Entinostat and Tucidinostat Potentiate Temozolomide Response in Glioblastoma Models

Glioblastoma (GBM) frequently develops resistance to temozolomide (TMZ), limiting the effectiveness of standard therapy. Histone deacetylase (HDAC) inhibitors have emerged as potential chemosensitizers; however, the comparative performance of these inhibitors and the associated mechanistic impact remain incompletely characterized.

In vitro, this study systematically evaluated the effects of the Class I-selective HDAC inhibitors Entinostat and Tucidinostat, and the pan-HDAC inhibitor Vorinostat on TMZ sensitivity in multiple GBM cell lines (U87, U251, LN229). Additionally, this study assessed cell viability, morphology, and the expression of selected markers glial fibrillary acidic protein (GFAP), microtubule-associated protein 2 (MAP2), class III β-tubulin (TUBB3), and synapsin I (SYN1). In vivo, the therapeutic efficacy of Entinostat combined with TMZ was evaluated in a U87 xenograft mouse model by measuring tumor growth, and the expression of GFAP and TUBB3 was examined in tumor tissues. Clustered Regularly Interspaced Short Palindromic Repeats associated protein 9 (CRISPR-Cas9)-mediated knockout of HDAC1/2/3 was used to test the dependence of these effects on canonical Class I HDACs.

Entinostat and Tucidinostat significantly enhanced TMZ-induced cytotoxicity across all cell lines, demonstrating greater TMZ-sensitizing activity than Vorinostat under the conditions tested. Treatment was associated with altered cell morphology and changes in marker expression, including reduced GFAP and increased MAP2 and TUBB3. In the U87 xenograft model, Entinostat combined with TMZ significantly suppressed tumor growth and was associated with decreased GFAP and increased TUBB3 expression. Importantly, both TMZ sensitization and the associated morphological and marker changes persisted in HDAC1/2/3-knockout cells, suggesting that the effects are not fully explained by HDAC1/2/3 depletion. These effects were consistent across multiple cell lines and in xenograft tumors.

Entinostat and Tucidinostat enhance TMZ sensitivity in GBM models and are associated with specific markers and morphological changes that persist despite HDAC1/2/3 knockout. These findings support further evaluation of Entinostat and Tucidinostat as TMZ-sensitizing agents in GBM models and highlight associated phenotypic and molecular changes that warrant further mechanistic validation.