Glioblastoma multiforme (GBM) is the most aggressive brain tumor. It remains incurable with a median survival of around 15 months. The main limitation of standard therapy is the development of resistance to temozolomide (TMZ), often associated with tumor heterogeneity and enrichment of stem-like glioma initiating cells (GICs). This underlines the need for new therapeutic strategies. Bromodomain and extraterminal (BET) proteins are epigenetic readers that regulate proliferation, stemness and survival and their pharmacological inhibition with BET inhibitors (BETi) jeopardizes these properties. Proteolysis-targeting chimeras (PROTACs) have emerged as a tool for selectively degrading proteins, and they could potentially overcome the limitations of pharmacological inhibition. This study aims to determine whether acquired TMZ resistance generates BET dependent state that can be therapeutically exploited.

BET protein expression and its association with prognosis and response were analysed using GlioVis and Roc Plotter databases. A TMZ-resistant cell line (U87R) was generated from the TMZ-sensitive cell line (U87S) using a pulsed-strategy over 6-7 months. Resistance was monitored by MTT assays. Doubling time assays, Western blot (WB), in vivo implantation (subcutaneous and intracranial), and RNAseq were performed to characterize the resistant model. The impact of BETi and TMZ, alone or in combination, as well as BET-PROTACs were evaluated by MTT, flow cytometry, and WB. Patient-derived 3D models (GH2 and 12O12) were used to evaluate the effect of BETi and PROTAC sphere formation, proliferation, and stemness.

High expression of BET proteins correlates with poor prognosis in GBM patients. TMZ-resistant models exhibited an increased in proliferation rate and enhanced tumor growth in vivo. Molecular characterization showed elevated levels of BRD4 and increased expression of stemness markers (SOX2, SOX9) and RNAseq identified pathways consistent with transcriptional reprogramming and cellular plasticity. The combination of BETi and TMZ in U87R cells reduced proliferation compared to single treatments, demonstrating synergy. This combination enhanced cell death, reducing BCL2 proteins and stemness markers. Notably, BET-PROTACs alone were able to reduce cell proliferation and increase cell death in resistant cells, efficiently degrading BET proteins and reducing stemness markers. PROTACs also reduced sphere formation and proliferation in 3D patient-derived models.

Acquired TMZ resistance is associated with elevated BET proteins levels and a stem-like state in GBM models. Pharmacological inhibition and proteolytic degradation of BET proteins reduce viability and stemness features, supporting that TMZ-resistant cells exhibit increased BET dependence that can be therapeutically exploited.