HDAC8 AND COHESIN: 'OMICS' ANALYSES FOR THE IDENTIFICATION AND FUNCTIONAL VALIDATION OF THEIR TARGETS USING ZEBRAFISH (DANIO RERIO) AND IN VITRO MODEL SYSTEMS

The use of pan-HDACi for disease treatment has gained an interest in recent years, albeit exhibiting low specificity, variable efficacy and side effects. Each HDAC with its own activity could be considered as an independent pharmacological target to develop an effective therapy that circumvents the adverse effects of pan-HDACi treatment. HDAC8 belongs to class I HDAC and is known to modulate cohesin complex activity through deacetylation of SMC3. It possesses a unique structure among HDACs, which allowed the development of highly specific inhibitors, such as the PCI-34051. However, HDAC8 function and its involvement in pathological conditions is still largely unknown. To examine in depth HDAC8 physiological and pathological roles and assess whether it can represent a valuable pharmacological target, we analysed its function and the effect of its inhibition by using both in vitro (cell lines) and in vivo (zebrafish) models. In particular, we assessed HDAC8 function in three different tissues and related disorders: i) haematopoietic stem and progenitor cells (HSPC) and acute myeloid leukemia (AML); ii) neural stem cells (NSC) and Cornelia de Lange syndrome (CdLS); iii) skeletal muscle and Duchenne muscular dystrophy (DMD). We found that HDAC8 overexpression increased the proliferation of HSPCs in zebrafish and that its inhibition with PCI treatment restored normal phenotype, favouring cell cycle arrest, and induced apoptosis of AML cells. By contrast, HDAC8 knockdown lead to impairment of both central nervous system development and skeletal muscle differentiation. Furthermore, we found that HDAC8 overexpression is also associated with DMD phenotype and demonstrated that treatment with PCI inhibitor almost restored normal condition. Such positive effects were underlined by multiple mechanisms, which included cell cycle arrest, apoptosis induction and modulation of canonical Wnt pathway. Moreover, by acetylome profiling we identified α-tubulin as HDAC8 target thus revealing HDAC8 involvement in regulation of microtubule structure. Additionally, to confirm the involvement of HDAC8 in the aforementioned pathologies, we investigated also the role of its partner NIPBL. By RNA-seq analysis we assessed the effect of NIPBL knockdown on gene expression revealing a number of differentially expressed genes linked to pathways altered in CdLS or AML.