Systems analysis uncovers early temozolomide responses and peptide antigens in glioblastoma.

Temozolomide (TMZ) is the standard treatment for nearly all glioblastoma (GBM) patients, as it is the only chemotherapy shown to extend overall survival. However, this benefit is limited to a few months, underscoring the need for combination strategies to improve its efficacy. While TMZ-induced DNA damage can both mediate cytotoxicity and promote resistance, DNA damage more broadly can also stimulate immune activation. To evaluate its immunomodulatory potential, we characterized the previously unexplored early, cell-intrinsic consequences of TMZ in GBM cells, spanning DNA damage, stress responses, and antigen presentation. A multi-omics approach combining RNA sequencing and quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) profiled changes in gene expression, nascent protein translation, steady-state protein levels, kinase-substrate phosphorylation patterns, and MHC-I peptide presentation in GBM cells within 72 hours of TMZ exposure. This analysis revealed rapid activation of DNA damage signaling and p53-associated stress pathways, alongside dynamic changes in protein synthesis and antigen presentation. A set of TMZ treatment-associated peptide antigens (TAPAs) was identified, including peptides derived from stress response proteins, phosphorylated MHC-I peptides, and those induced by other genotoxic treatments such as radiation. Several of these peptides were also detected in recurrent GBM patient tumors. Our findings suggest that TMZ not only triggers early adaptive and potentially resistance-associated stress programs but may also enhance the immune visibility of GBM cells. These data highlight potential windows for combination therapies with TMZ that bolster immune recognition of GBM, while the systems approach provides a framework to examine how genotoxic therapies across cancers alter tumor immunogenicity.