Simple SummaryGlioblastoma, IDH-wild type (GBM) is the most common malignant primary brain tumor. Advances in cancer therapy remain unsuccessful in the treatment of GBM patients and have not extended the median survival beyond 12–18 months with the current treatment of surgery, chemotherapy, and radiotherapy. A central issue to finding a curative treatment option is the radioresistant nature of GBM. The goal of our study was to validate the therapeutic efficacy of enriching GBM tumor cells in the phase of the cell cycle where they are most vulnerable to radiotherapy, mitosis, using a small molecule inhibitor to the mitotic kinesin, KIF11. We confirmed that KIF11 inhibition radiosensitized GBM cells and improved overall survival in preclinical mouse models of GBM. These findings offer a new therapeutic modality that can increase the efficacy of radiotherapy for GBM with the ultimate goal of improving patient outcomes.AbstractGlioblastoma, IDH-wild type (GBM) is the most common and lethal malignant primary brain tumor. Standard of care includes surgery, radiotherapy, and chemotherapy with the DNA alkylating agent temozolomide (TMZ). Despite these intensive efforts, current GBM therapy remains mainly palliative with only modest improvement achieved in overall survival. With regards to radiotherapy, GBM is ranked as one of the most radioresistant tumor types. In this study, we wanted to investigate if enriching cells in the most radiosensitive cell cycle phase, mitosis, could improve localized radiotherapy for GBM. To achieve cell cycle arrest in mitosis we used ispinesib, a small molecule inhibitor to the mitotic kinesin, KIF11. Cell culture studies validated that ispinesib radiosensitized patient-derived GBM cells. In vivo, we validated that ispinesib increased the fraction of tumor cells arrested in mitosis as well as increased apoptosis. Critical for the translation of this approach, we validated that combination therapy with ispinesib and irradiation led to the greatest increase in survival over either monotherapy alone. Our data highlight KIF11 inhibition in combination with radiotherapy as a new combinatorial approach that reduces the overall radioresistance of GBM and which can readily be moved into clinical trials.