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1.
EMBO J ; 43(12): 2397-2423, 2024 Jun.
Article En | MEDLINE | ID: mdl-38760575

The nucleoside analogue decitabine (or 5-aza-dC) is used to treat several haematological cancers. Upon its triphosphorylation and incorporation into DNA, 5-aza-dC induces covalent DNA methyltransferase 1 DNA-protein crosslinks (DNMT1-DPCs), leading to DNA hypomethylation. However, 5-aza-dC's clinical outcomes vary, and relapse is common. Using genome-scale CRISPR/Cas9 screens, we map factors determining 5-aza-dC sensitivity. Unexpectedly, we find that loss of the dCMP deaminase DCTD causes 5-aza-dC resistance, suggesting that 5-aza-dUMP generation is cytotoxic. Combining results from a subsequent genetic screen in DCTD-deficient cells with the identification of the DNMT1-DPC-proximal proteome, we uncover the ubiquitin and SUMO1 E3 ligase, TOPORS, as a new DPC repair factor. TOPORS is recruited to SUMOylated DNMT1-DPCs and promotes their degradation. Our study suggests that 5-aza-dC-induced DPCs cause cytotoxicity when DPC repair is compromised, while cytotoxicity in wild-type cells arises from perturbed nucleotide metabolism, potentially laying the foundations for future identification of predictive biomarkers for decitabine treatment.


DNA (Cytosine-5-)-Methyltransferase 1 , Decitabine , Ubiquitin-Protein Ligases , Decitabine/pharmacology , Humans , DNA (Cytosine-5-)-Methyltransferase 1/metabolism , DNA (Cytosine-5-)-Methyltransferase 1/genetics , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/genetics , DNA Methylation/drug effects , Antimetabolites, Antineoplastic/pharmacology , Animals , Sumoylation/drug effects
2.
Nat Cell Biol ; 26(5): 797-810, 2024 May.
Article En | MEDLINE | ID: mdl-38600235

Covalent DNA-protein cross-links (DPCs) are toxic DNA lesions that block replication and require repair by multiple pathways. Whether transcription blockage contributes to the toxicity of DPCs and how cells respond when RNA polymerases stall at DPCs is unknown. Here we find that DPC formation arrests transcription and induces ubiquitylation and degradation of RNA polymerase II. Using genetic screens and a method for the genome-wide mapping of DNA-protein adducts, DPC sequencing, we discover that Cockayne syndrome (CS) proteins CSB and CSA provide resistance to DPC-inducing agents by promoting DPC repair in actively transcribed genes. Consequently, CSB- or CSA-deficient cells fail to efficiently restart transcription after induction of DPCs. In contrast, nucleotide excision repair factors that act downstream of CSB and CSA at ultraviolet light-induced DNA lesions are dispensable. Our study describes a transcription-coupled DPC repair pathway and suggests that defects in this pathway may contribute to the unique neurological features of CS.


Cockayne Syndrome , DNA Helicases , DNA Repair Enzymes , DNA Repair , Poly-ADP-Ribose Binding Proteins , RNA Polymerase II , Transcription, Genetic , Ubiquitination , Poly-ADP-Ribose Binding Proteins/metabolism , Poly-ADP-Ribose Binding Proteins/genetics , DNA Repair Enzymes/metabolism , DNA Repair Enzymes/genetics , Humans , DNA Helicases/metabolism , DNA Helicases/genetics , RNA Polymerase II/metabolism , RNA Polymerase II/genetics , Cockayne Syndrome/genetics , Cockayne Syndrome/metabolism , Cockayne Syndrome/pathology , DNA Damage , Ultraviolet Rays , DNA/metabolism , DNA/genetics , DNA Adducts/metabolism , DNA Adducts/genetics , Excision Repair , Transcription Factors , Receptors, Interleukin-17
3.
Mol Cell ; 83(23): 4290-4303.e9, 2023 Dec 07.
Article En | MEDLINE | ID: mdl-37951216

Reactive aldehydes are abundant endogenous metabolites that challenge homeostasis by crosslinking cellular macromolecules. Aldehyde-induced DNA damage requires repair to prevent cancer and premature aging, but it is unknown whether cells also possess mechanisms that resolve aldehyde-induced RNA lesions. Here, we establish photoactivatable ribonucleoside-enhanced crosslinking (PAR-CL) as a model system to study RNA crosslinking damage in the absence of confounding DNA damage in human cells. We find that such RNA damage causes translation stress by stalling elongating ribosomes, which leads to collisions with trailing ribosomes and activation of multiple stress response pathways. Moreover, we discovered a translation-coupled quality control mechanism that resolves covalent RNA-protein crosslinks. Collisions between translating ribosomes and crosslinked mRNA-binding proteins trigger their modification with atypical K6- and K48-linked ubiquitin chains. Ubiquitylation requires the E3 ligase RNF14 and leads to proteasomal degradation of the protein adduct. Our findings identify RNA lesion-induced translational stress as a central component of crosslinking damage.


