PROTAC-mediated conditional degradation of the WRN helicase as a potential strategy for selective killing of cancer cells with microsatellite instability.

Multiple studies have demonstrated that cancer cells with microsatellite instability (MSI) are intolerant to loss of the Werner syndrome helicase (WRN), whereas microsatellite-stable (MSS) cancer cells are not. Therefore, WRN represents a promising new synthetic lethal target for developing drugs to treat cancers with MSI. Given the uncertainty of how effective inhibitors of WRN activity will prove in clinical trials, and the likelihood of tumours developing resistance to WRN inhibitors, alternative strategies for impeding WRN function are needed. Proteolysis-targeting chimeras (PROTACs) are heterobifunctional small molecules that target specific proteins for degradation. Here, we engineered the WRN locus so that the gene product is fused to a bromodomain (Bd)-tag, enabling conditional WRN degradation with the AGB-1 PROTAC specific for the Bd-tag. Our data revealed that WRN degradation is highly toxic in MSI but not MSS cell lines. In MSI cells, WRN degradation caused G2/M arrest, chromosome breakage and ATM kinase activation. We also describe a multi-colour cell-based platform for facile testing of selective toxicity in MSI versus MSS cell lines. Together, our data show that a degrader approach is a potentially powerful way of targeting WRN in MSI cancers and paves the way for the development of WRN-specific PROTAC compounds.

Chemo-Phosphoproteomic Profiling with ATR Inhibitors Berzosertib and Gartisertib Uncovers New Biomarkers and DNA Damage Response Regulators.

The ATR kinase protects cells against DNA damage and replication stress and represents a promising anti-cancer drug target. The ATR inhibitors (ATRi) berzosertib and gartisertib are both in clinical trials for the treatment of advanced solid tumors as monotherapy or in combination with genotoxic agents. We carried out quantitative phospho-proteomic screening for ATR biomarkers that are highly sensitive to berzosertib and gartisertib, using an optimized mass spectrometry pipeline. Screening identified a range of novel ATR-dependent phosphorylation events, which were grouped into three broad classes: (i) targets whose phosphorylation is highly sensitive to ATRi and which could be the next generation of ATR biomarkers; (ii) proteins with known genome maintenance roles not previously known to be regulated by ATR; (iii) novel targets whose cellular roles are unclear. Class iii targets represent candidate DNA damage response proteins and, with this in mind, proteins in this class were subjected to secondary screening for recruitment to DNA damage sites. We show that one of the proteins recruited, SCAF1, interacts with RNAPII in a phospho-dependent manner and recruitment requires PARP activity and interaction with RNAPII. We also show that SCAF1 deficiency partly rescues RAD51 loading in cells lacking the BRCA1 tumor suppressor. Taken together these data reveal potential new ATR biomarkers and new genome maintenance factors.

Functional characterization of C21ORF2 association with the NEK1 kinase mutated in human in diseases.

The NEK1 kinase controls ciliogenesis, mitosis, and DNA repair, and NEK1 mutations cause human diseases including axial spondylometaphyseal dysplasia and amyotrophic lateral sclerosis. C21ORF2 mutations cause a similar pattern of human diseases, suggesting close functional links with NEK1 Here, we report that endogenous NEK1 and C21ORF2 form a tight complex in human cells. A C21ORF2 interaction domain "CID" at the C-terminus of NEK1 is necessary for its association with C21ORF2 in cells, and pathogenic mutations in this region disrupt the complex. AlphaFold modelling predicts an extended binding interface between a leucine-rich repeat domain in C21ORF2 and the NEK1-CID, and our model may explain why pathogenic mutations perturb the complex. We show that NEK1 mutations that inhibit kinase activity or weaken its association with C21ORF2 severely compromise ciliogenesis, and that C21ORF2, like NEK1 is required for homologous recombination. These data enhance our understanding of how the NEK1 kinase is regulated, and they shed light on NEK1-C21ORF2-associated diseases.

CDKL5 kinase controls transcription-coupled responses to DNA damage.

