Systematic profiling of conditional pathway activation identifies context-dependent synthetic lethalities.

The paradigm of cancer-targeted therapies has focused largely on inhibition of critical pathways in cancer. Conversely, conditional activation of signaling pathways as a new source of selective cancer vulnerabilities has not been deeply characterized. In this study, we sought to systematically identify context-specific gene-activation-induced lethalities in cancer. To this end, we developed a method for gain-of-function genetic perturbations simultaneously across ~500 barcoded cancer cell lines. Using this approach, we queried the pan-cancer vulnerability landscape upon activating ten key pathway nodes, revealing selective activation dependencies of MAPK and PI3K pathways associated with specific biomarkers. Notably, we discovered new pathway hyperactivation dependencies in subsets of APC-mutant colorectal cancers where further activation of the WNT pathway by APC knockdown or direct ß-catenin overexpression led to robust antitumor effects in xenograft and patient-derived organoid models. Together, this study reveals a new class of conditional gene-activation dependencies in cancer.

Complement C4 Prevents Viral Infection through Capsid Inactivation.

The complement system is vital for anti-microbial defense. In the classical pathway, pathogen-bound antibody recruits the C1 complex (C1qC1r2C1s2) that initiates a cleavage cascade involving C2, C3, C4, and C5 and triggering microbial clearance. We demonstrate a C4-dependent antiviral mechanism that is independent of downstream complement components. C4 inhibits human adenovirus infection by directly inactivating the virus capsid. Rapid C4 activation and capsid deposition of cleaved C4b are catalyzed by antibodies via the classical pathway. Capsid-deposited C4b neutralizes infection independent of C2 and C3 but requires C1q antibody engagement. C4b inhibits capsid disassembly, preventing endosomal escape and cytosolic access. C4-deficient mice exhibit heightened viral burdens. Additionally, complement synergizes with the Fc receptor TRIM21 to block transduction by an adenovirus gene therapy vector but is partially restored by Fab virus shielding. These results suggest that the complement system could be altered to prevent virus infection and enhance virus gene therapy efficacy.

Bcl9 and Pygo synergise downstream of Apc to effect intestinal neoplasia in FAP mouse models.

Bcl9 and Pygo are Wnt enhanceosome components that effect ß-catenin-dependent transcription. Whether they mediate ß-catenin-dependent neoplasia is unclear. Here we assess their roles in intestinal tumourigenesis initiated by Apc loss-of-function (ApcMin), or by Apc1322T encoding a partially-functional Apc truncation commonly found in colorectal carcinomas. Intestinal deletion of Bcl9 extends disease-free survival in both models, and essentially cures Apc1322T mice of their neoplasia. Loss-of-Bcl9 synergises with loss-of-Pygo to shift gene expression within Apc-mutant adenomas from stem cell-like to differentiation along Notch-regulated secretory lineages. Bcl9 loss also promotes tumour retention in ApcMin mice, apparently via relocating nuclear ß-catenin to the cell surface, but this undesirable effect is not seen in Apc1322T mice whose Apc truncation retains partial function in regulating ß-catenin. Our results demonstrate a key role of the Wnt enhanceosome in ß-catenin-dependent intestinal tumourigenesis and reveal the potential of BCL9 as a therapeutic target during early stages of colorectal cancer.

TRIM21 mediates antibody inhibition of adenovirus-based gene delivery and vaccination.

Adenovirus has enormous potential as a gene-therapy vector, but preexisting immunity limits its widespread application. What is responsible for this immune block is unclear because antibodies potently inhibit transgene expression without impeding gene transfer into target cells. Here we show that antibody prevention of adenoviral gene delivery in vivo is mediated by the cytosolic antibody receptor TRIM21. Genetic KO of TRIM21 or a single-antibody point mutation is sufficient to restore transgene expression to near-naïve immune levels. TRIM21 is also responsible for blocking cytotoxic T cell induction by vaccine vectors, preventing a protective response against subsequent influenza infection and an engrafted tumor. Furthermore, adenoviral preexisting immunity can lead to an augmented immune response upon i.v. administration of the vector. Transcriptomic analysis of vector-transduced tissue reveals that TRIM21 is responsible for the specific up-regulation of hundreds of immune genes, the majority of which are components of the intrinsic or innate response. Together, these data define a major mechanism underlying the preimmune block to adenovirus gene therapy and demonstrate that TRIM21 efficiently blocks gene delivery in vivo while simultaneously inducing a rapid program of immune transcription.

Constitutive scaffolding of multiple Wnt enhanceosome components by Legless/BCL9.

Wnt/ß-catenin signaling elicits context-dependent transcription switches that determine normal development and oncogenesis. These are mediated by the Wnt enhanceosome, a multiprotein complex binding to the Pygo chromatin reader and acting through TCF/LEF-responsive enhancers. Pygo renders this complex Wnt-responsive, by capturing ß-catenin via the Legless/BCL9 adaptor. We used CRISPR/Cas9 genome engineering of Drosophila legless (lgs) and human BCL9 and B9L to show that the C-terminus downstream of their adaptor elements is crucial for Wnt responses. BioID proximity labeling revealed that BCL9 and B9L, like PYGO2, are constitutive components of the Wnt enhanceosome. Wnt-dependent docking of ß-catenin to the enhanceosome apparently causes a rearrangement that apposes the BCL9/B9L C-terminus to TCF. This C-terminus binds to the Groucho/TLE co-repressor, and also to the Chip/LDB1-SSDP enhanceosome core complex via an evolutionary conserved element. An unexpected link between BCL9/B9L, PYGO2 and nuclear co-receptor complexes suggests that these ß-catenin co-factors may coordinate Wnt and nuclear hormone responses.

Cell-derived vesicles exposing coagulant tissue factor in saliva.

On vascular damage, coagulation is initiated by extravascular tissue factor (TF). Intravascular TF, which is present on circulating cell-derived vesicles, is noncoagulant under physiologic conditions but prothrombotic under pathologic conditions. Human saliva triggers coagulation, but the mechanism and physiologic relevance are unknown. Because saliva is known to contain TF, we hypothesized that this TF may also be associated with cell-derived vesicles to facilitate coagulation when saliva directly contacts blood. The saliva-induced shortening of the clotting time of autologous plasma and whole blood from healthy subjects (n = 10) proved TF-dependent. This TF was associated with various types of cell-derived vesicles, including microparticles and exosomes. The physiologic function was shown by adding saliva to human pericardial wound blood collected from patients undergoing cardiac surgery. Addition of saliva shortened the clotting time from 300 ± 96 to 186 ± 24 seconds (P = .03). Our results show that saliva triggers coagulation, thereby reducing blood loss and the risk of pathogens entering the blood. We postulate that our reflex to lick a wound may be a mechanism to enable TF-exposing vesicles, present in saliva, to aid in the coagulation process and thus protect the organism from entering pathogens. This unique compartmentalization may be highly conserved because also animals lick their wounds.