506, a novel and highly selective inhibitor of SRC with a unique mode of action. Whole-genome RNA sequencing following bosutinib treatment in ILK knockout cells identified broad changes in the expression of genes regulating cell adhesion and cell–extracellular matrix. Increased sensitivity to SRC inhibition in ILK knockout cells was associated with defective adhesion, resulting in reduced cell number as well as increased G(1) arrest and apoptosis. These findings support the potential of ILK loss as an exploitable therapeutic vulnerability in breast cancer, enhancing the effectiveness of clinical SRC inhibitors. SIGNIFICANCE: A CRISPR-Cas9 screen reveals that loss of integrin-linked kinase synergizes with SRC inhibition, providing a new opportunity for enhancing the clinical effectiveness of SRC inhibitors in breast cancer

PRIM1 Deficiency Causes a Distinctive Primordial Dwarfism Syndrome

DNA replication is fundamental for cell proliferation in all organisms. Nonetheless, components of the replisome have been implicated in human disease, and here we report PRIM1 encoding the catalytic subunit of DNA primase as a novel disease gene. Using a variant classification agnostic approach, biallelic mutations in PRIM1 were identified in five individuals. PRIM1 protein levels were markedly reduced in patient cells, accompanied by replication fork asymmetry, increased interorigin distances, replication stress, and prolonged S-phase duration. Consequently, cell proliferation was markedly impaired, explaining the patients' extreme growth failure. Notably, phenotypic features distinct from those previously reported with DNA polymerase genes were evident, highlighting differing developmental requirements for this core replisome component that warrant future investigation

RNase H2, mutated in Aicardi-Goutières syndrome, promotes LINE-1 retrotransposition

Long INterspersed Element class 1 (LINE-1) elements are a type of abundant retrotransposons active in mammalian genomes. An average human genome contains ~100 retrotransposition-competent LINE-1s, whose activity is influenced by the combined action of cellular repressors and activators. TREX1, SAMHD1 and ADAR1 are known LINE-1 repressors and when mutated cause the autoinflammatory disorder Aicardi-Goutières syndrome (AGS). Mutations in RNase H2 are the most common cause of AGS, and its activity was proposed to similarly control LINE-1 retrotransposition. It has therefore been suggested that increased LINE-1 activity may be the cause of aberrant innate immune activation in AGS. Here, we establish that, contrary to expectations, RNase H2 is required for efficient LINE-1 retrotransposition. As RNase H1 overexpression partially rescues the defect in RNase H2 null cells, we propose a model in which RNase H2 degrades the LINE-1 RNA after reverse transcription, allowing retrotransposition to be completed. This also explains how LINE-1 elements can retrotranspose efficiently without their own RNase H activity. Our findings appear to be at odds with LINE-1-derived nucleic acids driving autoinflammation in AGS.M.B.-G. is funded by a “Formacion Profesorado Universitario” (FPU) PhD fellowship from the Government of Spain (MINECO, Ref FPU15/03294), and this paper is part of her thesis project (“Epigenetic control of the mobility of a human retrotransposon”). R.V.-A. is funded by a PFIS Fellowship from the Government of Spain (ISCiii, FI16/00413). O.M. is funded by an EMBO Long-Term Fellowship (ALTF 7-2015), the European Commission FP7 (Marie Curie Actions, LTFCOFUND2013, GA-2013-609409) and the Swiss National Science Foundation (P2ZHP3_158709). S.R.H. is funded by the Government of Spain (MINECO, RYC-2016-21395 and SAF2015-71589-P). A.P.J’s laboratory is supported by the UK Medical Research Council (MRC University Unit grant U127527202). J.L.G.P’s laboratory is supported by CICEFEDER- P12-CTS-2256, Plan Nacional de I+D+I 2008-2011 and 2013-2016 (FISFEDER- PI14/02152), PCIN-2014-115-ERA-NET NEURON II, the European Research Council (ERC-Consolidator ERC-STG-2012-233764), by an International Early Career Scientist grant from the Howard Hughes Medical Institute (IECS-55007420), by The Wellcome Trust-University of Edinburgh Institutional Strategic Support Fund (ISFF2) and by a private donation from Ms Francisca Serrano (Trading y Bolsa para Torpes, Granada, Spain)

TRAIP promotes DNA damage response during genome replication and is mutated in primordial dwarfism.

