ER-directed TREX1 limits cGAS activation at micronuclei.

Micronuclei are aberrant nuclear compartments that can form as a result of chromosome mis-segregation. Frequent loss of micronuclear envelope integrity exposes DNA to the cytoplasm, leading to chromosome fragmentation and immune activation. Here, we use micronuclei purification to show that the endoplasmic reticulum (ER)-associated nuclease TREX1 inhibits cGAS activation at micronuclei by degrading micronuclear DNA upon micronuclear envelope rupture. We demonstrate that the ER accesses ruptured micronuclei and plays a critical role in enabling TREX1 nucleolytic attack. TREX1 mutations, previously implicated in immune disease, untether TREX1 from the ER, disrupt TREX1 localization to micronuclei, diminish micronuclear DNA damage, and enhance cGAS activation. These results establish ER-directed resection of micronuclear DNA by TREX1 as a critical regulator of cytosolic DNA sensing in chromosomally unstable cells and provide a mechanistic basis for the importance of TREX1 ER tethering in preventing autoimmunity.

Mechanisms of APOBEC3 mutagenesis in human cancer cells.

The APOBEC3 family of cytosine deaminases has been implicated in some of the most prevalent mutational signatures in cancer1-3. However, a causal link between endogenous APOBEC3 enzymes and mutational signatures in human cancer genomes has not been established, leaving the mechanisms of APOBEC3 mutagenesis poorly understood. Here, to investigate the mechanisms of APOBEC3 mutagenesis, we deleted implicated genes from human cancer cell lines that naturally generate APOBEC3-associated mutational signatures over time4. Analysis of non-clustered and clustered signatures across whole-genome sequences from 251 breast, bladder and lymphoma cancer cell line clones revealed that APOBEC3A deletion diminished APOBEC3-associated mutational signatures. Deletion of both APOBEC3A and APOBEC3B further decreased APOBEC3 mutation burdens, without eliminating them. Deletion of APOBEC3B increased APOBEC3A protein levels, activity and APOBEC3A-mediated mutagenesis in some cell lines. The uracil glycosylase UNG was required for APOBEC3-mediated transversions, whereas the loss of the translesion polymerase REV1 decreased overall mutation burdens. Together, these data represent direct evidence that endogenous APOBEC3 deaminases generate prevalent mutational signatures in human cancer cells. Our results identify APOBEC3A as the main driver of these mutations, indicate that APOBEC3B can restrain APOBEC3A-dependent mutagenesis while contributing its own smaller mutation burdens and dissect mechanisms that translate APOBEC3 activities into distinct mutational signatures.

Magnesium accumulation upon cyclin M4 silencing activates microsomal triglyceride transfer protein improving NASH.

BACKGROUND & AIMS: Perturbations of intracellular magnesium (Mg2+) homeostasis have implications for cell physiology. The cyclin M family, CNNM, perform key functions in the transport of Mg2+ across cell membranes. Herein, we aimed to elucidate the role of CNNM4 in the development of non-alcoholic steatohepatitis (NASH). METHODS: Serum Mg2+ levels and hepatic CNNM4 expression were characterised in clinical samples. Primary hepatocytes were cultured under methionine and choline deprivation. A 0.1% methionine and choline-deficient diet, or a choline-deficient high-fat diet were used to induce NASH in our in vivo rodent models. Cnnm4 was silenced using siRNA, in vitro with DharmaFECT and in vivo with Invivofectamine® or conjugated to N-acetylgalactosamine. RESULTS: Patients with NASH showed hepatic CNNM4 overexpression and dysregulated Mg2+ levels in the serum. Cnnm4 silencing ameliorated hepatic lipid accumulation, inflammation and fibrosis in the rodent NASH models. Mechanistically, CNNM4 knockdown in hepatocytes induced cellular Mg2+ accumulation, reduced endoplasmic reticulum stress, and increased microsomal triglyceride transfer activity, which promoted hepatic lipid clearance by increasing the secretion of VLDLs. CONCLUSIONS: CNNM4 is overexpressed in patients with NASH and is responsible for dysregulated Mg2+ transport. Hepatic CNNM4 is a promising therapeutic target for the treatment of NASH. LAY SUMMARY: Cyclin M4 (CNNM4) is overexpressed in non-alcoholic steatohepatitis (NASH) and promotes the export of magnesium from the liver. The liver-specific silencing of Cnnm4 ameliorates NASH by reducing endoplasmic reticulum stress and promoting the activity of microsomal triglyceride transfer protein.

Adding New Dimensions to 3D Cancer Models.

Understanding patient-specific responses to anticancer therapies and how individual tumors interact with their tumor microenvironment (TME) is a challenging task. To measure the impact of the TME on diverse and clinically relevant treatments, Ramos Zapatero and colleagues coupled patient-derived organoid (PDO) and cancer-associated fibroblast (CAF) cocultures with high-throughput mass cytometry-based assessment of cell state. Using a newly developed "Trellis" algorithm enabled integration and analysis of highly complex, multidimensional treatment response data. This work showed that tumor cell response to chemotherapy was associated with both intrinsic and nonintrinsic signaling states, whereby proliferative rate, growth factor signaling, and CAFs interaction influenced chemoprotection. Furthermore, the work suggests a potential role for the TME in promoting lineage plasticity associated with drug resistance. In all, the pipeline described provides a blueprint for exploring the intricate interplay of factors influencing cancer treatment response.

Base editing sensor libraries for high-throughput engineering and functional analysis of cancer-associated single nucleotide variants.

Base editing can be applied to characterize single nucleotide variants of unknown function, yet defining effective combinations of single guide RNAs (sgRNAs) and base editors remains challenging. Here, we describe modular base-editing-activity 'sensors' that link sgRNAs and cognate target sites in cis and use them to systematically measure the editing efficiency and precision of thousands of sgRNAs paired with functionally distinct base editors. By quantifying sensor editing across >200,000 editor-sgRNA combinations, we provide a comprehensive resource of sgRNAs for introducing and interrogating cancer-associated single nucleotide variants in multiple model systems. We demonstrate that sensor-validated tools streamline production of in vivo cancer models and that integrating sensor modules in pooled sgRNA libraries can aid interpretation of high-throughput base editing screens. Using this approach, we identify several previously uncharacterized mutant TP53 alleles as drivers of cancer cell proliferation and in vivo tumor development. We anticipate that the framework described here will facilitate the functional interrogation of cancer variants in cell and animal models.

APOBEC3-dependent kataegis and TREX1-driven chromothripsis during telomere crisis.

Chromothripsis and kataegis are frequently observed in cancer and may arise from telomere crisis, a period of genome instability during tumorigenesis when depletion of the telomere reserve generates unstable dicentric chromosomes1-5. Here we examine the mechanism underlying chromothripsis and kataegis by using an in vitro telomere crisis model. We show that the cytoplasmic exonuclease TREX1, which promotes the resolution of dicentric chromosomes4, plays a prominent role in chromothriptic fragmentation. In the absence of TREX1, the genome alterations induced by telomere crisis primarily involve breakage-fusion-bridge cycles and simple genome rearrangements rather than chromothripsis. Furthermore, we show that the kataegis observed at chromothriptic breakpoints is the consequence of cytosine deamination by APOBEC3B. These data reveal that chromothripsis and kataegis arise from a combination of nucleolytic processing by TREX1 and cytosine editing by APOBEC3B.