Targeting destabilized DNA G-quadruplexes and aberrant splicing in drug-resistant glioblastoma

Temozolomide (TMZ) is a chemotherapy agent that adds mutagenic adducts to guanine, and is first-line standard of care for the aggressive brain cancer glioblastoma (GBM). Methyl guanine methyl transferase (MGMT) is a DNA repair enzyme that can remove O6-methyl guanine adducts prior to the development of catastrophic mutations, and is associated with TMZ resistance. However, inhibition of MGMT fails to reverse TMZ resistance. Guanines are essential nucleotides in many DNA and RNA secondary structures. In several neurodegenerative diseases (NDs), disruption of these secondary structures is pathogenic. We therefore took a structural view of TMZ resistance, seeking to establish the role of guanine mutations in disrupting critical nucleotide secondary structures. To test whether these have functional impacts on TMZ-resistant GBM, we focused on two specific guanine-rich regions: G-quadruplexes (G4s) and splice sites. Here we report broad sequence- and conformation-based changes in G4s in acquired or intrinsic TMZ resistant vs. sensitive GBM cells, accompanied by nucleolar stress and enrichment of nucleolar RNA:DNA hybrids (r-loops). We further show widespread splice-altering mutations, exon skipping, and deregulation of splicing-regulatory serine/arginine rich (SR) protein phosphorylation in TMZ-resistant GBM cells. The G4-stabilizing ligand TMPyP4 and a novel inhibitor of cdc2-like kinases (CLKs) partially normalize G4 structure and SR protein phosphorylation, respectively, and are preferentially growth-inhibitory in TMZ-resistant cells. Lastly, we report that the G4- and RNA-binding protein EWSR1 forms aberrant cytoplasmic aggregates in response to acute TMZ treatment, and these aggregates are abundant in TMZ resistant cells. Preliminary evidence suggests these cytoplasmic EWSR1 aggregates are also present in GBM clinical samples. This work supports altered nucleotide secondary structure and splicing deregulation as pathogenic features of TMZ-resistant GBM. It further positions cytoplasmic aggregation of EWSR1 as a potential indicator for TMZ resistance, establishes the possibility of successful intervention with splicing modulatory or G4-targeting agents, and provides a new context in which to study aggregating RNA binding proteins.