Triple-negative breast cancer (TNBC) remains the most aggressive breast cancer subtype, accounting for over half of breast cancer-related deaths. In the era of precision oncology, epigenetic dysregulation has emerged as a key driver of breast tumorigenesis. Among epigenetic targets, histone deacetylase 6 (HDAC6) has attracted increasing attention due to its cytoplasmic localization and regulation of non-histone substrates such as Hsp90, α-tubulin, and cortactin. We recently identified a novel class of spirocyclic molecules as potent and selective HDAC6 inhibitors. Here, we investigated the pharmacological profile of Mesinostat, one of the most selective compounds of this series, in human TNBC cell lines, 3D spheroids, and patient-derived organoids (PDOs) from lymph node metastases. In TNBC cells, using western blot analysis, we demonstrated that Mesinostat (1-30 µM) selectively inhibited HDAC6 by increasing acetylated α-tubulin levels, with no effect on the histone H3 acetylation. Yet, Mesinostat markedly reduced cell proliferation through apoptosis, cell cycle arrest, and autophagy. Furthermore, it inhibited epithelial-mesenchymal transition, decreased cell migration, and disrupted spheroid integrity. Treatment of PDOs led to a dose-dependent reduction in viability and clonogenic capacity, accompanied by downregulation of stemness and proliferation markers. Overall, these findings demonstrate that selective HDAC6 inhibition by Mesinostat modulates key oncogenic pathways in TNBC, supporting its potential as a promising therapeutic approach for HDAC6-overexpressing breast cancers.