Mutations in the gene encoding the CDKL5 kinase are among the most common genetic causes of childhood epilepsy and can also give rise to the severe neurodevelopmental condition CDD (CDKL5 deficiency disorder). Despite its importance for human health, the phosphorylation targets and cellular roles of CDKL5 are poorly understood, especially in the cell nucleus. Here, we report that CDKL5 is recruited to sites of DNA damage in actively transcribed regions of the nucleus. A quantitative phosphoproteomic screen for nuclear CDKL5 substrates reveals a network of transcriptional regulators including Elongin A (ELOA), phosphorylated on a specific CDKL5 consensus motif. Recruitment of CDKL5 and ELOA to damaged DNA, and subsequent phosphorylation of ELOA, requires both active transcription and the synthesis of poly(ADP-ribose) (PAR), to which CDKL5 can bind. Critically, CDKL5 kinase activity is essential for the transcriptional silencing of genes induced by DNA double-strand breaks. Thus, CDKL5 is a DNA damage-sensing, PAR-controlled transcriptional modulator, a finding with implications for understanding the molecular basis of CDKL5-related diseases.

Endogenous DNA 3' Blocks Are Vulnerabilities for BRCA1 and BRCA2 Deficiency and Are Reversed by the APE2 Nuclease.

The APEX2 gene encodes APE2, a nuclease related to APE1, the apurinic/apyrimidinic endonuclease acting in base excision repair. Loss of APE2 is lethal in cells with mutated BRCA1 or BRCA2, making APE2 a prime target for homologous recombination-defective cancers. However, because the function of APE2 in DNA repair is poorly understood, it is unclear why BRCA-deficient cells require APE2 for viability. Here we present the genetic interaction profiles of APE2, APE1, and TDP1 deficiency coupled to biochemical and structural dissection of APE2. We conclude that the main role of APE2 is to reverse blocked 3' DNA ends, problematic lesions that preclude DNA synthesis. Our work also suggests that TOP1 processing of genomic ribonucleotides is the main source of 3'-blocking lesions relevant to APEX2-BRCA1/2 synthetic lethality. The exquisite sensitivity of BRCA-deficient cells to 3' blocks indicates that they represent a tractable vulnerability in homologous recombination-deficient tumor cells.

Phosphoproteomic screening identifies physiological substrates of the CDKL5 kinase.

Mutations in the gene encoding the protein kinase CDKL5 cause a debilitating neurodevelopmental disease termed CDKL5 disorder. The impact of these mutations on CDKL5 function is poorly understood because the substrates and cellular processes controlled by CDKL5 are unclear. Here, we describe a quantitative phosphoproteomic screening which identified MAP1S, CEP131 and DLG5-regulators of microtubule and centrosome function-as cellular substrates of CDKL5. Antibodies against MAP1S phospho-Ser900 and CEP131 phospho-Ser35 confirmed CDKL5-dependent phosphorylation of these targets in human cells. The phospho-acceptor serine residues in MAP1S, CEP131 and DLG5 lie in the motif RPXSA, although CDKL5 can tolerate residues other than Ala immediately C-terminal to the phospho-acceptor serine. We provide insight into the control of CDKL5 activity and show that pathogenic mutations in CDKL5 cause a major reduction in CDKL5 activity in vitro and in cells. These data reveal the first cellular substrates of CDKL5, which may represent important biomarkers in the diagnosis and treatment of CDKL5 disorder, and illuminate the functions of this poorly characterized kinase.

Comparing initial diagnostic excision biopsy of cutaneous malignant melanoma in primary versus secondary care: A study of Irish National data.

BACKGROUND: The incidence of melanoma is rising worldwide. Current Irish guidelines from the National Cancer Control Programme state suspicious pigmented lesions should not be removed in primary care. There are conflicting guidelines and research advising who should remove possible melanomas. OBJECTIVES: To determine whether initial diagnostic excision biopsy of cutaneous malignant melanoma in primary versus secondary care leads to poorer survival. METHODS: Analysis of data comprising 7116 cases of cutaneous malignant melanoma from the National Cancer Registry Ireland between January 2002 and December 2011. Single predictor variables were examined by the chi-square or Mann-Whitney U test. The effects of single predictor variables on survival were examined by Cox proportionate hazards modelling and a multivariate Cox model of survival based on excision in a non-hospital setting versus hospital setting was derived with adjusted and unadjusted hazard ratios. RESULTS: Over a 10-year period 8.5% of melanomas in Ireland were removed in a non-hospital setting. When comparing melanoma death between the hospital and non-hospital groups, the adjusted hazard ratio was 1.56 (95%CI: 1.08-2.26); (P = .02), indicating a non-inferior outcome for the melanoma cases initially treated in the non-hospital group, after adjustment for significant covariates. CONCLUSION: This study suggests that initial excision biopsy carried out in general practice does not lead to a poorer outcome. [Box: see text].

