The innate immune response is essential for combating infectious disease. Macrophages and other cells respond to infection by releasing cytokines such as interleukin-1β (IL-1β), which in turn activate a well-described myeloid differentiation factor 88 (MYD88) -mediated, nuclear factor-κB (NF-κB) -dependent transcriptional pathway that results in inflammatory cell activation and recruitment1–4. Endothelial cells, which usually serve as a barrier to the movement of inflammatory cells out of the blood and into tissue, are also critical mediators of the inflammatory response5,6. Paradoxically, the same cytokines vital to a successful immune defense also have disruptive effects on endothelial cell-cell interactions and can trigger degradation of barrier function and dissociation of tissue architecture7–9. The mechanism of this barrier dissolution and its relationship to the canonical NF-κB pathway remains ill defined. Here we show that the direct, immediate, and disruptive effects of IL-1β on endothelial stability are NF-κB independent and are instead the result of signaling via the small GTPase, ADP-ribosylation factor 6 (ARF6), and its activator, ARF nucleotide binding site opener (ARNO). Moreover, we show that ARNO binds directly to the adaptor protein MYD88, and thus propose MYD88-ARNO-ARF6 as a proximal IL-1β signaling pathway distinct from that mediated by NF-κB (Supplementary Fig. 1). Finally, we show that SecinH3, an inhibitor of ARF guanine nucleotide-exchange factors (GEFs) such as ARNO, enhances vascular stability and significantly improves outcomes in animal models of inflammatory arthritis and acute inflammation.

SUMMARY Activating mutations in Gαq proteins, which form the a subunit of certain heterotrimeric G proteins, drive uveal melanoma oncogenesis by triggering multiple downstream signaling pathways, including PLC/PKC, Rho/Rac, and YAP. Here we show that the small GTPase ARF6 acts as a proximal node of oncogenic Gαq signaling to induce all of these downstream pathways as well as β-catenin signaling. ARF6 activates these diverse pathways through a common mechanism—the trafficking of GNAQ and β-catenin from the plasma membrane to cytoplasmic vesicles and the nucleus, respectively. Blocking ARF6 with a small molecule reduces uveal melanoma cell proliferation and tumorigenesis in a mouse model, confirming the functional relevance of this pathway and suggesting a therapeutic strategy for Gα-mediated diseases.

β-Catenin has a dual function in cells: fortifying cadherin-based adhesion at the plasma membrane and activating transcription in the nucleus. We found that in melanoma cells, WNT5A stimulated the disruption of N-cadherin and β-catenin complexes by activating the guanosine triphosphatase adenosine diphosphate ribosylation factor 6 (ARF6). Binding of WNT5A to the Frizzled 4–LRP6 (low-density lipoprotein receptor–related protein 6) receptor complex activated ARF6, which liberated β-catenin from N-cadherin, thus increasing the pool of free β-catenin, enhancing β-catenin–mediated transcription, and stimulating invasion. In contrast to WNT5A, the guidance cue SLIT2 and its receptor ROBO1 inhibited ARF6 activation and, accordingly, stabilized the interaction of N-cadherin with β-catenin and reduced transcription and invasion. Thus, ARF6 integrated competing signals in melanoma cells, thereby enabling plasticity in the response to external cues. Moreover, small-molecule inhibition of ARF6 stabilized adherens junctions, blocked β-catenin signaling and invasiveness of melanoma cells in culture, and reduced spontaneous pulmonary metastasis in mice, suggesting that targeting ARF6 may provide a means of inhibiting WNT/β-catenin signaling in cancer.

Background Cerebral cavernous malformation (CCM) is a hemorrhagic stroke disease affecting up to 0.5% of North Americans with no approved non-surgical treatment. A subset of patients have a hereditary form of the disease due primarily to loss-of-function mutations in KRIT1, CCM2, or PDCD10. We sought to identify known drugs that could be repurposed to treat CCM. Methods and Results We developed an unbiased screening platform based on both cellular and animal models of loss-of-function of CCM2. Our discovery strategy consisted of four steps: an automated immunofluorescence and machine-learning-based primary screen of structural phenotypes in human endothelial cells deficient in CCM2; a secondary screen of functional changes in endothelial stability in these same cells; a rapid in vivo tertiary screen of dermal microvascular leak in mice lacking endothelial Ccm2; and finally a quaternary screen of CCM lesion burden in these same mice. We screened 2,100 known drugs and bioactive compounds, and identified two candidates for further study, cholecalciferol (Vitamin D3) and tempol (a scavenger of superoxide). Each drug decreased lesion burden in a mouse model of CCM vascular disease by approximately 50%. Conclusions By identifying known drugs as potential therapeutics for CCM, we have decreased the time, cost, and risk of bringing treatments to patients. Each drug also prompts additional exploration of biomarkers of CCM disease. We further suggest that the structure-function screening platform presented here may be adapted and scaled to facilitate drug discovery for diverse loss-of-function genetic vascular disease.