RNA , Ubiquitin , Humans , RNA/metabolism , Ubiquitination , Ubiquitin/metabolism , Ribosomes/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Aldehydes , Protein Biosynthesis
4.
Nat Commun ; 14(1): 352, 2023 01 21.
Article En | MEDLINE | ID: mdl-36681662

DNA-protein crosslinks (DPCs) are pervasive DNA lesions that are induced by reactive metabolites and various chemotherapeutic agents. Here, we develop a technique for the Purification of x-linked Proteins (PxP), which allows identification and tracking of diverse DPCs in mammalian cells. Using PxP, we investigate DPC repair in cells genetically-engineered to express variants of the SPRTN protease that cause premature ageing and early-onset liver cancer in Ruijs-Aalfs syndrome patients. We find an unexpected role for SPRTN in global-genome DPC repair, that does not rely on replication-coupled detection of the lesion. Mechanistically, we demonstrate that replication-independent DPC cleavage by SPRTN requires SUMO-targeted ubiquitylation of the protein adduct and occurs in addition to proteasomal DPC degradation. Defective ubiquitin binding of SPRTN patient variants compromises global-genome DPC repair and causes synthetic lethality in combination with a reduction in proteasomal DPC repair capacity.


DNA Damage , DNA-Binding Proteins , Animals , Humans , DNA Damage/genetics , DNA Repair/genetics , DNA-Binding Proteins/metabolism , Mammals/genetics , Proteasome Endopeptidase Complex/metabolism
5.
Cancer Res ; 78(8): 2096-2114, 2018 04 15.
Article En | MEDLINE | ID: mdl-29382705

The myotonic dystrophy-related Cdc42-binding kinases MRCKα and MRCKß contribute to the regulation of actin-myosin cytoskeleton organization and dynamics, acting in concert with the Rho-associated coiled-coil kinases ROCK1 and ROCK2. The absence of highly potent and selective MRCK inhibitors has resulted in relatively little knowledge of the potential roles of these kinases in cancer. Here, we report the discovery of the azaindole compounds BDP8900 and BDP9066 as potent and selective MRCK inhibitors that reduce substrate phosphorylation, leading to morphologic changes in cancer cells along with inhibition of their motility and invasive character. In over 750 human cancer cell lines tested, BDP8900 and BDP9066 displayed consistent antiproliferative effects with greatest activity in hematologic cancer cells. Mass spectrometry identified MRCKα S1003 as an autophosphorylation site, enabling development of a phosphorylation-sensitive antibody tool to report on MRCKα status in tumor specimens. In a two-stage chemical carcinogenesis model of murine squamous cell carcinoma, topical treatments reduced MRCKα S1003 autophosphorylation and skin papilloma outgrowth. In parallel work, we validated a phospho-selective antibody with the capability to monitor drug pharmacodynamics. Taken together, our findings establish an important oncogenic role for MRCK in cancer, and they offer an initial preclinical proof of concept for MRCK inhibition as a valid therapeutic strategy.Significance: The development of selective small-molecule inhibitors of the Cdc42-binding MRCK kinases reveals their essential roles in cancer cell viability, migration, and invasive character. Cancer Res; 78(8); 2096-114. ©2018 AACR.


Antineoplastic Agents/therapeutic use , Carcinoma, Squamous Cell/drug therapy , Drug Discovery , Myotonin-Protein Kinase/antagonists & inhibitors , Protein Kinase Inhibitors/therapeutic use , Pyridines/therapeutic use , Pyrimidines/therapeutic use , Pyrroles/therapeutic use , Skin Neoplasms/drug therapy , Animals , Antineoplastic Agents/pharmacology , Carcinoma, Squamous Cell/enzymology , Cell Line, Tumor , Disease Models, Animal , HEK293 Cells , Humans , Mice , Mice, Nude , Phosphorylation , Protein Kinase Inhibitors/pharmacology , Pyridines/pharmacology , Pyrimidines/pharmacology , Pyrroles/pharmacology , Skin Neoplasms/enzymology , Xenograft Model Antitumor Assays
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