LINE-1 activation in the cerebellum drives ataxia.

Dysregulation of long interspersed nuclear element 1 (LINE-1, L1), a dominant class of transposable elements in the human genome, has been linked to neurodegenerative diseases, but whether elevated L1 expression is sufficient to cause neurodegeneration has not been directly tested. Here, we show that the cerebellar expression of L1 is significantly elevated in ataxia telangiectasia patients and strongly anti-correlated with the expression of epigenetic silencers. To examine the role of L1 in the disease etiology, we developed an approach for direct targeting of the L1 promoter for overexpression in mice. We demonstrated that L1 activation in the cerebellum led to Purkinje cell dysfunctions and degeneration and was sufficient to cause ataxia. Treatment with a nucleoside reverse transcriptase inhibitor blunted ataxia progression by reducing DNA damage, attenuating gliosis, and reversing deficits of molecular regulators for calcium homeostasis in Purkinje cells. Our study provides the first direct evidence that L1 activation can drive neurodegeneration.

Oncometabolites suppress DNA repair by disrupting local chromatin signalling.

Deregulation of metabolism and disruption of genome integrity are hallmarks of cancer1. Increased levels of the metabolites 2-hydroxyglutarate, succinate and fumarate occur in human malignancies owing to somatic mutations in the isocitrate dehydrogenase-1 or -2 (IDH1 or IDH2) genes, or germline mutations in the fumarate hydratase (FH) and succinate dehydrogenase genes (SDHA, SDHB, SDHC and SDHD), respectively2-4. Recent work has made an unexpected connection between these metabolites and DNA repair by showing that they suppress the pathway of homology-dependent repair (HDR)5,6 and confer an exquisite sensitivity to inhibitors of poly (ADP-ribose) polymerase (PARP) that are being tested in clinical trials. However, the mechanism by which these oncometabolites inhibit HDR remains poorly understood. Here we determine the pathway by which these metabolites disrupt DNA repair. We show that oncometabolite-induced inhibition of the lysine demethylase KDM4B results in aberrant hypermethylation of histone 3 lysine 9 (H3K9) at loci surrounding DNA breaks, masking a local H3K9 trimethylation signal that is essential for the proper execution of HDR. Consequently, recruitment of TIP60 and ATM, two key proximal HDR factors, is substantially impaired at DNA breaks, with reduced end resection and diminished recruitment of downstream repair factors. These findings provide a mechanistic basis for oncometabolite-induced HDR suppression and may guide effective strategies to exploit these defects for therapeutic gain.

Mitochondrial DNA Stress Signalling Protects the Nuclear Genome.

The mammalian genome comprises nuclear DNA (nDNA) derived from both parents and mitochondrial DNA (mtDNA) that is maternally inherited and encodes essential proteins required for oxidative phosphorylation. Thousands of copies of the circular mtDNA are present in most cell types that are packaged by TFAM into higher-order structures called nucleoids1. Mitochondria are also platforms for antiviral signalling2 and, due to their bacterial origin, mtDNA and other mitochondrial components trigger innate immune responses and inflammatory pathology2,3. We showed previously that instability and cytoplasmic release of mtDNA activates the cGAS-STING-TBK1 pathway resulting in interferon stimulated gene (ISG) expression that promotes antiviral immunity4. Here, we find that persistent mtDNA stress is not associated with basally activated NF-κB signalling or interferon gene expression typical of an acute antiviral response. Instead, a specific subset of ISGs, that includes Parp9, remains activated by the unphosphorylated form of ISGF3 (U-ISGF3) that enhances nDNA damage and repair responses. In cultured primary fibroblasts and cancer cells, the chemotherapeutic drug doxorubicin causes mtDNA damage and release, which leads to cGAS-STING-dependent ISG activation. In addition, mtDNA stress in TFAM-deficient mouse melanoma cells produces tumours that are more resistant to doxorubicin in vivo. Finally, Tfam +/- mice exposed to ionizing radiation exhibit enhanced nDNA repair responses in spleen. Therefore, we propose that damage to and subsequent release of mtDNA elicits a protective signalling response that enhances nDNA repair in cells and tissues, suggesting mtDNA is a genotoxic stress sentinel.

