DDEL-10. Mild inhibition of microtubule assembly disrupts mitochondria function in glioblastoma in interphase via Drp1 activation

Abstract Microtubule targeting agents (MTAs) disrupt the mitotic process and kill cancer cells by triggering apoptosis and autophagy. ST-401 is a mild inhibitor of microtubule (MT) assembly that does not disrupt mitosis and kills glioblastoma (GBM) cells in interphase by disrupting the molecular machinery involved in mitochondrial energy supply. ST-401 passes the blood brain barrier and exhibits significant in vivo antitumor activity in GBM mouse models. Here we sought to identify the small molecule chemical characteristics required to mildly inhibit MT assembly and kill GBM cells in interphase by disrupting energy supply as a foundation for drug development. The solved Cryo-EM structure of ST-403, the active enantiomer of ST-401, bound to the colchicine site of a,b-tubulin and ensuing computational analysis of its docking revealed its unique structural stabilization mode of a,b-tubulin. Molecular dynamic analysis of related compounds that bind the colchicine site with comparable mild inhibitory activities provided a model depicting the structural stabilization induced by mild inhibitors of MT. Accordingly, ST-403 and these mild inhibitors of MT assembly similarly kill GBM cells in culture and reduce their mitochondrial energy supply. These results outline the fundamental chemical determinants required for brain penetrant mild inhibitors of MT assembly to kill GBM cells by reducing mitochondrial energy supply. We then used an unbiased phospho-proteomics approach to identify signaling networks that are modulated by mild inhibitors of MT assembly and identified mitochondrial fission induced by Drp1 as a key mediator validated by its genetic knockout and overexpression. Accordingly, low Drp1 expression represents a potential prognostic biomarker of increased survival in GBM patients. Our study unravels a previously unappreciated molecular mechanism that links brain-penetrant mild inhibitors of MT assembly to the killing GBM cells in interphase by disrupting mitochondria energy supply.