An innate immune response to adeno-associated virus genomes decreases cortical dendritic complexity and disrupts synaptic transmission.

Recombinant adeno-associated viruses (AAVs) allow rapid and efficient gene delivery to the nervous system, are widely used in neuroscience research, and are the basis of FDA-approved neuron-targeting gene therapies. Here we find that an innate immune response to the AAV genome reduces dendritic length and complexity and disrupts synaptic transmission in mouse somatosensory cortex. Dendritic loss is apparent 3 weeks after injection of experimentally relevant viral titers, is not restricted to a particular capsid serotype, transgene, promoter, or production facility, and cannot be explained by responses to surgery or transgene expression. AAV-associated dendritic loss is accompanied by a decrease in the frequency and amplitude of miniature excitatory postsynaptic currents and an increase in the proportion of GluA2-lacking, calcium-permeable AMPA receptors. The AAV genome is rich in unmethylated CpG DNA, which is recognized by the innate immunoreceptor Toll-like receptor 9 (TLR9), and acutely blocking TLR9 preserves dendritic complexity and AMPA receptor subunit composition in AAV-injected mice. These results reveal unexpected impacts of an immune response to the AAV genome on neuronal structure and function and identify approaches to improve the safety and efficacy of AAV-mediated gene delivery in the nervous system.

Substratum stiffness signals through integrin-linked kinase and ß1-integrin to regulate midbody proteins and abscission during EMT.

Abscission is the final stage of cytokinesis during which the parent cell physically separates to yield two identical daughters. Failure of abscission results in multinucleation (MNC), a sign of genomic instability and a precursor to aneuploidy, enabling characteristics of neoplastic progression. Induction of epithelial-mesenchymal transition (EMT) causes MNC in mammary epithelial cells cultured on stiff microenvironments that have mechanical properties similar to those found in breast tumors, but not on soft microenvironments reminiscent of the normal mammary gland. Here we report that on stiff microenvironments, EMT signaling through Snail up-regulates the midbody-associated proteins septin-6, Mklp1, and anillin, leading to abscission failure and MNC. To uncover the mechanism by which stiff microenvironments promote MNC in cells undergoing EMT, we investigated the role of cell-matrix adhesion through ß1-integrin and integrin-linked kinase (ILK). We found that ILK expression, but not kinase activity, is required for EMT-associated MNC in cells on stiff microenvironments. Conversely, increasing focal adhesions by expressing an autoclustering mutant of ß1-integrin promotes MNC in cells on soft microenvironments. Our data suggest that signaling through focal adhesions causes failure of cytokinesis in cells actively undergoing EMT. These results highlight the importance of tissue mechanics and adhesion in regulating the cellular response to EMT inducers.

Cooperation of the ATM and Fanconi Anemia/BRCA Pathways in Double-Strand Break End Resection.

Cells deficient in ataxia telangiectasia mutated (ATM) are hypersensitive to ionizing radiation and other anti-cancer agents that induce double-strand DNA breaks. ATM inhibitors may therefore sensitize cancer cells to these agents. Some cancers may also have underlying genetic defects predisposing them to an ATM inhibitor monotherapy response. We have conducted a genome-wide CRISPR screen to identify genetic vulnerabilities that sensitize lung cancer cells to ATM inhibitors. Knockout of genes in the Fanconi anemia (FA)/BRCA pathway results in hypersensitivity to the ATM inhibitor M3541. Knockdown of either an FA gene or of ATM results in reduced double-strand break end resection, enhanced non-homologous end joining (NHEJ) repair, and decreased homologous recombination repair. Knockout of both the FA/BRCA pathway and ATM strongly inhibits end resection and generates toxic levels of NHEJ, thereby elucidating a mechanism of cellular death by synthetic lethality. ATM inhibitors may therefore be useful for the treatment of tumors with a defective FA/BRCA pathway.

The CHK1 Inhibitor Prexasertib Exhibits Monotherapy Activity in High-Grade Serous Ovarian Cancer Models and Sensitizes to PARP Inhibition.

PURPOSE: PARP inhibitors are approved for the treatment of high-grade serous ovarian cancers (HGSOC). Therapeutic resistance, resulting from restoration of homologous recombination (HR) repair or replication fork stabilization, is a pressing clinical problem. We assessed the activity of prexasertib, a checkpoint kinase 1 (CHK1) inhibitor known to cause replication catastrophe, as monotherapy and in combination with the PARP inhibitor olaparib in preclinical models of HGSOC, including those with acquired PARP inhibitor resistance. EXPERIMENTAL DESIGN: Prexasertib was tested as a single agent or in combination with olaparib in 14 clinically annotated and molecularly characterized luciferized HGSOC patient-derived xenograft (PDX) models and in a panel of ovarian cancer cell lines. The ability of prexasertib to impair HR repair and replication fork stability was also assessed. RESULTS: Prexasertib monotherapy demonstrated antitumor activity across the 14 PDX models. Thirteen models were resistant to olaparib monotherapy, including 4 carrying BRCA1 mutation. The combination of olaparib with prexasertib was synergistic and produced significant tumor growth inhibition in an olaparib-resistant model and further augmented the degree and durability of response in the olaparib-sensitive model. HGSOC cell lines, including those with acquired PARP inhibitor resistance, were also sensitive to prexasertib, associated with induction of DNA damage and replication stress. Prexasertib also sensitized these cell lines to PARP inhibition and compromised both HR repair and replication fork stability. CONCLUSIONS: Prexasertib exhibits monotherapy activity in PARP inhibitor-resistant HGSOC PDX and cell line models, reverses restored HR and replication fork stability, and synergizes with PARP inhibition.