Mcp1 Promotes Macrophage-Dependent Cyst Expansion in Autosomal Dominant Polycystic Kidney Disease.

BACKGROUND: In patients with autosomal dominant polycystic kidney disease (ADPKD), most of whom have a mutation in PKD1 or PKD2, abnormally large numbers of macrophages accumulate around kidney cysts and promote their growth. Research by us and others has suggested that monocyte chemoattractant protein-1 (Mcp1) may be a signal for macrophage-mediated cyst growth. METHODS: To define the role of Mcp1 and macrophages in promoting cyst growth, we used mice with inducible knockout of Pkd1 alone (single knockout) or knockout of both Pkd1 and Mcp1 (double knockout) in the murine renal tubule. Levels of Mcp1 RNA expression were measured in single-knockout mice and controls. RESULTS: In single-knockout mice, upregulation of Mcp1 precedes macrophage infiltration. Macrophages accumulating around nascent cysts (0-2 weeks after induction) are initially proinflammatory and induce tubular cell injury with morphologic flattening, oxidative DNA damage, and proliferation-independent cystic dilation. At 2-6 weeks after induction, macrophages switch to an alternative activation phenotype and promote further cyst growth because of an additional three-fold increase in tubular cell proliferative rates. In double-knockout mice, there is a marked reduction in Mcp1 expression and macrophage numbers, resulting in less initial tubular cell injury, slower cyst growth, and improved renal function. Treatment of single-knockout mice with an inhibitor to the Mcp1 receptor Ccr2 partially reproduced the morphologic and functional improvement seen with Mcp1 knockout. CONCLUSIONS: Mcp1 is upregulated after knockout of Pkd1 and promotes macrophage accumulation and cyst growth via both proliferation-independent and proliferation-dependent mechanisms in this orthologous mouse model of ADPKD.

Krebs-cycle-deficient hereditary cancer syndromes are defined by defects in homologous-recombination DNA repair.

The hereditary cancer syndromes hereditary leiomyomatosis and renal cell cancer (HLRCC) and succinate dehydrogenase-related hereditary paraganglioma and pheochromocytoma (SDH PGL/PCC) are linked to germline loss-of-function mutations in genes encoding the Krebs cycle enzymes fumarate hydratase and succinate dehydrogenase, thus leading to elevated levels of fumarate and succinate, respectively1-3. Here, we report that fumarate and succinate both suppress the homologous recombination (HR) DNA-repair pathway required for the resolution of DNA double-strand breaks (DSBs) and for the maintenance of genomic integrity, thus rendering tumor cells vulnerable to synthetic-lethal targeting with poly(ADP)-ribose polymerase (PARP) inhibitors. These results identify HLRCC and SDH PGL/PCC as familial DNA-repair deficiency syndromes, providing a mechanistic basis to explain their cancer predisposition and suggesting a potentially therapeutic approach for advanced HLRCC and SDH PGL/PCC, both of which are incurable when metastatic.

PTEN Regulates Nonhomologous End Joining By Epigenetic Induction of NHEJ1/XLF.

DNA double-strand breaks (DSB) are the most cytotoxic DNA lesions, and up to 90% of DSBs require repair by nonhomologous end joining (NHEJ). Functional and genomic analyses of patient-derived melanomas revealed that PTEN loss is associated with NHEJ deficiency. In PTEN-null melanomas, PTEN complementation rescued the NHEJ defect; conversely, suppression of PTEN compromised NHEJ. Mechanistic studies revealed that PTEN promotes NHEJ through direct induction of expression of XRCC4-like factor (NHEJ1/XLF), which functions in DNA end bridging and ligation. PTEN was found to occupy the NHEJ1 gene promoter and to recruit the histone acetyltransferases, PCAF and CBP, inducing XLF expression. This recruitment activity was found to be independent of its phosphatase activity, but dependent on K128, a site of regulatory acetylation on PTEN. These findings define a novel function for PTEN in regulating NHEJ DSB repair, and therefore may assist in the design of individualized strategies for cancer therapy.Implications: PTEN is the second most frequently lost tumor suppressor gene. Here it is demonstrated that PTEN has a direct and novel regulatory role in NHEJ, a key DNA repair pathway in response to radiation and chemotherapy. Mol Cancer Res; 16(8); 1241-54. ©2018 AACR.

