MTH1 Inhibitor TH588 Disturbs Mitotic Progression and Induces Mitosis-Dependent Accumulation of Genomic 8-oxodG.

Reactive oxygen species (ROS) oxidize nucleotide triphosphate pools (e.g., 8-oxodGTP), which may kill cells if incorporated into DNA. Whether cancers avoid poisoning from oxidized nucleotides by preventing incorporation via the oxidized purine diphosphatase MTH1 remains under debate. Also, little is known about DNA polymerases incorporating oxidized nucleotides in cells or how oxidized nucleotides in DNA become toxic. Here we show that replacement of one of the main DNA replicases in human cells, DNA polymerase delta (Pol δ), with an error-prone variant allows increased 8-oxodG accumulation into DNA following treatment with TH588, a dual MTH1 inhibitor and microtubule targeting agent. The resulting elevated genomic 8-oxodG correlated with increased cytotoxicity of TH588. Interestingly, no substantial perturbation of replication fork progression was observed, but rather mitotic progression was impaired and mitotic DNA synthesis triggered. Reducing mitotic arrest by reversin treatment prevented accumulation of genomic 8-oxodG and reduced cytotoxicity of TH588, in line with the notion that mitotic arrest is required for ROS buildup and oxidation of the nucleotide pool. Furthermore, delayed mitosis and increased mitotic cell death was observed following TH588 treatment in cells expressing the error-prone but not wild-type Pol δ variant, which is not observed following treatments with antimitotic agents. Collectively, these results link accumulation of genomic oxidized nucleotides with disturbed mitotic progression. SIGNIFICANCE: These findings uncover a novel link between accumulation of genomic 8-oxodG and perturbed mitotic progression in cancer cells, which can be exploited therapeutically using MTH1 inhibitors.See related commentary by Alnajjar and Sweasy, p. 3459.

Nuclear F-actin and myosins drive relocalization of heterochromatic breaks.

Heterochromatin mainly comprises repeated DNA sequences that are prone to ectopic recombination. In Drosophila cells, 'safe' repair of heterochromatic double-strand breaks by homologous recombination relies on the relocalization of repair sites to the nuclear periphery before strand invasion. The mechanisms responsible for this movement were unknown. Here we show that relocalization occurs by directed motion along nuclear actin filaments assembled at repair sites by the Arp2/3 complex. Relocalization requires nuclear myosins associated with the heterochromatin repair complex Smc5/6 and the myosin activator Unc45, which is recruited to repair sites by Smc5/6. ARP2/3, actin nucleation and myosins also relocalize heterochromatic double-strand breaks in mouse cells. Defects in this pathway result in impaired heterochromatin repair and chromosome rearrangements. These findings identify de novo nuclear actin filaments and myosins as effectors of chromatin dynamics for heterochromatin repair and stability in multicellular eukaryotes.

Germline variation in the oxidative DNA repair genes NUDT1 and OGG1 is not associated with hereditary colorectal cancer or polyposis.

The causal association of NUDT1 (=MTH1) and OGG1 with hereditary colorectal cancer (CRC) remains unclear. Here, we sought to provide additional evidence for or against the causal contribution of NUDT1 and OGG1 mutations to hereditary CRC and/or polyposis. Mutational screening was performed using pooled DNA amplification and targeted next-generation sequencing in 529 families (441 uncharacterized MMR-proficient familial nonpolyposis CRC and 88 polyposis cases). Cosegregation, in silico analyses, in vitro functional assays, and case-control associations were carried out to characterize the identified variants. Five heterozygous carriers of novel (n = 1) or rare (n = 4) NUDT1 variants were identified. In vitro deleterious effects were demonstrated for c.143G>A p.G48E (catalytic activity and protein stability) and c.403G>T p.G135W (protein stability), although cosegregation data in the carrier families were inconclusive or nonsupportive. The frequency of missense, loss-of-function, and splice-site NUDT1 variants in our familial CRC cohort was similar to the one observed in cancer-free individuals, suggesting lack of association with CRC predisposition. No OGG1 pathogenic mutations were identified. Our results suggest that the contribution of NUDT1 and OGG1 germline mutations to hereditary CRC and to polyposis is inexistent or, at most, negligible. The inclusion of these genes in routine genetic testing is not recommended.

The Aurora-B-dependent NoCut checkpoint prevents damage of anaphase bridges after DNA replication stress.

Anaphase chromatin bridges can lead to chromosome breakage if not properly resolved before completion of cytokinesis. The NoCut checkpoint, which depends on Aurora B at the spindle midzone, delays abscission in response to chromosome segregation defects in yeast and animal cells. How chromatin bridges are detected, and whether abscission inhibition prevents their damage, remain key unresolved questions. We find that bridges induced by DNA replication stress and by condensation or decatenation defects, but not dicentric chromosomes, delay abscission in a NoCut-dependent manner. Decatenation and condensation defects lead to spindle stabilization during cytokinesis, allowing bridge detection by Aurora B. NoCut does not prevent DNA damage following condensin or topoisomerase II inactivation; however, it protects anaphase bridges and promotes cellular viability after replication stress. Therefore, the molecular origin of chromatin bridges is critical for activation of NoCut, which plays a key role in the maintenance of genome stability after replicative stress.