Temozolomide-induced guanine mutations create exploitable vulnerabilities of guanine-rich DNA and RNA regions in drug-resistant gliomas

Temozolomide (TMZ) is a chemotherapeutic agent that has been the first-line standard of care for the aggressive brain cancer glioblastoma (GBM) since 2005. Although initially beneficial, TMZ resistance is universal and second-line interventions are an unmet clinical need. Here, we took advantage of the known mechanism of action of TMZ to target guanines (G) and investigated G-rich G-quadruplex (G4) and splice site changes that occur upon TMZ resistance. We report that TMZ-resistant GBM has guanine mutations that disrupt the G-rich DNA G4s and splice sites that lead to deregulated alternative splicing. These alterations create vulnerabilities, which are selectively targeted by either the G4-stabilizing drug TMPyP4 or a novel splicing kinase inhibitor of cdc2-like kinase. Last, we show that the G4 and RNA binding protein EWSR1 aggregates in the cytoplasm in TMZ-resistant GBM cells and patient samples. Together, our findings provide insight into targetable vulnerabilities of TMZ-resistant GBM and present cytoplasmic EWSR1 as a putative biomarker.

Automated summary

Temozolomide (TMZ) is a chemotherapeutic agent used for the treatment of glioblastoma (GBM), but TMZ resistance is common. This study identified guanine mutations and splice site changes in TMZ-resistant GBM that disrupt G-rich DNA G-quadruplexes and alternative splicing. The vulnerabilities created by these alterations can be targeted by G-quadruplex stabilizers or a splicing kinase inhibitor. Furthermore, cytoplasmic aggregates of the RNA binding protein EWSR1 were observed in TMZ-resistant GBM, suggesting it may serve as a biomarker.