Insights from multi-omic modeling of neurodegeneration in xeroderma pigmentosum using an induced pluripotent stem cell system.

Xeroderma pigmentosum (XP) is caused by defective nucleotide excision repair of DNA damage. This results in hypersensitivity to ultraviolet light and increased skin cancer risk, as sunlight-induced photoproducts remain unrepaired. However, many XP patients also display early-onset neurodegeneration, which leads to premature death. The mechanism of neurodegeneration is unknown. Here, we investigate XP neurodegeneration using pluripotent stem cells derived from XP patients and healthy relatives, performing functional multi-omics on samples during neuronal differentiation. We show substantially increased levels of 5',8-cyclopurine and 8-oxopurine in XP neuronal DNA secondary to marked oxidative stress. Furthermore, we find that the endoplasmic reticulum stress response is upregulated and reversal of the mutant genotype is associated with phenotypic rescue. Critically, XP neurons exhibit inappropriate downregulation of the protein clearance ubiquitin-proteasome system (UPS). Chemical enhancement of UPS activity in XP neuronal models improves phenotypes, albeit inadequately. Although more work is required, this study presents insights with intervention potential.

The chemotherapeutic drug CX-5461 is a potent mutagen in cultured human cells.

The chemotherapeutic agent CX-5461, or pidnarulex, has been fast-tracked by the United States Food and Drug Administration for early-stage clinical studies of BRCA1-, BRCA2- and PALB2-mutated cancers. It is under investigation in phase I and II trials. Here, we find that, although CX-5461 exhibits synthetic lethality in BRCA1-/BRCA2-deficient cells, it also causes extensive, nonselective, collateral mutagenesis in all three cell lines tested, to magnitudes that exceed known environmental carcinogens.

Substitution mutational signatures in whole-genome-sequenced cancers in the UK population.

Whole-genome sequencing (WGS) permits comprehensive cancer genome analyses, revealing mutational signatures, imprints of DNA damage and repair processes that have arisen in each patient's cancer. We performed mutational signature analyses on 12,222 WGS tumor-normal matched pairs, from patients recruited via the UK National Health Service. We contrasted our results to two independent cancer WGS datasets, the International Cancer Genome Consortium (ICGC) and Hartwig Foundation, involving 18,640 WGS cancers in total. Our analyses add 40 single and 18 double substitution signatures to the current mutational signature tally. Critically, we show for each organ, that cancers have a limited number of 'common' signatures and a long tail of 'rare' signatures. We provide a practical solution for utilizing this concept of common versus rare signatures in future analyses.

All Driven by Energy Demand? Integrative Comparison of Metabolism of Enterococcus faecalis Wildtype and a Glutamine Synthase Mutant.

Lactic acid bacteria (LAB) play a significant role in biotechnology, e.g., food industry and also in human health. Many LAB genera have developed a multidrug resistance in the past few years, causing a serious problem in controlling hospital germs worldwide. Enterococcus faecalis accounts for a large part of the human infections caused by LABs. Therefore, studying its adaptive metabolism under various environmental conditions is particularly important to promote the development of new therapeutic approaches. In this study, we investigated the effect of glutamine auxotrophy (ΔglnA mutant) on metabolic and proteomic adaptations of E. faecalis in response to a changing pH in its environment. Changing pH values are part of the organism's natural environment in the human body and play a role in the food industry. We compared the results with those of the wildtype. Using a genome-scale metabolic model constrained by metabolic and proteomic data, our integrative method allows us to understand the bigger picture of the adaptation strategies of this bacterium. The study showed that energy demand is the decisive factor in adapting to a new environmental pH. The energy demand of the mutant was higher at all conditions. It has been reported that ΔglnA mutants of bacteria are energetically less effective. With the aid of our data and model we are able to explain this phenomenon as a consequence of a failure to regulate glutamine uptake and the costs for the import of glutamine and the export of ammonium. Methodologically, it became apparent that taking into account the nonspecificity of amino acid transporters is important for reproducing metabolic changes with genome-scale models because it affects energy balance. IMPORTANCE The integration of new pH-dependent experimental data on metabolic uptake and release fluxes, as well as of proteome data with a genome-scale computational model of a glutamine synthetase mutant of E. faecalis is used and compared with those of the wildtype to understand why glutamine auxotrophy results in a less efficient metabolism and how-in comparison with the wildtype-the glutamine synthetase knockout impacts metabolic adjustments during acidification or simply exposure to lower pH. We show that forced glutamine auxotrophy causes more energy demand and that this is likely due to a disregulated glutamine uptake. Proteome changes during acidification observed for the mutant resemble those of the wildtype with the exception of glycolysis-related genes, as the mutant is already energetically stressed at a higher pH and the respective proteome changes were in effect.

A systematic CRISPR screen defines mutational mechanisms underpinning signatures caused by replication errors and endogenous DNA damage.

Mutational signatures are imprints of pathophysiological processes arising through tumorigenesis. We generated isogenic CRISPR-Cas9 knockouts (Δ) of 43 genes in human induced pluripotent stem cells, cultured them in the absence of added DNA damage, and performed whole-genome sequencing of 173 subclones. ΔOGG1, ΔUNG, ΔEXO1, ΔRNF168, ΔMLH1, ΔMSH2, ΔMSH6, ΔPMS1, and ΔPMS2 produced marked mutational signatures indicative of being critical mitigators of endogenous DNA modifications. Detailed analyses revealed mutational mechanistic insights, including how 8-oxo-dG elimination is sequence-context-specific while uracil clearance is sequence-context-independent. Mismatch repair (MMR) deficiency signatures are engendered by oxidative damage (C>A transversions), differential misincorporation by replicative polymerases (T>C and C>T transitions), and we propose a 'reverse template slippage' model for T>A transversions. ΔMLH1, ΔMSH6, and ΔMSH2 signatures were similar to each other but distinct from ΔPMS2. Finally, we developed a classifier, MMRDetect, where application to 7,695 WGS cancers showed enhanced detection of MMR-deficient tumors, with implications for responsiveness to immunotherapies.