HEDGEHOG/HDAC6 INHIBITION AND CHEMOTHERAPY: ASSESSMENT OF NEW DRUG COMBINATION IN ACUTE MYELOID LEUKEMIA

In Acute Myeloid Leukemia (AML), the dysregulation of the Hh signaling is involved in the development and expansion of leukemic cancer cells and influences the response to therapeutic agents. Notably, the FDA approved only one Hh inhibitor (glasdegib) as a therapeutic strategy for AML treatment, and the majority of patients eventually relapse, underlying the urgency of discovering new therapeutic targets. One characteristic of the Hh pathway is its localization on the primary cilium membrane (PC), a microtubule-based organelle expressed by almost all non-proliferating mammalian cells. Indeed, centrosomes participate in a mutually exclusive manner in the formation of PC or mitotic spindle. Cancer cells, including AML cell lines, are characterized by a high rate of proliferation and fail to present the PC on their surface. Novel approaches to restore the PC on the surface of cancer cells are emerging, and most of them target the histone deacetylase HDAC6. Indeed, HDAC6 inhibition prevents the reabsorption of the PC and blocks cell proliferation. Since HDAC6 inhibitors (i.e., TubastatinA) are already used to treat other tumors, they might also be promising in AML treatment. In this work, analyzing the blood samples of 36 adult AML patients, we demonstrated that Hh target genes (GLI1, PTCH1), HDAC6, and the Multi- Drug-Resistant genes (MDRs) ABCC1 and ASXL1 were more expressed than in healthy donors (HD). In addition, through in silico analyses, we verified that in AML patients the expression of Hh/HDAC6 and MDRs genes were positively correlated. We also detected the same genetic regulation in in vitro models of AML, as cell lines with higher Hh expression (U937 and THP-1) showed higher levels of HDAC6 and MDRs than cell lines with low Hh expression (NB-4 and OCI-AML2). We generated a zebrafish model with Hh hyperactivation through the injection of the shh mRNA to functionally investigate the effect of Hh/HDAC6 dysregulation, and we confirmed the increased expression of hdac6 and MDRs. Moreover, in the zebrafish reporter line for the hematopoietic stem precursor cells (HSPCs) the Tg(CD41:GFP) line, we found that Hh hyperactivation induces the hyperproliferation and expansion of the HSPCs in the caudal hematopoietic tissue, a phenotype that resembles the expansion of leukemic blast of AML patients. Interestingly, we rescued this hematopoietic defect by treating the embryos with the HDAC6 inhibitor TubastatinA but not with the Hh inhibitor cyclopamine. By the generation of a zebrafish model carrying the overexpression of the human HDAC6 mRNA, we demonstrated that HDAC6 alone can induce the hyperproliferation of the HSPCs population and that this phenotype is specific as, through its inhibition, we rescued the hematopoietic defect. Since we observed that an increased proliferation rate elicited the expansion of HSPCs, we hypothesized an implication of the PC. Indeed, we demonstrated that zebrafish HSPCs present the PC, therefore suggesting that HDAC6 controls HSPCs proliferation, through PC's stabilization on their surface. For the first time, we described a role for HDAC6 in HSPCs expansion and identified it as a promising target for AML patients. Indeed, HDAC6 inhibition was also efficient in reducing the viability of leukemic cell lines and HSPCs expansion in well-established AML zebrafish models, carrying the overexpression of genes frequently mutated in AML patients: NPMc+ and FLT3-ITD. Moreover, in these zebrafish models, we demonstrated the efficacy of combination therapy with the standard chemotherapeutic agent cytarabine and HDAC6 inhibition. In conclusion, we identified a positive correlation between the Hh signaling, HDAC6 and the MDR genes in AML patients. In the zebrafish model, we reported that both Hh or HDAC6 overexpression drive the hyperproliferation of the HSPCs population, a phenotype that is rescued only through HDAC6 inhibition. HSPCs hyperproliferation and the rescue through the specific HDAC6 inhibition, might be explained by alteration in the PC, that we described to present in the HSPCs. As reporter for Hh inhibition, we demonstrated that also HDAC6 inhibition efficiently reduces the expression of MDR genes. Therefore, we hypothesize the use of HDAC6 inhibitor to counteract AML resistance mechanisms. Finally, we described that HDAC6 inhibition shows high potency in specific AML condition, and that can be a suitable target for the assessment of new combination therapies with standard chemotherapic agents.