EHMT1/2-LIG1 axis enhances Temozolomide Sensitivity in Experimental Glioma

Background Temozolomide (TMZ) is a standard treatment for glioblastoma. The efficacy is limited by acquired resistance, particularly in tumors with an unmethylated MGMT promoter. Identifying synthetic lethal interactions may inform future therapeutic strategies. Methods We performed genome-wide CRISPR-Cas9 knockout screens in MGMT-expressing glioma cell lines under TMZ treatment. Candidate hits were validated by genetic depletion and pharmacological inhibition and assessed in publicly available datasets. Mechanistic investigation included RNA-seq, immunoblotting and immunofluorescence. Therapeutic efficacy was evaluated in patient-derived glioblastoma cultures and an orthotopic xenograft glioma model. Results CRISPR screening identified euchromatic histone-lysine N-methyltransferases (EHMT1/EHMT2) and DNA Ligase 1 (LIG1) as synthetic lethal interactors of TMZ. Their expression is associated with overall survival in publicly available glioma datasets. Genetic depletion of EHMT1, EHMT2 and LIG1 enhanced TMZ sensitivity and increased DNA damage. Pharmacological EHMT1/2 inhibition using UNC0642 demonstrated robust synergism with TMZ across multiple glioma cell lines and patient-derived primary cultures. Transcriptomic profiling revealed rapid DNA mismatch repair (MMR) pathway induction upon TMZ, whereas synthetic lethal interactors were not consistently upregulated. Combined TMZ and UNC0642 treatment disrupted the EHMT1/2-LIG1K126-UHRF1-DNMT1 DNA methylation maintenance axis, resulting in enhanced DNA damage. In an orthotopic xenograft model, combination therapy prolonged survival compared to monotherapies in late treatment intervals. Conclusion We discovered and validated EHMT1/2 and LIG1 as actionable targets synergizing with TMZ through disruption of the DNA methylation maintenance pathway. These data provide a preclinical rationale for the investigation of EHMT1/2 inhibitors in combination with TMZ, particularly for MGMT-unmethylated glioblastoma.