DNA lesions encountered by replicative polymerases threaten genome stability and cell cycle progression. Here we report the identification of mutations in TRAIP, encoding an E3 RING ubiquitin ligase, in patients with microcephalic primordial dwarfism. We establish that TRAIP relocalizes to sites of DNA damage, where it is required for optimal phosphorylation of H2AX and RPA2 during S-phase in response to ultraviolet (UV) irradiation, as well as fork progression through UV-induced DNA lesions. TRAIP is necessary for efficient cell cycle progression and mutations in TRAIP therefore limit cellular proliferation, providing a potential mechanism for microcephaly and dwarfism phenotypes. Human genetics thus identifies TRAIP as a component of the DNA damage response to replication-blocking DNA lesions.This work was supported by funding from the Medical Research Council and the European Research Council (ERC, 281847) (A.P.J.), the Lister Institute for Preventative Medicine (A.P.J. and G.S.S.), Medical Research Scotland (L.S.B.), German Federal Ministry of Education and Research (BMBF, 01GM1404) and E-RARE network EuroMicro (B.W), Wellcome Trust (M. Hurles), CMMC (P.N.), Cancer Research UK (C17183/A13030) (G.S.S. and M.R.H), Swiss National Science Foundation (P2ZHP3_158709) (O.M.), AIRC (12710) and ERC/EU FP7 (CIG_303806) (S.S.), Cancer Research UK (C6/A11224) and ERC/EU FP7 (HEALTH-F2- 2010-259893) (A.N.B. and S.P.J.).This is the author accepted manuscript. The final version is available from NPG via http://dx.doi.org/10.1038/ng.345

Mutations in DONSON disrupt replication fork stability and cause microcephalic dwarfism

To ensure efficient genome duplication, cells have evolved numerous factors that promote unperturbed DNA replication and protect, repair and restart damaged forks. Here we identify downstream neighbor of SON (DONSON) as a novel fork protection factor and report biallelic DONSON mutations in 29 individuals with microcephalic dwarfism. We demonstrate that DONSON is a replisome component that stabilizes forks during genome replication. Loss of DONSON leads to severe replication-associated DNA damage arising from nucleolytic cleavage of stalled replication forks. Furthermore, ATM- and Rad3-related (ATR)-dependent signaling in response to replication stress is impaired in DONSON-deficient cells, resulting in decreased checkpoint activity and the potentiation of chromosomal instability. Hypomorphic mutations in DONSON substantially reduce DONSON protein levels and impair fork stability in cells from patients, consistent with defective DNA replication underlying the disease phenotype. In summary, we have identified mutations in DONSON as a common cause of microcephalic dwarfism and established DONSON as a critical replication fork protein required for mammalian DNA replication and genome stability

