The DNA Structure-Specific Endonuclease MUS81 Mediates DNA Sensor STING-Dependent Host Rejection of Prostate Cancer Cells.

Self-DNA is present in the cytosol of many cancer cells and can promote effective immune rejection of tumor cells, but the mechanisms leading to the presence of cytosolic DNA are unknown. Here, we report that the cleavage of genomic DNA by DNA structure-specific endonuclease MUS81 and PARP-dependent DNA repair pathways leads to the accumulation of cytosolic DNA in prostate cancer cells. The number of nuclear MUS81 foci and the amount of cytosolic dsDNA increased in tandem from hyperplasia to clinical stage II prostate cancers and decreased at stage III. Cytosolic DNA generated by MUS81 stimulated DNA sensor STING-dependent type I interferon (IFN) expression and promoted phagocytic and T cell responses, resulting in type I and II IFN-mediated rejection of prostate tumor cells via mechanisms that partly depended on macrophages. Our results demonstrate that the tumor suppressor MUS81 alerts the immune system to the presence of transformed host cells.

A Human Pleiotropic Multiorgan Condition Caused by Deficient Wnt Secretion.

BACKGROUND: Structural birth defects occur in approximately 3% of live births; most such defects lack defined genetic or environmental causes. Despite advances in surgical approaches, pharmacologic prevention remains largely out of reach. METHODS: We queried worldwide databases of 20,248 families that included children with neurodevelopmental disorders and that were enriched for parental consanguinity. Approximately one third of affected children in these families presented with structural birth defects or microcephaly. We performed exome or genome sequencing of samples obtained from the children, their parents, or both to identify genes with biallelic pathogenic or likely pathogenic mutations present in more than one family. After identifying disease-causing variants, we generated two mouse models, each with a pathogenic variant "knocked in," to study mechanisms and test candidate treatments. We administered a small-molecule Wnt agonist to pregnant animals and assessed their offspring. RESULTS: We identified homozygous mutations in WLS, which encodes the Wnt ligand secretion mediator (also known as Wntless or WLS) in 10 affected persons from 5 unrelated families. (The Wnt ligand secretion mediator is essential for the secretion of all Wnt proteins.) Patients had multiorgan defects, including microcephaly and facial dysmorphism as well as foot syndactyly, renal agenesis, alopecia, iris coloboma, and heart defects. The mutations affected WLS protein stability and Wnt signaling. Knock-in mice showed tissue and cell vulnerability consistent with Wnt-signaling intensity and individual and collective functions of Wnts in embryogenesis. Administration of a pharmacologic Wnt agonist partially restored embryonic development. CONCLUSIONS: Genetic variations affecting a central Wnt regulator caused syndromic structural birth defects. Results from mouse models suggest that what we have named Zaki syndrome is a potentially preventable disorder. (Funded by the National Institutes of Health and others.).

Author Correction: RSPO2 inhibition of RNF43 and ZNRF3 governs limb development independently of LGR4/5/6.

In this Letter, the surname of author Lena Vlaminck was misspelled 'Vlaeminck'. In addition, author Kris Vleminckx should have been associated with affiliation 16 (Center for Medical Genetics, Ghent University, Ghent, Belgium). These have been corrected online.

RSPO2 inhibition of RNF43 and ZNRF3 governs limb development independently of LGR4/5/6.

The four R-spondin secreted ligands (RSPO1-RSPO4) act via their cognate LGR4, LGR5 and LGR6 receptors to amplify WNT signalling1-3. Here we report an allelic series of recessive RSPO2 mutations in humans that cause tetra-amelia syndrome, which is characterized by lung aplasia and a total absence of the four limbs. Functional studies revealed impaired binding to the LGR4/5/6 receptors and the RNF43 and ZNRF3 transmembrane ligases, and reduced WNT potentiation, which correlated with allele severity. Unexpectedly, however, the triple and ubiquitous knockout of Lgr4, Lgr5 and Lgr6 in mice did not recapitulate the known Rspo2 or Rspo3 loss-of-function phenotypes. Moreover, endogenous depletion or addition of exogenous RSPO2 or RSPO3 in triple-knockout Lgr4/5/6 cells could still affect WNT responsiveness. Instead, we found that the concurrent deletion of rnf43 and znrf3 in Xenopus embryos was sufficient to trigger the outgrowth of supernumerary limbs. Our results establish that RSPO2, without the LGR4/5/6 receptors, serves as a direct antagonistic ligand to RNF43 and ZNRF3, which together constitute a master switch that governs limb specification. These findings have direct implications for regenerative medicine and WNT-associated cancers.

