Glioblastoma multiforme (GBM) is the most aggressive brain tumor, characterized by a high recurrence rate and resistance to current treatments. Available therapies, such as radiotherapy and chemotherapy with temozolomide (TMZ), offer a median survival of only 15 months after diagnosis. Glioma-initiating cells (GICs) contribute to TMZ resistance, highlighting the need for novel therapeutic strategies. Bromodomain and extraterminal (BET) proteins regulate the transcription of key genes involved in oncogenesis and treatment resistance. BET inhibitors (BETi) have been shown to modulate genes associated with proliferation, metastasis and cancer stem cells and have demonstrated efficacy in other solid tumors such as breast cancer. CDK9 is an essential kinase in gene transcription and its overexpression is associated with poor prognosis in several cancers. CDK9 inhibition induces apoptosis, and studies suggest that the combination of BETi and CDK9 inhibitors (CDK9i) may enhance their antitumor effects, providing new treatment options for GBM. The aim of this study is to evaluate the therapeutic potential of BETi alone or in combination with CDK9i in GBM models.

TMZ-resistant cell line (U87R) were generated from TMZ-sensitive immortalized cell line (U87) using pulse treatments. BET and CDK9 expression and prognosis were analyzed using the GlioVis and Roc Plotter databases. In addition, BET and CDK9 protein expression was determined in the GBM cell lines (A172, T98, U87, U87R) by Western blot (WB). The efficacy of BETi and CDK9i alone or in combination was assessed using MTT, flow cytometry and WB assays. Tumor characteristics such as migration, invasion and recurrence were also analyzed. The treatment's efficacy was confirmed in a subcutaneous xenograft model in mice using the resistant model (U87R). RNAseq analyses were performed.

Increased expression of BRD4 and CDK9 was observed in GBM tissues compared to non-tumor tissues. A positive correlation was found between BRD4 and CDK9, suggesting that both may be effective therapeutic targets. We confirmed by WB that the resistant model showed elevated levels of BRD4 and CDK9 proteins. Combination treatment with BETi and CDK9i induced a strong synergistic effect leading to increased cell death as confirmed by flow cytometry and WB. In addition, a reduction in tumor cell migration, invasion and recurrence capacity was observed with the combination treatment compared to the single treatments. The in vivo experiments also showed a reduction in tumor growth with the combination therapy. Finally, RNAseq analyses were performed to elucidate how the combined pharmacological inhibition works, leading to the identification of several deregulated genes and pathways that require further analysis.

Combined inhibition of BRD4 and CDK9 shows promise as a potential therapeutic strategy for the treatment of GBM.