Molecular and cellular aspects of DNA-end resection by human CtlP

Die Erbinformation (DNA) in unseren Zellen wird durch eine Vielzahl von Umwelteinflüssen konstant beschädigt. DNA Doppelstrangbrüche (DSB) gehören zu den gefährlichsten aller DNA Schäden, weil sie ohne Reparatur zum Tod einer Zelle führen können. Zudem besteht eine direkte Verbindung zwischen falscher DSB Reparatur und Krebs. Zellen besitzen zwei verschiedene Systeme für die DSB Reparatur: Nicht- homologe Enden-Verknüpfung (NHEJ) und Homologe Rekombination (HR). Im Unterschied zu NHEJ, müssen für HR die Enden der Brüche zuerst prozessiert werden, sodass einzelsträngige DNA Überhänge entstehen. An diesem Schritt, der als 'DNA- Endresektion' bezeichnet wird, ist das CtIP Protein massgeblich beteiligt. Der erste Teil meiner Doktorarbeit beschäftigt sich mit der Frage ob CtIP auch in der Reparatur von kovalent-quervernetzten DNA Strängen, sogenannten 'interstrand crosslinks' (ICL), eine Rolle spielt und ob ein Zusammenspiel zwischen DNA- Endresektion und dem Fanconi anemia (FA) Signalweg existiert. Die FA-Proteine steuern die komplexe Reparatur von ICLs, indem sie diese im Chromatin erkennen, prozessieren und, in Zusammenarbeit mit anderen Reparatursystemen inklusive der HR, korrigieren. Wir zeigen, dass die Bindung von CtIP an ICL-geschädigtes Chromatin von der Funktion des FA-Kernkomplexes und der Mono-Ubiquitinierung des FANCD2 Proteins abhängig ist. Wir legen außerdem dar, dass die Lokalisierung von CtIP an ICLs durch die direkte Interaktion von CtIP mit FANCD2 vermittelt und möglicherweise durch die Fähigkeit von CtIP ubiquitinierte Substrate zu binden verstärkt wird. Bemerkenswerterweise haben Zellen in denen FANCD2 inaktiviert ist, einen ähnlichen Defekt in der DNA-Endresektion wie CtIP-defiziente Zellen. Zudem haben wir eine FANCD2-Bindestelle innerhalb des CtIP Proteins identifizieren können, welche für eine fehlerfreie Reparatur von ICLs erforderlich ist und somit potentielle, durch NHEJ- verursachte, chromosomale Aberrationen verhindert. Interessant ist auch, dass charakteristische Phänotypen von FANCD2-defizienten Zellen, wie genomische Instabilität und Hypersensitivität gegenüber ICL-induzierenden Stoffen, durch eine zusätzlichen Verlust von CtIP verschlimmert werden. Daraus schließen wir, dass CtIP auch in Abwesenheit von FANCD2 eine bedeutende Funktion für die ICL Reparatur hat. Insgesamt zeigen unsere Ergebnisse, dass FANCD2 ein wichtiger Regulator der CtIP- vermittelten DNA-Endresektion darstellt und dass CtIP eine essenzielle Rolle in der Aufrechterhaltung der Stabilität unseres Genoms spielt. Im zweiten Teil meiner Doktorarbeit untersuchen wir eine spezielle Klasse von Mutationen des menschlichen RAD50 Proteins, genannt RAD50S, die in Hefe zu einer Trennung verschiedener Funktionalitäten des Proteins führt. Bei RAD50 handelt es sich um eine Untereinheit des MRE11-RAD50-NBS1 (MRN) Komplexes, der mit CtIP interagiert und mehrere Aufgaben in der DSB Reparatur spielt. Wir zeigen, dass RAD50S Mutationen die DNA-Endresektion und DSB Reparatur beeinträchtigt, vor allem wenn Zellen mit DNA Topoisomerase-hemmenden Substanzen behandelt werden. RAD50S Mutationen führten jedoch weder zu einer veränderten Struktur des MRN Komplexes, noch zu einer Beeinträchtigung MRN-abhängiger Signalwege nach Behandlung von Zellen mit anderen DNA-schädigenden Substanzen. Basierend auf unseren biochemischen Daten kommen wir zum Schluss, dass die von uns beobachteten zellulären Phänotypen höchstwahrscheinlich durch eine Störung der Interaktion zwischen RAD50S und CtIP hervorgerufen werden, welche besonders für die