Septin inhibitors exert opposing effects on distinct higher-order septin structures in melanoma cells

Septins are cytoskeletal GTPases that assemble into diverse higher-order structures with context-dependent mechanical and signaling functions. Recent work identified a septin-dependent pro-survival pathway in metastatic cancer cells, termed bleb signaling, in which plasma membrane blebbing promotes the formation of septin signaling hubs that support survival during cellular detachment and dissemination. Disrupting these hubs may offer a novel therapeutic strategy targeting disseminated cancer cells. Here, we compare the effects of three septin-targeting compounds, forchlorfenuron (FCF), UR214-9, and REM127, on septin organization and viability in melanoma cells using confocal microscopy and quantitative computer vision analysis. FCF and UR214-9 both effectively destabilized septin hubs, while REM127 had no appreciable effect. FCF also reproduced the context-specific viability reduction previously seen in detached melanoma cells, while UR214-9 caused broad off-target toxicity and cell death in all growth conditions. As with hub disruption, REM127 produced no effect on viability. Surprisingly, though none of the compounds caused an increase in higher-order septin structures during detachment, all three increased end-on-end polymerization of filamentous septins at stress fibers when grown adhered to substrate. These findings demonstrate that septin-targeting compounds exert divergent effects on different classes of septin higher-order structures within the same cell type, promoting filamentous assembly while disrupting or failing to promote membrane-associated signaling hubs. This highlights not only the structural distinctiveness of such classes, but that pharmacological targeting can produce opposite effects on them. Moreover, this study emphasizes the need for next-generation septin inhibitors capable of selectively targeting disease-relevant septin assemblies.