Karyomegalic interstitial nephritis and DNA damage-induced polyploidy in Fan1 nuclease-defective knock-in mice.

The Fan1 endonuclease is required for repair of DNA interstrand cross-links (ICLs). Mutations in human Fan1 cause karyomegalic interstitial nephritis (KIN), but it is unclear whether defective ICL repair is responsible or whether Fan1 nuclease activity is relevant. We show that Fan1 nuclease-defective (Fan1(nd/nd)) mice develop a mild form of KIN. The karyomegalic nuclei from Fan1(nd/nd) kidneys are polyploid, and fibroblasts from Fan1(nd/nd) mice become polyploid upon ICL induction, suggesting that defective ICL repair causes karyomegaly. Thus, Fan1 nuclease activity promotes ICL repair in a manner that controls ploidy, a role that we show is not shared by the Fanconi anemia pathway or the Slx4-Slx1 nuclease also involved in ICL repair.

Ubiquitinated Fancd2 recruits Fan1 to stalled replication forks to prevent genome instability.

Mono-ubiquitination of Fancd2 is essential for repairing DNA interstrand cross-links (ICLs), but the underlying mechanisms are unclear. The Fan1 nuclease, also required for ICL repair, is recruited to ICLs by ubiquitinated (Ub) Fancd2. This could in principle explain how Ub-Fancd2 promotes ICL repair, but we show that recruitment of Fan1 by Ub-Fancd2 is dispensable for ICL repair. Instead, Fan1 recruitment--and activity--restrains DNA replication fork progression and prevents chromosome abnormalities from occurring when DNA replication forks stall, even in the absence of ICLs. Accordingly, Fan1 nuclease-defective knockin mice are cancer-prone. Moreover, we show that a Fan1 variant in high-risk pancreatic cancers abolishes recruitment by Ub-Fancd2 and causes genetic instability without affecting ICL repair. Therefore, Fan1 recruitment enables processing of stalled forks that is essential for genome stability and health.

Prospective diagnostic test accuracy comparison of computed tomography during arterial portography and Primovist magnetic resonance imaging in the pre-operative assessment of colorectal cancer liver metastases.

OBJECTIVES: To assess and compare the accuracy and inter-observer agreement for the detection of liver lesions using Primovist magnetic resonance imaging (pMRI) and computed tomography during arterial portography (CTAP). METHODS: Patients evaluated at St George Hospital Liver Unit for colorectal liver metastases (CRCLM) underwent CTAP as part of standard staging. pMRI was added to the pre-operative assessment. Two radiologists reported CTAP and two reported pMRI. The sensitivity and specificity of CTAP and pMRI were calculated using histopathology as the gold standard. RESULTS: Complete data were available for 62 patients corresponding to 219 lesions confirmed on histopathology. Agreement on the detection of lesions between the two radiologists that reported pMRI was higher than for CTAP (Kappa = 0.80 versus 0.74). Specificity of lesion detection for pMRI was 0.88 and 0.83 for CTAP (P = 0.112). Sensitivity for pMRI was 0.83 and 0.81 for CTAP. For patients who had chemotherapy before evaluation, pMRI had a significantly higher specificity than CTAP (0.79 versus 0.63, P = 0.011). CONCLUSIONS: pMRI is less invasive, has a good inter-observer agreement, has comparable sensitivity and specificity to CTAP in the pre-chemotherapy population and demonstrates better specificity in patients assessed post-chemotherapy. pMRI is a valid alternative to CTAP in the assessment of CRCLM.

USP45 deubiquitylase controls ERCC1-XPF endonuclease-mediated DNA damage responses.