Suppression of homology-dependent DNA double-strand break repair induces PARP inhibitor sensitivity in VHL-deficient human renal cell carcinoma.

The von Hippel-Lindau (VHL) tumor suppressor gene is inactivated in the vast majority of human clear cell renal carcinomas. The pathogenesis of VHL loss is currently best understood to occur through stabilization of the hypoxia-inducible factors, activation of hypoxia-induced signaling pathways, and transcriptional reprogramming towards a pro-angiogenic and pro-growth state. However, hypoxia also drives other pro-tumorigenic processes, including the development of genomic instability via down-regulation of DNA repair gene expression. Here, we find that DNA repair genes involved in double-strand break repair by homologous recombination (HR) and in mismatch repair, which are down-regulated by hypoxic stress, are decreased in VHL-deficient renal cancer cells relative to wild type VHL-complemented cells. Functionally, this gene repression is associated with impaired DNA double-strand break repair in VHL-deficient cells, as determined by the persistence of ionizing radiation-induced DNA double-strand breaks and reduced repair activity in a homology-dependent plasmid reactivation assay. Furthermore, VHL deficiency conferred increased sensitivity to PARP inhibitors, analogous to the synthetic lethality observed between hypoxia and these agents. Finally, we discovered a correlation between VHL inactivation and reduced HR gene expression in a large panel of human renal carcinoma samples. Together, our data elucidate a novel connection between VHL-deficient renal carcinoma and hypoxia-induced down-regulation of DNA repair, and identify potential opportunities for targeting DNA repair defects in human renal cell carcinoma.

Nickel induces transcriptional down-regulation of DNA repair pathways in tumorigenic and non-tumorigenic lung cells.

The heavy metal nickel is a known carcinogen, and occupational exposure to nickel compounds has been implicated in human lung and nasal cancers. Unlike many other environmental carcinogens, however, nickel does not directly induce DNA mutagenesis, and the mechanism of nickel-related carcinogenesis remains incompletely understood. Cellular nickel exposure leads to signaling pathway activation, transcriptional changes and epigenetic remodeling, processes also impacted by hypoxia, which itself promotes tumor growth without causing direct DNA damage. One of the mechanisms by which hypoxia contributes to tumor growth is the generation of genomic instability via down-regulation of high-fidelity DNA repair pathways. Here, we find that nickel exposure similarly leads to down-regulation of DNA repair proteins involved in homology-dependent DNA double-strand break repair (HDR) and mismatch repair (MMR) in tumorigenic and non-tumorigenic human lung cells. Functionally, nickel induces a defect in HDR capacity, as determined by plasmid-based host cell reactivation assays, persistence of ionizing radiation-induced DNA double-strand breaks and cellular hypersensitivity to ionizing radiation. Mechanistically, we find that nickel, in contrast to the metalloid arsenic, acutely induces transcriptional repression of HDR and MMR genes as part of a global transcriptional pattern similar to that seen with hypoxia. Finally, we find that exposure to low-dose nickel reduces the activity of the MLH1 promoter, but only arsenic leads to long-term MLH1 promoter silencing. Together, our data elucidate novel mechanisms of heavy metal carcinogenesis and contribute to our understanding of the influence of the microenvironment on the regulation of DNA repair pathways.

2-Hydroxyglutarate produced by neomorphic IDH mutations suppresses homologous recombination and induces PARP inhibitor sensitivity.

