Dottorato in MORFOGENESI E INGEGNERIA TISSUTALE

Titolo della tesi: Characterization of the AML Bone Marrow Microenvironment Response to Azacitidine + Venetoclax Treatment

Despite significant advances in targeted therapies for acute myeloid leukemia (AML), drug resistance and relapse remain major challenges, largely due to clonal selection and the protective effect of the bone marrow microenvironment. This project aimed to elucidate the molecular mechanisms by which bone marrow stromal cells (BMSCs) and BM mesenchymal stem cells (MSCs) confer resistance to FLT3-ITD+ AML cells treated with Azacitidine and Venetoclax (AV), a new standard of care for AML patients unfit for intensive chemotherapy, with the goal of identifying potential targets to overcome this protection. Using co-culture systems of the AML cell line MOLM-13 with murine BMSCs (MS5) or human MSCs (hMSC/BM-MSC TERT 292), we evaluated cell interactions and survival upon AV treatment through flow cytometry and confocal microscopy. Proteomic analyses of AML cells and hMSCs, cultured alone or together, with and without AV treatment, revealed modulation of pathways connected to cell-cell communication, inflammation, and extracellular matrix organization. Functional assays confirmed that both mBMSCs and hMSCs protect AML cells from AV-induced apoptosis, with partial dependence on direct cell contact in the case of hMSCs. Based on proteomic data and previous findings implicating mechanotransduction, and particularly the transcriptional co-activator YAP, in the protective mechanisms executed by BMSCs, we further investigated its role. We first observed dynamic localization of YAP between the nucleus and cytoplasm in hMSCs upon direct contact with AML cells and AV treatment, suggesting its involvement in the cross-talk. Subsequently, we found that YAP silencing in hMSCs restored AML sensitivity to AV treatment and induced a cytostatic effect in AML cells. Furthermore, YAP depletion triggered activation of an inflammatory response through phosphorylation of STAT1 and STAT2, observed only under AML-hMSC direct contact conditions. In conclusion, our findings demonstrate that BMSCs protect AML cells from AV treatment through mechanisms involving YAP. Targeting YAP in stromal cells impairs the MSCs’ protective effect and re-sensitizes AML cells to AV treatment, likely through the activation of the inflammatory response.