Abstract 2887: ROCK and mDia have dynamic roles in glioblastoma tumor microtube invasion

Invasive motility limits treatment efficacy and is a significant contributor to poor outcomes in glioblastoma (GBM). GBM tumor microtubes are actin- and microtubule-enriched membrane tubes that facilitate invasive motility and underlie many components of GBM pathophysiology. Rho-GTPases mediate GBM invasion through localized activation of cytoskeletal effector proteins, such as mammalian Diaphanous-related formins (mDia) and Rho-associated protein kinase (ROCK). Active mDia nucleates and polymerizes F-actin and independently stabilizes microtubules, while active ROCK phosphorylates myosin phosphatase and myosin light chain to induce the actomyosin crosslinking required for contractility. GBM invasion depends on a delicate balance between mDia-mediated extension of leading-edge structures (such as tumor microtubes) and ROCK-mediated contraction of trailing cell bodies. In this study, we assessed the roles of ROCK and mDia in tumor microtube-associated GBM invasion using a 3D patient-derived neurosphere model of GBM invasion. Neurospheres were embedded in 3D matrices and treated with the small molecule inhibitor of ROCK (Y-27632) and the small molecule agonist of mDia (IMM-02). Treatment with Y-27632 alone reduced the total distance of cell body migration and increased tumor microtube length without effecting the total area of neurosphere invasion. Tumor microtubes extending from Y-27632 treated neurospheres displayed an atypical undulant morphology, further suggesting that ROCK inhibition modifies primary invasion programs. Treatment with IMM-02 alone profoundly reduced total area of neurosphere invasion, distance of cell body migration, and length of tumor microtubes. Combination treatment effects (Y-27632 + IMM-02) were time dependent. At 24 hours, combination treatment did not significantly reduce total area of invasion over either individual treatment. However, tumor microtubes were shorter with combination treatment than Y-27632 alone and longer than IMM-02 alone. At 96 hours, combination treatment reduced total area invaded and length of tumor microtubes in comparison to Y-27632 alone, but no significant difference was observed in comparison to IMM-02 alone. Western blot analysis of both free-floating patient-derived 3D neurospheres and semi-adherent “2.5D” monolayer cultures demonstrated that agonism of mDia formins with IMM-02 (96-hrs) results in the progressive loss of mDia1 and mDia2 protein expression. Therefore, endogenous mDia regulatory mechanisms triggered in response to continuous agonist-mediated mDia activation may inhibit mDia function more effectively than direct antagonism strategies in GBM. Conclusively, both IMM-02 and Y-27632 treatment disrupt the tumor microtube mechanism of GBM neurosphere invasion. Further studies are warranted to evaluate these small molecule compounds for potential anti-tumor microtube therapeutic effect in vivo. Citation Format: Kathryn N. Becker, Krista M. Pettee, Amanda Sugrue, Kevin A. Reinard, Jason L. Schroeder, Kathryn M. Eisenmann. ROCK and mDia have dynamic roles in glioblastoma tumor microtube invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2887.