Mutations in the ATM tumor suppressor gene confer hypersensitivity to DNA-damaging chemotherapeutic agents. To explore genetic resistance mechanisms, we performed genome-wide CRISPR-Cas9 screens in ...cells treated with the DNA topoisomerase I inhibitor topotecan. Thus, we here establish that inactivating terminal components of the non-homologous end-joining (NHEJ) machinery or of the BRCA1-A complex specifically confer topotecan resistance to ATM-deficient cells. We show that hypersensitivity of ATM-mutant cells to topotecan or the poly-(ADP-ribose) polymerase (PARP) inhibitor olaparib reflects delayed engagement of homologous recombination at DNA-replication-fork associated single-ended double-strand breaks (DSBs), allowing some to be subject to toxic NHEJ. Preventing DSB ligation by NHEJ, or enhancing homologous recombination by BRCA1-A complex disruption, suppresses this toxicity, highlighting a crucial role for ATM in preventing toxic LIG4-mediated chromosome fusions. Notably, suppressor mutations in ATM-mutant backgrounds are different to those in BRCA1-mutant scenarios, suggesting new opportunities for patient stratification and additional therapeutic vulnerabilities for clinical exploitation.
BRCA1 deficiencies cause breast, ovarian, prostate and other cancers, and render tumours hypersensitive to poly(ADP-ribose) polymerase (PARP) inhibitors. To understand the resistance mechanisms, we ...conducted whole-genome CRISPR-Cas9 synthetic-viability/resistance screens in BRCA1-deficient breast cancer cells treated with PARP inhibitors. We identified two previously uncharacterized proteins, C20orf196 and FAM35A, whose inactivation confers strong PARP-inhibitor resistance. Mechanistically, we show that C20orf196 and FAM35A form a complex, 'Shieldin' (SHLD1/2), with FAM35A interacting with single-stranded DNA through its C-terminal oligonucleotide/oligosaccharide-binding fold region. We establish that Shieldin acts as the downstream effector of 53BP1/RIF1/MAD2L2 to promote DNA double-strand break (DSB) end-joining by restricting DSB resection and to counteract homologous recombination by antagonizing BRCA2/RAD51 loading in BRCA1-deficient cells. Notably, Shieldin inactivation further sensitizes BRCA1-deficient cells to cisplatin, suggesting how defining the SHLD1/2 status of BRCA1-deficient tumours might aid patient stratification and yield new treatment opportunities. Highlighting this potential, we document reduced SHLD1/2 expression in human breast cancers displaying intrinsic or acquired PARP-inhibitor resistance.
The SPRTN metalloprotease is essential for DNA-protein crosslink (DPC) repair and DNA replication in vertebrate cells. Cells deficient in SPRTN protease exhibit DPC-induced replication stress and ...genome instability, manifesting as premature ageing and liver cancer. Here, we provide a body of evidence suggesting that SPRTN activates the ATR-CHK1 phosphorylation signalling cascade during physiological DNA replication by proteolysis-dependent eviction of CHK1 from replicative chromatin. During this process, SPRTN proteolyses the C-terminal/inhibitory part of CHK1, liberating N-terminal CHK1 kinase active fragments. Simultaneously, CHK1 full length and its N-terminal fragments phosphorylate SPRTN at the C-terminal regulatory domain, which stimulates SPRTN recruitment to chromatin to promote unperturbed DNA replication fork progression and DPC repair. Our data suggest that a SPRTN-CHK1 cross-activation loop plays a part in DNA replication and protection from DNA replication stress. Finally, our results with purified components of this pathway further support the proposed model of a SPRTN-CHK1 cross-activation loop.