A GLI3 variant leading to polydactyly in heterozygotes and Pallister-Hall-like syndrome in a homozygote.

Variants in transcriptional activator Gli Kruppel Family Member 3 (GLI3) have been reported to be associated with several phenotypes including Greig cephalopolysyndactyly syndrome (MIM #175700), Pallister-Hall syndrome (PHS) (MIM #146510), postaxial polydactyly types A1 (PAPA1) and B (PAPB) (MIM #174200), and preaxial polydactyly type 4 (MIM #174700). All these disorders follow an autosomal dominant pattern of inheritance. Hypothalamic hamartomas (MIM 241800) is associated with somatic variants in GLI3. We report a related couple with parents having PAPA1 and PAPB, who had a fetus with a phenotype most compatible with PHS. Molecular analyses demonstrated homozygosity for a pathogenic GLI3 variant (c.1927C > T; p. Arg643*) in the fetus and heterozygosity in the parents. The genetic analysis in this family demonstrates that heterozygosity and homozygosity for the same GLI3 variant can cause a different phenotype. Furthermore, the occurrence of Pallister-Hall-like syndrome in a homozygous patient should be taken into account in genetic counseling of families with PAPA1/PAPB.

RNA polymerase III regulates cytosolic RNA:DNA hybrids and intracellular microRNA expression.

RNA:DNA hybrids form in the nuclei and mitochondria of cells as transcription-induced R-loops or G-quadruplexes, but exist only in the cytosol of virus-infected cells. Little is known about the existence of RNA:DNA hybrids in the cytosol of virus-free cells, in particular cancer or transformed cells. Here, we show that cytosolic RNA:DNA hybrids are present in various human cell lines, including transformed cells. Inhibition of RNA polymerase III (Pol III), but not DNA polymerase, abrogated cytosolic RNA:DNA hybrids. Cytosolic RNA:DNA hybrids bind to several components of the microRNA (miRNA) machinery-related proteins, including AGO2 and DDX17. Furthermore, we identified miRNAs that are specifically regulated by Pol III, providing a potential link between RNA:DNA hybrids and the miRNA machinery. One of the target genes, exportin-1, is shown to regulate cytosolic RNA:DNA hybrids. Taken together, we reveal previously unknown mechanism by which Pol III regulates the presence of cytosolic RNA:DNA hybrids and miRNA biogenesis in various human cells.

Genome-derived cytosolic DNA contributes to type I interferon expression and immunogenicity of B-cell lymphoma cells.

We recently provided evidence that genome-derived DNA is present in the cytosol of many tumor cells. Genomic loci that give rise to cytosolic DNA can potentially form non-B DNA structures including triple-stranded RNA:DNA structures (R-loops). The RNA:DNA-specific endonuclease RNaseh1 reduced the levels of cytosolic DNA and type I interferon-dependent rejection of B-cell lymphoma suggesting that cytosolic DNA may contribute to immune surveillance of B-cell lymphoma.

Discovery of a genetic module essential for assigning left-right asymmetry in humans and ancestral vertebrates.

