Combining interstrand crosslinking agents with histone deacetylase inhibitors against high grade IDH mutant gliomas

Abstract

Mismatch repair (MMR) deficiency contributes to temozolomide (TMZ) resistance in a subset of isocitrate dehydrogenase 1/2 mutant (IDHmut) gliomas, creating a need for therapies that retain activity despite MMR loss. We evaluated the novel interstrand crosslinking agent KL-50, alone and in combination with the histone deacetylase inhibitor belinostat, in patient-derived IDHmut glioma models. MSH6 knockout or constitutive expression established four matched MMR-proficient and MMR-deficient culture models. Drug responses were assessed using live cell count and cytotoxicity assays, and KL-50 monotherapy was tested in intracranial BT142 orthotopic xenografts. LOEWE synergy analysis, RNA sequencing, and a genome-wide CRISPR knockout screen assessed combination activity and potential mechanisms. MMR deficiency increased TMZ GI values 2.2- to 30.4-fold, whereas KL-50 retained antiproliferative activity and showed enhanced cytotoxicity in selected MMR-deficient models. In TMZ-naive, MMR-proficient BT142 xenografts, KL-50 increased median survival from 107.5 to 194 days. Two of eight treated mice had no histologically detectable residual tumor after 220 days post-engraftment. KL-50 and belinostat exhibited synergistic cytotoxicity in BT142, 905, and TS603 IDHmut glioma cultures. Transcriptomic gene ontology analyses identified downregulation of DNA repair pathways following histone deacetylase inhibition, including terms for homologous recombination and double-strand break repair. The CRISPR screen identified depletion of PKMYT1-targeting gRNAs during KL-50 treatment, implicating this mitotic checkpoint kinase as a candidate determinant of drug sensitivity. PKMYT1 expression was also reduced by histone deacetylase inhibition. These findings support further preclinical development of KL-50, alone and in combination with belinostat, for IDHmut gliomas including tumors with MMR-associated TMZ resistance.