Reversible protein ubiquitylation plays important roles in various processes including DNA repair. Here, we identify the deubiquitylase USP45 as a critical DNA repair regulator. USP45 associates with ERCC1, a subunit of the DNA repair endonuclease XPF-ERCC1, via a short acidic motif outside of the USP45 catalytic domain. Wild-type USP45, but not a USP45 mutant defective in ERCC1 binding, efficiently deubiquitylates ERCC1 in vitro, and the levels of ubiquitylated ERCC1 are markedly enhanced in USP45 knockout cells. Cells lacking USP45 are hypersensitive specifically to UV irradiation and DNA interstrand cross-links, similar to cells lacking ERCC1. Furthermore, the repair of UV-induced DNA damage is markedly reduced in USP45-deficient cells. ERCC1 translocation to DNA damage-induced subnuclear foci is markedly impaired in USP45 knockout cells, possibly accounting for defective DNA repair. Finally, USP45 localises to sites of DNA damage in a manner dependent on its deubiquitylase activity, but independent of its ability to bind ERCC1-XPF. Together, these results establish USP45 as a new regulator of XPF-ERCC1 crucial for efficient DNA repair.

Distinct functional roles for the two SLX4 ubiquitin-binding UBZ domains mutated in Fanconi anemia.

Defects in SLX4, a scaffold for DNA repair nucleases, result in Fanconi anemia (FA), due to the defective repair of inter-strand DNA crosslinks (ICLs). Some FA patients have an SLX4 deletion removing two tandem UBZ4-type ubiquitin-binding domains that are implicated in protein recruitment to sites of DNA damage. Here, we show that human SLX4 is recruited to sites of ICL induction but that the UBZ-deleted form of SLX4 in cells from FA patients is not. SLX4 recruitment does not require either the ubiquitylation of FANCD2 or the E3 ligases RNF8, RAD18 and BRCA1. We show that the first (UBZ-1) but not the second UBZ domain of SLX4 binds to ubiquitin polymers, with a preference for K63-linked chains. Furthermore, UBZ-1 is required for SLX4 recruitment to ICL sites and for efficient ICL repair in murine fibroblasts. The SLX4 UBZ-2 domain does not bind to ubiquitin in vitro or contribute to ICL repair, but it is required for the resolution of Holliday junctions in vivo. These data shed light on SLX4 recruitment, and they point to the existence of currently unidentified ubiquitylated ligands and E3 ligases that are crucial for ICL repair.

Localization-dependent and -independent roles of SLX4 in regulating telomeres.

SLX4, a scaffold for structure-specific DNA repair nucleases, is important for several types of DNA repair. Many repair proteins bind to sites of DNA damage, resulting in subnuclear "foci," but SLX4 forms foci in human cells even without DNA damage. Using several approaches, we show that most, but not all, SLX4 foci localize to telomeres in a range of human cell lines irrespective of the mechanisms used to maintain telomere length. The SLX1 Holliday-junction-processing enzyme is recruited to telomeres by SLX4, and SLX4, in turn, is recruited by a motif that binds to the shelterin subunit TRF2 directly. We also show that TRF2-dependent recruitment of SLX4 prevents telomere damage. Furthermore, SLX4 prevents telomere lengthening and fragility in a manner that appears to be independent of telomere association. These findings reveal that SLX4 plays multiple roles in regulating telomere homeostasis.

DVC1 (C1orf124) recruits the p97 protein segregase to sites of DNA damage.

Ubiquitin-binding domains (UBDs) are crucial for recruiting many proteins to sites of DNA damage. Here we characterize C1orf124 (Spartan; referred to as DVC1), which has an UBZ4-type UBD found predominantly in DNA repair proteins. DVC1 associates with DNA replication factories and localizes to sites of DNA damage in human cells, in a manner that requires the ability of the DVC1 UBZ domain to bind to ubiquitin polymers in vitro and a conserved PCNA-interacting motif. DVC1 interacts with the p97 protein 'segregase'. We show that DVC1 recruits p97 to sites of DNA damage, where we propose that p97 facilitates the extraction of the translesion synthesis (TLS) polymerase (Pol) η during DNA repair to prevent excessive TLS and limit the incidence of mutations induced by DNA damage. We introduce DVC1 as a regulator of cellular responses to DNA damage that prevents mutations when DNA damage occurs.

