Rewiring mitotic checkpoint control via Mps1 inhibition overcomes DNA repair-mediated resistance in glioblastoma.

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain cancer in adults, and treatment is frequently limited by tumor resistance to temozolomide (TMZ), the standard-of-care chemotherapy. This resistance is often driven by the tumor cell's enhanced capacity to repair TMZ-induced DNA damage. Cell division is normally controlled by two quality-control systems: the spindle assembly checkpoint (SAC), which ensures accurate chromosome segregation during mitosis, and the DNA damage response, which detects and repairs genomic damage. Growing evidence suggests these two systems are functionally connected, but whether this connection can be exploited pharmacologically in cancer remains unclear. Here, we redesigned a brain-penetrant chemical scaffold to develop G17, a small molecule that selectively inhibits monopolar spindle 1 (Mps1), the central kinase controlling SAC signaling. Characterization of G17 in biochemical and cellular models showed that Mps1 inhibition forces GBM cells to exit mitosis prematurely, resulting in persistent DNA damage and impaired long-term tumor cell growth. Notably, G17 remained active in TMZ-resistant glioblastoma cells that express O6-methylguanine-DNA methyltransferase (MGMT), the enzyme primarily responsible for TMZ resistance, indicating that its activity does not depend on MGMT-mediated DNA repair. Together, these findings provide pharmacological evidence that disrupting SAC signaling can expose a DNA repair vulnerability in glioblastoma and identify Mps1 inhibition as a candidate strategy warranting further investigation in treatment-resistant disease.