HDAC6 and HDAC8 combined targeting to counteract Glioblastoma Multiforme progression and treatment resistance

Glioblastoma Multiforme (GBM) is the most common and lethal primary brain tumor in adults. Standard care, comprising surgical resection followed by radio and chemotherapy with Temozolomide (TMZ), shows limited efficacy, highlighting the need for new therapeutic strategies. Among the aberrant pathways involved in GBM, histone deacetylases (HDACs) are frequently upregulated; in particular, HDAC6 and HDAC8 have emerged as potential therapeutic targets, paving the way for their combined inhibition as an alternative treatment approach. In addition, deregulation of sphingolipid metabolism and signaling has been implicated in GBM growth and drug resistance. In this work, we investigated the effects of HDAC6 and HDAC8 combined targeting by pharmacological inhibition with the selective compounds Tubastatin A and PCI34051 respectively. We employed two human GBM cell lines, U87-MG and T98G, which differ in their genetic profile and sensitivity to TMZ treatment. Additionally, we took advantage of the in vivo GBM zebrafish model zic:RAS, in which tumor development is induced by the expression of specific oncogenes in neural cells. Our results revealed that the combined inhibition of HDAC6 and HDAC8 significantly reduced GBM cell viability, as well as tumor growth in the in vivo zebrafish model. Co-treatment with TMZ further enhanced the reduction in cell viability and was also effective in vivo. Mechanistically, in T89G cells HDAC6 inhibition induced lysosomal stress, ultimately resulting in autophagic disruption and apoptosis, whereas HDAC8 inhibition determined cell-cycle alterations. Moreover, sphingolipidomic analysis indicated that both inhibitors synergistically modulated dihydroceramide/ceramide ratio, a critical regulator of tumor cell fate. In U87-MG cells, we observed a similar response to HDAC6 inhibition on the lysosomal compartment and an increase in acidic sphingomyelinase content. Interestingly, HDAC8 inhibition affected the acidic sphingomyelinase too, as revealed by a preliminary proteomic study. Furthermore, HDAC8 inhibition impacted on dihydroceramide/ceramide ratio and decreased glucosylceramide level, a well characterized feature of reduced drug resistance in GBM. The therapeutic relevance of the combined targeting is further supported by the negative impact of HDAC6/HDAC8 inhibition on the side population, a cellular subset related to cancer stemness and known to mediate treatment resistance and disease relapse. Therefore, taken together, our findings support the potential of the combined inhibition as a promising therapeutic approach for GBM and other HDAC6/HDAC8 overexpressing cancers.