Family with sequence similarity 60A (FAM60A) protein is a cell cycle-fluctuating regulator of the SIN3-HDAC1 histone deacetylase complex.

The SIN3A-HDAC complex deacetylates histones thereby repressing gene transcription. Here we describe family with sequence similarity 60A (FAM60A), a cell cycle-regulated protein that binds to the SIN3-HDAC complex. FAM60A expression peaks during G(1) and S phases of the cell cycle in U2OS cells, in a manner similar to the G(1) regulator cyclin D1, which is a known target of SIN3-HDAC. In this light we found that FAM60A binds to SIN3-HDAC-regulated promoters such as cyclin D1 in G(1) and S phases. Cells depleted of FAM60A show increased histone acetylation at the cyclin D1 promoter and elevated levels of cyclin D1 mRNA and protein. Furthermore, depletion of FAM60A altered the periodic association of HDAC1 with the cyclin D1 promoter, increased cyclin D1 expression at all cell cycle phases, and caused premature S phase entry. The data in this study introduce FAM60A as a novel regulator of SIN3-HDAC function and gene expression.

SLX4, a coordinator of structure-specific endonucleases, is mutated in a new Fanconi anemia subtype.

DNA interstrand crosslink repair requires several classes of proteins, including structure-specific endonucleases and Fanconi anemia proteins. SLX4, which coordinates three separate endonucleases, was recently recognized as an important regulator of DNA repair. Here we report the first human individuals found to have biallelic mutations in SLX4. These individuals, who were previously diagnosed as having Fanconi anemia, add SLX4 as an essential component to the FA-BRCA genome maintenance pathway.

Identification of KIAA1018/FAN1, a DNA repair nuclease recruited to DNA damage by monoubiquitinated FANCD2.

DNA interstrand crosslinks (ICLs) are highly toxic because they block the progression of replisomes. The Fanconi Anemia (FA) proteins, encoded by genes that are mutated in FA, are important for repair of ICLs. The FA core complex catalyzes the monoubiquitination of FANCD2, and this event is essential for several steps of ICL repair. However, how monoubiquitination of FANCD2 promotes ICL repair at the molecular level is unknown. Here, we describe a highly conserved protein, KIAA1018/MTMR15/FAN1, that interacts with, and is recruited to sites of DNA damage by, the monoubiquitinated form of FANCD2. FAN1 exhibits endonuclease activity toward 5' flaps and has 5' exonuclease activity, and these activities are mediated by an ancient VRR_nuc domain. Depletion of FAN1 from human cells causes hypersensitivity to ICLs, defects in ICL repair, and genome instability. These data at least partly explain how ubiquitination of FANCD2 promotes DNA repair.

Vertebroplasty for painful acute osteoporotic vertebral fractures: recent Medical Journal of Australia editorial is not relevant to the patient group that we treat with vertebroplasty.

We use vertebroplasty for patients with the most severe pain caused by osteoporotic vertebral fractures less than 6 weeks old, and have observed dramatic pain relief in this acute setting. A recent editorial in the Journal, written by the authors of two recent vertebroplasty trials, suggested that vertebroplasty is not an effective therapy for acute osteoporotic vertebral fractures. The trials described in the editorial sampled a very different patient cohort to the one that we treat with vertebroplasty. Our clinical experience and most of the published literature relating to the benefits of vertebroplasty are in striking contrast to the opinions presented in that editorial.

Biochemical characterisation of the SWI/SNF family member HLTF.

HLTF is highly similar in domain organisation to yeast Rad5. We identify PTIP and RPA70, both involved in DNA replication and DNA repair, as HLTF-interacting proteins although cells depleted of HLTF did not show defects in cellular responses to DNA damage. In vitro, HLTF has ATPase activity and E3 ubiquitin ligase activity with a range of E2 ubiquitin conjugating enzymes. HLTF expression is severely reduced in a range of cancer cells, and we suggest that the HLTF antibodies generated in this study could be used for cancer diagnostic purposes.