ROLE OF NOTCH SIGNALING IN STROMA-DEPENDENT ACUTE MYELOID LEUKEMIA CELL CHEMORESISTANCE.

Both preclinical and clinical investigations suggest that Notch signaling is critical for the development of many cancers and for their response to chemotherapy. We previously showed that Notch inhibition abrogates stromal-induced chemoresistance in lymphoid neoplasms.However, the role of Notch in acute myeloid leukemia (AML) and its contribution to the crosstalk between leukemia cells and bone marrow stromal cells remain controversial.In this study, we evaluated the role of the Notch pathway in the proliferation, survival and chemoresistance of AML cells in co-culture with bone marrow mesenchymal stromal cells expanded from both healthy donors (hBM-MSCs) and AML patients (hBM-MSCs*). hBMMSCs and hBM-MSCs* showed similar expression of Notch receptors and ligands, except for Notch1 and Jagged1 that were expressed at higher level by hBM-MSCs*. In addition, at least 50% of AML samples showed basal Notch activation, and the expression and activation pattern was modulated after co-culture with hBM-MSCs*. Notably, hBM-MSCs* were more efficient in supporting AML proliferation and inducing AML chemoresistance, as comparedto hBM-MSCs. Treatment with either Notch inhibitors or combinations of Notch receptorblocking antibodies significantly lowered the supportive role of hBM-MSCs and hBMMSCs* towards AML blast cells in presence of chemotherapeutic agents. The specific blockade of Notch1, 2, 3 or Jagged1, 2 reduced partially the chemoresistance, while Notch4 blockade restored completely primary AML cell chemosensitivity when in co-culture withhBM-MSCs. With the aim to unravel the mechanisms underlying Notch activation, we silenced the main Notch transcription factor RBP-jK through shRNA plasmids in two AML cells lines, namely HL-60 and THP1. Consistently, silenced cells were more sensible to drug treatment than cells infected with non-specific shRNA. Interestingly, RBP-jk knock-down was associated with low levels of pro-survival proteins, such as AKT, STAT3 and NF-κB.Accordingly, we found that Notch pharmacological blockade through gamma-secretase inhibitors in synergy with chemotherapy activates Bax/caspase-3 axis by lowering the protein level of AKT, STAT3 and NF-κB in AML cells in co-culture with hBM-MSCs.Our results suggest that Notch signaling inhibition, by overcoming the stromal-mediated promotion of chemoresistance, may represent a potential therapeutic target not only for lymphoid neoplasms but also for AML.