2-Hydroxyglutarate (2HG) exists as two enantiomers, (R)-2HG and (S)-2HG, and both are implicated in tumor progression via their inhibitory effects on α-ketoglutarate (αKG)-dependent dioxygenases. The former is an oncometabolite that is induced by the neomorphic activity conferred by isocitrate dehydrogenase 1 (IDH1) and IDH2 mutations, whereas the latter is produced under pathologic processes such as hypoxia. We report that IDH1/2 mutations induce a homologous recombination (HR) defect that renders tumor cells exquisitely sensitive to poly(adenosine 5'-diphosphate-ribose) polymerase (PARP) inhibitors. This "BRCAness" phenotype of IDH mutant cells can be completely reversed by treatment with small-molecule inhibitors of the mutant IDH1 enzyme, and conversely, it can be entirely recapitulated by treatment with either of the 2HG enantiomers in cells with intact IDH1/2 proteins. We demonstrate mutant IDH1-dependent PARP inhibitor sensitivity in a range of clinically relevant models, including primary patient-derived glioma cells in culture and genetically matched tumor xenografts in vivo. These findings provide the basis for a possible therapeutic strategy exploiting the biological consequences of mutant IDH, rather than attempting to block 2HG production, by targeting the 2HG-dependent HR deficiency with PARP inhibition. Furthermore, our results uncover an unexpected link between oncometabolites, altered DNA repair, and genetic instability.

In vivo correction of anaemia in ß-thalassemic mice by γPNA-mediated gene editing with nanoparticle delivery.

The blood disorder, ß-thalassaemia, is considered an attractive target for gene correction. Site-specific triplex formation has been shown to induce DNA repair and thereby catalyse genome editing. Here we report that triplex-forming peptide nucleic acids (PNAs) substituted at the γ position plus stimulation of the stem cell factor (SCF)/c-Kit pathway yielded high levels of gene editing in haematopoietic stem cells (HSCs) in a mouse model of human ß-thalassaemia. Injection of thalassemic mice with SCF plus nanoparticles containing γPNAs and donor DNAs ameliorated the disease phenotype, with sustained elevation of blood haemoglobin levels into the normal range, reduced reticulocytosis, reversal of splenomegaly and up to 7% ß-globin gene correction in HSCs, with extremely low off-target effects. The combination of nanoparticle delivery, next generation γPNAs and SCF treatment may offer a minimally invasive treatment for genetic disorders of the blood that can be achieved safely and simply by intravenous administration.

miR-155 Overexpression Promotes Genomic Instability by Reducing High-fidelity Polymerase Delta Expression and Activating Error-Prone DSB Repair.

UNLABELLED: miR-155 is an oncogenic miRNA that is often overexpressed in cancer and is associated with poor prognosis. miR-155 can target several DNA repair factors, including RAD51, MLH1, and MSH6, and its overexpression results in an increased mutation frequency in vitro, although the mechanism has yet to be fully understood. Here, we demonstrate that overexpression of miR-155 drives an increased mutation frequency both in vitro and in vivo, promoting genomic instability by affecting multiple DNA repair pathways. miR-155 overexpression causes a decrease in homologous recombination, but yields a concurrent increase in the error-prone nonhomologous end-joining pathway. Despite repressing established targets MLH1 and MSH6, the identified mutation pattern upon miR-155 overexpression does not resemble that of a mismatch repair-deficient background. Further investigation revealed that all four subunits of polymerase delta, a high-fidelity DNA replication, and repair polymerase are downregulated at the mRNA level in the context of miR-155 overexpression. FOXO3a, a transcription factor and known target of miR-155, has one or more putative binding site(s) in the promoter of all four polymerase delta subunits. Finally, suppression of FOXO3a by miR-155 or by siRNA knockdown is sufficient to repress the expression of the catalytic subunit of polymerase delta, POLD1, at the protein level, indicating that FOXO3a contributes to the regulation of polymerase delta levels. IMPLICATIONS: Taken together, miR-155 overexpression drives an increase in mutation frequency via multifaceted impact on DNA damage response and DNA repair pathways.