Evaluation of Histone Deacetylase 6 inhibitors as possible neuroprotectors against Paclitaxel-induced peripheral neurotoxicity

About 70% of treated cancer patients might develop Chemotherapy-Induced Peripheral Neuropathy (CIPN) during or after chemotherapy treatment. CIPN is an important therapeutic problem to solve, and it is mandatory identifying drugs able to counteract it, studying the mechanisms at the basis of the pathology. We focused our attention on the NMNAT2/SARM1 pathway, suggested by several data to be associated with peripheral neuropathy and neurite degeneration. We investigated Paclitaxel, a microtubule-stabilizing agent that is widely used against breast cancer, inducing CIPN with high frequency; by binding microtubules, it reduces axonal transport (including NMNAT2 transport), consequently promoting activation of SARM1, a pro-degenerative protein. Histone deacetylase 6 (HDAC6) localizes predominantly in cytoplasm and exerts the deacetylase enzymatic activity mainly on non-histone substrates. It is mainly involved in deacetylating tubulin, playing a crucial role in microtubule stability and therefore emerging as a potential target to reduce Paclitaxel-induced neurotoxicity. The aim of this work was to investigate NMNAT2/SARM1 pathway as the possible molecular mechanism through which HDAC6 inhibitors protect against Paclitaxel-induced neurotoxicity. The potential effect of three selected HDAC6 inhibitors (Ricolinostat, SW-100, Tubastatin A), was tested, evaluating neuronal viability and neurite length, in an in vitro model based on adult mice primary sensory neurons cultures. Neurons were treated with HDAC6 inhibitors individually, as well as in combination with Paclitaxel, with increasing concentrations of drugs; NMNAT2 and SARM1 protein expressions were analyzed through Western blotting. Lastly, acetylated SARM1 levels were evaluated using immunoprecipitation assay, and NMNAT2 and SARM1 localization was evaluated by immunofluorescence. Data demonstrate that Paclitaxel reduces neuronal survival rate and neurite elongation in a dose-dependent manner, whilst the co-treatment with Paclitaxel and each of the three HDAC6 inhibitors protects against Paclitaxel-induced neurotoxicity. Furthermore, no HDAC6 inhibitor showed interference with Paclitaxel antitumoral activity on MCF-7 cells. Paclitaxel decreases NMNAT2 and SARM1 protein expression, which are significantly restored upon HDAC6 inhibitor co-treatment. Immunofluorescence experiments demonstrate that Paclitaxel induces neurite fragmentation and a redistribution of NMNAT2. Ricolinostat, although neuroprotective, does not restore the localization of NMNAT2 in control neurons. However, Ricolinostat treatment (alone or in combination with Paclitaxel), increases SARM1 acetylation, thus contributing to its inhibition. Our results demonstrate that HDAC6 inhibitors modulate NMNAT2/SARM1 pathway suggesting that their protective effect against Paclitaxel-induced neurotoxicity may be mediated by this pathway.