Small-molecule Mediated Activation of Mdia Formins Triggers Global Changes in Serine/threonine Kinase Activity Before Glioblastoma Tumor Microtube Collapse

Introduction: Tumor microtubes are a cytoskeleton-dependent mechanism that glioblastoma cells use to avoid therapeutic lethality. These neurite-like membrane tubes facilitate diffuse central nervous system invasion and interconnect tumor cells into a functional syncytial network that is highly resistant to current clinical standards of GBM care. Identifying targetable signaling nodes that critically maintain tumor microtube networks has the potential to meaningfully impact treatment outcomes in GBM. Targeting mDia formins with the small-molecule agonist IMM-02 triggers the collapse of existing tumor microtubes and their accompanying syncytial networks. Here we investigate the alterations in serine/threonine protein kinase activity triggered by IMM-02 treatment.

Methods: Pat27 patient-derived 2.5D semi-adherent networked culture systems were treated with IMM-02 or DMSO for 18 or 30 hours. The PamStation12 instrument was used with serine-threonine kinase (STK) PamChips to measure kinase activity. Upstream kinase activity was identified using Kinome Random Sampling Analyzer (KRSA).

Results: We observed a global change in kinase activity across multiple distinct protein kinase families including AUR, PKA, ERK, JNK, and PKC between 18-30 hours of IMM-02 treatment. After 18 hours of IMM-02 treatment, serine/threonine kinase activity was increased. In contrast, serine/threonine kinase activity was globally reduced across all observed kinase families after 30 hours of IMM-02 treatment. The ERK and PKA kinase families showed the greatest magnitude of change in activation dynamics in response to IMM-02 treatment.

Conclusion : The small-molecule mDia agonist IMM-02 triggers an initial increase, followed by a profound and global suppression of serine/threonine protein kinase activity after 18 hours and 30 hours of treatment in a patient-derived model of glioblastoma tumor microtube networks.