The coordinated activity of DNA replication factors is a highly dynamic process that involves ubiquitin-dependent regulation. In this context, the ubiquitin-directed ATPase CDC-48/p97 recently ...emerged as a key regulator of chromatin-associated degradation in several of the DNA metabolic pathways that assure genome integrity. However, the spatiotemporal control of distinct CDC-48/p97 substrates in the chromatin environment remained unclear. Here, we report that progression of the DNA replication fork is coordinated by UBXN-3/FAF1. UBXN-3/FAF1 binds to the licensing factor CDT-1 and additional ubiquitylated proteins, thus promoting CDC-48/p97-dependent turnover and disassembly of DNA replication factor complexes. Consequently, inactivation of UBXN-3/FAF1 stabilizes CDT-1 and CDC-45/GINS on chromatin, causing severe defects in replication fork dynamics accompanied by pronounced replication stress and eventually resulting in genome instability. Our work identifies a critical substrate selection module of CDC-48/p97 required for chromatin-associated protein degradation in both Caenorhabditis elegans and humans, which is relevant to oncogenesis and aging.
Nature Communications 7: Article number: 10612 (2016); Published: 4 February 2016; Updated: 17 May 2016. This Article contains errors in the labelling of Fig. 5 and Supplementary Fig. 6, the former ...of which was introduced during the production process. In Fig. 5b, an ‘x’ indicating the presence of UBXN-3(SGG)-HIS in lane 6 was inadvertently omitted.
The DNA damage response (DDR) fulfils essential roles to preserve genome integrity. Targeting the DDR in tumors has had remarkable success over the last decade, exemplified by the licensing of PARP ...inhibitors for cancer therapy. Recent studies suggest that the application of DDR inhibitors impacts on cellular innate and adaptive immune responses, wherein key DNA repair factors have roles in limiting chronic inflammatory signaling. Antitumor immunity plays an emerging part in cancer therapy, and extensive efforts have led to the development of immune checkpoint inhibitors overcoming immune suppressive signals in tumors. Here, we review the current understanding of the molecular mechanisms underlying DNA damage-triggered immune responses, including cytosolic DNA sensing via the cGAS/STING pathway. We highlight the implications of DDR components for therapeutic outcomes of immune checkpoint inhibitors or their use as biomarkers. Finally, we discuss the rationale for novel combinations of DDR inhibitors with antagonists of immune checkpoints and current hindrances limiting their broader therapeutic applications.
We evaluated the properties and activity of AZD9574, a blood-brain barrier (BBB) penetrant selective inhibitor of PARP1, and assessed its efficacy and safety alone and in combination with ...temozolomide (TMZ) in preclinical models.
AZD9574 was interrogated in vitro for selectivity, PARylation inhibition, PARP-DNA trapping, the ability to cross the BBB, and the potential to inhibit cancer cell proliferation. In vivo efficacy was determined using subcutaneous as well as intracranial mouse xenograft models. Mouse, rat, and monkey were used to assess AZD9574 BBB penetration and rat models were used to evaluate potential hematotoxicity for AZD9574 monotherapy and the TMZ combination.
AZD9574 demonstrated PARP1-selectivity in fluorescence anisotropy, PARylation, and PARP-DNA trapping assays and in vivo experiments demonstrated BBB penetration. AZD9574 showed potent single agent efficacy in preclinical models with homologous recombination repair deficiency in vitro and in vivo. In an O6-methylguanine-DNA methyltransferase (MGMT)-methylated orthotopic glioma model, AZD9574 in combination with TMZ was superior in extending the survival of tumor-bearing mice compared with TMZ alone.
The combination of three key features-PARP1 selectivity, PARP1 trapping profile, and high central nervous system penetration in a single molecule-supports the development of AZD9574 as the best-in-class PARP inhibitor for the treatment of primary and secondary brain tumors. As documented by in vitro and in vivo studies, AZD9574 shows robust anticancer efficacy as a single agent as well as in combination with TMZ. AZD9574 is currently in a phase I trial (NCT05417594). See related commentary by Lynce and Lin, p. 1217.