Reparatur blockierter Topoisomerase Moleküle an DSB Enden von Bedeutung ist. Beide Studien eingeschlossen, belegen unsere Ergebnisse dass CtIP durch Interaktionen mit FANCD2 und RAD50 eine Schlüsselrolle bei der Reparatur von DNA Schäden spielt und somit womöglich ein wichtiger Faktor zur Verhinderung von Krebs ist. SUMMARY Our genome is under constant threat from DNA damage that inflicts different kinds of lesions including DNA double-strand breaks (DSBs). Failure to correctly repair DSBs can cause gross chromosomal aberrations, which are a hallmark of cancer. Cells have evolved two major pathways to repair DSBs: non-homologous end-joining (NHEJ) and homologous recombination (HR). Human CtIP promotes DNA-end resection, which commits cells to error-free HR and prevents aberrant repair by NHEJ, hence it is a critical determinant of DSB repair pathway choice. However, the cellular response to DNA damage involves a complex interplay between different genome maintenance pathways and the role of CtIP in this multifaceted network is still poorly understood. In the first part of my PhD study, we addressed the functional interplay between CtIP- dependent resection and the Fanconi anemia (FA) pathway during the repair of DNA interstrand crosslinks (ICLs). Fanconi anemia is an inherited disorder associated with a high risk to develop cancer and is caused by mutations in sixteen FA genes. Together, the FA proteins orchestrate ICL incision, translesion synthesis and HR. We demonstrate that chromatin association of CtIP in response to ICL-induced damage is strictly dependent on a functional FA core complex and FANCD2 monoubiquitination. Furthermore, we show that CtIP recruitment to ICL lesions is mediated by its direct interaction with FANCD2 and might be further reinforced by the discovered ability of CtIP to recognize ubiquitinated substrates. Remarkably, cells lacking FANCD2 akin to CtIP-depleted cells are impaired in DNA-end resection. We have identified FANCD2-binding sites on CtIP and provide evidence that CtIP- FANCD2 complex is required for the faithful repair of ICLs, meanwhile counteracting mutagenic NHEJ pathway. Interestingly, the phenotypes of FA cells such as genome instability and ICL hypersensitivity are further aggravated by CtIP depletion, indicating the significance of CtIP even in the absence of proficient FA pathway. Taken together, our data establish FANCD2 as a critical regulator of CtIP-mediated DNA-end resection and emphasize the essential role of CtIP in maintaining genome stability in response to ICL damage. In the second part, we examined the phenotypes of the separation-of-function (S) mutations in human RAD50, a subunit of the MRE11-RAD50-NBS1 (MRN) complex that interacts with CtIP and plays crucial roles in DSB signaling and processing. We demonstrate that RAD50S mutants compromise resection and repair of DSBs induced specifically by DNA topoisomerase poisons, but do neither alter MRN complex integrity nor significantly affect MRN-dependent signaling in response to other types of DNA damaging agents. Based on our biochemical data we suggest that these phenotypes are caused by the impaired interaction between RAD50S mutants and CtIP, which is particularly important for the processing of topoisomerases trapped to DNA ends. Collectively, our results establish a key role for CtIP in the repair of ICLs and also highlight the significance of CtIP-MRN association for the processing of toxic protein-DNA adducts. Work presented in my thesis thus advances the understanding of how the DNA-end resection activity of CtIP is regulated to preserve genome stability