The vertebrate left-right axis is specified during embryogenesis by a transient organ: the left-right organizer (LRO). Species including fish, amphibians, rodents and humans deploy motile cilia in the LRO to break bilateral symmetry, while reptiles, birds, even-toed mammals and cetaceans are believed to have LROs without motile cilia. We searched for genes whose loss during vertebrate evolution follows this pattern and identified five genes encoding extracellular proteins, including a putative protease with hitherto unknown functions that we named ciliated left-right organizer metallopeptide (CIROP). Here, we show that CIROP is specifically expressed in ciliated LROs. In zebrafish and Xenopus, CIROP is required solely on the left side, downstream of the leftward flow, but upstream of DAND5, the first asymmetrically expressed gene. We further ascertained 21 human patients with loss-of-function CIROP mutations presenting with recessive situs anomalies. Our findings posit the existence of an ancestral genetic module that has twice disappeared during vertebrate evolution but remains essential for distinguishing left from right in humans.

cGAS-STING cytosolic DNA sensing pathway is suppressed by JAK2-STAT3 in tumor cells.

Deficiencies in DNA repair and DNA degrading nucleases lead to accumulation of cytosolic DNA. cGAS is a critical DNA sensor for the detection of cytosolic DNA and subsequent activation of the STING signaling pathway. Here, we show that the cGAS-STING pathway was unresponsive to STING agonists and failed to induce type I interferon (IFN) expression in many tested human tumor cells including DU145 prostate cancer cells. Inhibition of IL-6 or the downstream JAK2/STAT3 signaling restored responsiveness to STING agonists in DU145 cells. STING activity in murine TRAMP-C2 prostate cancer cells was critical for tumor rejection and immune cell infiltration. Endogenous STING agonists including double-stranded DNA and RNA:DNA hybrids present in TRAMP-C2 cells contribute to tumor rejection, but tumor growth was further suppressed by administration of cGAMP. Intratumoral co-injections of IL-6 significantly reduced the anti-tumor effects of cGAMP. In summary, STING in tumor cells contributes to tumor rejection in prostate cancer cells, but its functions are frequently suppressed in tumor cells in part via JAK2 and STAT3 pathways.

Sensing of dangerous DNA.

The presence of damaged and microbial DNA can pose a threat to the survival of organisms. Cells express various sensors that recognize specific aspects of such potentially dangerous DNA. Recognition of damaged or microbial DNA by sensors induces cellular processes that are important for DNA repair and inflammation. Here, we review recent evidence that the cellular response to DNA damage and microbial DNA are tightly intertwined. We also discuss insights into the parameters that enable DNA sensors to distinguish damaged and microbial DNA from DNA present in healthy cells.

Apoptotic Cells Release IL1 Receptor Antagonist in Response to Genotoxic Stress.

Apoptosis is a controlled means of eliminating damaged cells without causing an inflammatory response or tissue damage. The mechanisms that contribute to the suppression of an inflammatory response upon apoptosis of cells are poorly understood. Here, we report that apoptotic cells release the interleukin-1 receptor antagonist (IL1RA). The release of IL1RA depended on the DNA damage response, caspase 9, and caspase 3.De novotranslation, classical secretion pathways, or N-glycosylation was not required for the release of IL1RA. The amounts of IL1RA released by apoptotic cells impaired IL1-induced expression of IL6 In summary, we demonstrate that the release of IL1RA in response to genotoxic stress contributes to the immunosuppressive effects of apoptotic cells.

Genome-derived cytosolic DNA mediates type I interferon-dependent rejection of B cell lymphoma cells.

The DNA damage response (DDR) induces the expression of type I interferons (IFNs), but the underlying mechanisms are poorly understood. Here, we show the presence of cytosolic DNA in different mouse and human tumor cells. Treatment of cells with genotoxic agents increased the levels of cytosolic DNA in a DDR-dependent manner. Cloning of cytosolic DNA molecules from mouse lymphoma cells suggests that cytosolic DNA is derived from unique genomic loci and has the potential to form non-B DNA structures, including R-loops. Overexpression of Rnaseh1, which resolves R-loops, reduced the levels of cytosolic DNA, type I Ifn transcripts, and type I IFN-dependent rejection of lymphoma cells. Live-cell imaging showed a dynamic contact of cytosolic DNA with mitochondria, an important organelle for innate immune recognition of cytosolic nucleotides. In summary, we found that cytosolic DNA is present in many tumor cells and contributes to the immunogenicity of tumor cells.