The AAA+ ATPase VCP regulates the extraction of SUMO and ubiquitin-modified DNA replication factors from chromatin. We have previously described that active DNA synthesis is associated with a ...SUMO-high/ubiquitin-low environment governed by the deubiquitylase USP7. Here, we unveil a functional cooperation between USP7 and VCP in DNA replication, which is conserved from Caenorhabditis elegans to mammals. The role of VCP in chromatin is defined by its cofactor FAF1, which facilitates the extraction of SUMOylated and ubiquitylated proteins that accumulate after the block of DNA replication in the absence of USP7. The inactivation of USP7 and FAF1 is synthetically lethal both in C. elegans and mammalian cells. In addition, USP7 and VCP inhibitors display synergistic toxicity supporting a functional link between deubiquitylation and extraction of chromatin-bound proteins. Our results suggest that USP7 and VCPFAF1 facilitate DNA replication by controlling the balance of SUMO/Ubiquitin-modified DNA replication factors on chromatin.
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•VCP and USP7 control the SUMO/ubiquitin landscape during DNA replication•VCP is recruited by FAF1 to chromatin-associated SUMO/ubiquitylated proteins•FAF1 recognizes both SUMO and ubiquitin on target proteins•The control of DNA replication by VCPFAF1 and USP7 is evolutionarily conserved
Chromatin at DNA replication forks is SUMO-rich and ubiquitin-poor. Franz et al. report that FAF1 is a dual reader of SUMO and ubiquitin targeting VCP to extract DNA replication factors from chromatin in C. elegans and human cells. Together with USP7, VCPFAF1 controls the SUMO/ubiquitin landscape during replication.
Abstract
The Poly (ADP-ribose) polymerase (PARP) family has numerous essential functions in cellular processes such as transcription, chromatin remodelling, DNA damage response and repair as well as ...apoptosis. PARP inhibition blocks base excision repair and results in conversion of SSBs to DNA double-strand break (DSBs), the most deleterious form of DNA damage. DSBs can be repaired by homologous recombination repair (HRR) or non-homologous end joining (NHEJ). The physiological importance of HRR is underscored by the observation of genomic instability in HRR-deficient (HRD+) cells and, importantly, the association of cancer predisposition and developmental defects with mutations in HRR genes. PARP1 and PARP2 are required for SSB repair, while PARP1 is also involved in the repair of DNA double-strand breaks (DSBs) and replication fork damage. AZD9574 is a novel brain penetrant PARP1 inhibitor that acts by selectively inhibiting and trapping PARP1 at the sites of SSBs. While AZD9574 inhibited PARP1 enzymatic activity in all tested cell lines irrespective of the HRR status (IC50 range between 0.3 – 2 nM), colony formation assay in isogenic cell lines pairs confirmed higher potency and selectivity towards HRD+ models. In vivo, AZD9574 demonstrated dose-dependent efficacy in a BRCA1 mutant MDA-MB-436 subcutaneous xenograft model. Anti-tumour effects of AZD9574 were manifested by significant growth regressions that were durable after treatment withdrawal. An intracranial xenograft model of breast cancer brain metastases was developed to assess the efficacy of AZD9574 in the context of blood-brain barrier penetrance. Treatment of animals with established intracranial lesions showed sustained tumour growth suppression resulting in a significantly extended survival of tumour-bearing mice. Collectively, we believe that our data support the development of AZD9574 as a potential therapy for patients with HRD+ breast cancer whose disease has spread to the brain.This abstract was previously presented at AACR 2022 (Hamerlik et al, AACR 2022, Abs #3880)
The AAA
ATPase VCP regulates the extraction of SUMO and ubiquitin-modified DNA replication factors from chromatin. We have previously described that active DNA synthesis is associated with a ...SUMO-high/ubiquitin-low environment governed by the deubiquitylase USP7. Here, we unveil a functional cooperation between USP7 and VCP in DNA replication, which is conserved from Caenorhabditis elegans to mammals. The role of VCP in chromatin is defined by its cofactor FAF1, which facilitates the extraction of SUMOylated and ubiquitylated proteins that accumulate after the block of DNA replication in the absence of USP7. The inactivation of USP7 and FAF1 is synthetically lethal both in C. elegans and mammalian cells. In addition, USP7 and VCP inhibitors display synergistic toxicity supporting a functional link between deubiquitylation and extraction of chromatin-bound proteins. Our results suggest that USP7 and VCP
facilitate DNA replication by controlling the balance of SUMO/Ubiquitin-modified DNA replication factors on chromatin.
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