Molecular and Cellular Aspects of DNA-end Resection by Human CtIP

Our genome is under constant threat from DNA damage that inflicts different kinds of lesions including DNA double-strand breaks (DSBs). Failure to correctly repair DSBs can cause gross chromosomal aberrations, which are a hallmark of cancer. Cells have evolved two major pathways to repair DSBs: non-homologous end-joining (NHEJ) and homologous recombination (HR). Human CtIP promotes DNA-end resection, which commits cells to error-free HR and prevents aberrant repair by NHEJ, hence it is a critical determinant of DSB repair pathway choice. However, the cellular response to DNA damage involves a complex interplay between different genome maintenance pathways and the role of CtIP in this multifaceted network is still poorly understood. In the first part of my PhD study, we addressed the functional interplay between CtIP- dependent resection and the Fanconi anemia (FA) pathway during the repair of DNA interstrand crosslinks (ICLs). Fanconi anemia is an inherited disorder associated with a high risk to develop cancer and is caused by mutations in sixteen FA genes. Together, the FA proteins orchestrate ICL incision, translesion synthesis and HR. We demonstrate that chromatin association of CtIP in response to ICL-induced damage is strictly dependent on a functional FA core complex and FANCD2 monoubiquitination. Furthermore, we show that CtIP recruitment to ICL lesions is mediated by its direct interaction with FANCD2 and might be further reinforced by the discovered ability of CtIP to recognize ubiquitinated substrates. Remarkably, cells lacking FANCD2 akin to CtIP-depleted cells are impaired in DNA-end resection. We have identified FANCD2-binding sites on CtIP and provide evidence that CtIP-FANCD2 complex is required for the faithful repair of ICLs, meanwhile counteracting mutagenic NHEJ pathway. Interestingly, the phenotypes of FA cells such as genome instability and ICL hypersensitivity are further aggravated by CtIP depletion, indicating the significance of CtIP even in the absence of proficient FA pathway. Taken together, our data establish FANCD2 as a critical regulator of CtIP- mediated DNA-end resection and emphasize the essential role of CtIP in maintaining genome stability in response to ICL damage. In the second part, we examined the phenotypes of the separation-of-function (S) mutations in human RAD50, a subunit of the MRE11-RAD50-NBS1 (MRN) complex that interacts with CtIP and plays crucial roles in DSB signaling and processing. We demonstrate that RAD50S mutants compromise resection and repair of DSBs induced specifically by DNA topoisomerase poisons, but do neither alter MRN complex integrity nor significantly affect MRN-dependent signaling in response to other types of DNA damaging agents. Based on our biochemical data we suggest that these phenotypes are caused by the impaired interaction between RAD50S mutants and CtIP, which is particularly important for the processing of topoisomerases trapped to DNA ends. Collectively, our results establish a key role for CtIP in the repair of ICLs and also highlight the significance of CtIP-MRN association for the processing of toxic protein- DNA adducts. Work presented in my thesis thus advances the understanding of how the DNA-end resection activity of CtIP is regulated to preserve genome stability

The paradox of viable sup45 STOP mutations: a necessary equilibrium between translational readthrough, activity and stability of the protein.

International audienceThe mechanisms leading to non-lethality of nonsense mutations in essential genes are poorly understood. Here, we focus on the factors influencing viability of yeast cells bearing premature termination codons (PTCs) in the essential gene SUP45 encoding translation termination factor eRF1. Using a dual reporter system we compared readthrough efficiency of the natural termination codon of SUP45 gene, spontaneous sup45-n (nonsense) mutations, nonsense mutations obtained by site-directed mutagenesis (76Q --> TAA, 242R --> TGA, 317L --> TAG). The nonsense mutations in SUP45 gene were shown to be situated in moderate contexts for readthrough efficiency. We showed that readthrough efficiency of some of the mutations present in the sup45 mutants is not correlated with full-length Sup45 protein amount. This resulted from modification of both sup45 mRNA stability which varies 3-fold among sup45-n mutants and degradation rate of mutant Sup45 proteins. Our results demonstrate that some substitutions in the place of PTCs decrease Sup45 stability. The viability of sup45 nonsense mutants is therefore supported by diverse mechanisms that control the final amount of functional Sup45 in cells

Prolyl isomerase PIN1 regulates DNA double-strand break repair by Counteracting DNA end resection

The regulation of DNA double-strand break (DSB) repair by phosphorylation-dependent signaling pathways is crucial for the maintenance of genome stability; however, remarkably little is known about the molecular mechanisms by which phosphorylation controls DSB repair. Here, we show that PIN1, a phosphorylation-specific prolyl isomerase, interacts with key DSB repair factors and affects the relative contributions of homologous recombination (HR) and nonhomologous end-joining (NHEJ) to DSB repair. We find that PIN1-deficient cells display reduced NHEJ due to increased DNA end resection, whereas resection and HR are compromised in PIN1-overexpressing cells. Moreover, we identify CtIP as a substrate of PIN1 and show that DSBs become hyperresected in cells expressing a CtIP mutant refractory to PIN1 recognition. Mechanistically, we provide evidence that PIN1 impinges on CtIP stability by promoting its ubiquitylation and subsequent proteasomal degradation. Collectively, these data uncover PIN1-mediated isomerization as a regulatory mechanism coordinating DSB repair