Inhibition of Notch signalling overcomes bone marrow stromal-mediated anti-apoptotic and chemoresistance effects in AML.

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 may abro- gate stromal-induced chemoresistance in lymphoid neoplasms. How- ever, the role of Notch in acute myeloid leukemia (AML) and its contribution to the crosstalk between blast cells and bone marrow stro- mal cells remain controversial. AML blast cells were obtained from bone marrow samples (n=28) and peripheral blood (n=16) of AML patients. BM-MSCs were expanded from bone marrow of 12 healthy donors (BM-MSCs) and of 12 AML patients (BM-MSCs*). In vitro and in silico analysis showed that AML cells expressed Notch receptors and ligands, regardless the FAB classification or molecular pattern. Notably, 50% of AML samples showed basal Notch signalling activation as demonstrated by the detection, at mRNA and protein level, of HES-1. The activation of Notch signalling by using recombinant Jagged-1 or Jagged-2 did not lead to any change either in AML cell survival or in cell proliferation. On the contrary, the pan-blockade of Notch signalling, both at the mem- brane level by GSIs or the combination of Notch blocking antibodies, and at the transcriptional level using SAHM1, abolished AML cell pro- liferation in culture. Notably, BM-MSCs* showed higher level of Notch1 and Jagged1 as compared to BM-MSCs. Interestingly, we observed that BM-MSCs*, but not BM-MSCs, stimulated AML proliferation and the pan-Notch inhibition was sufficient to revert this phenomenon. In ad- dition BM-MSCs* were more efficient in rescuing AML cells from apop- tosis induced by chemotherapeutic agents. Pan-Notch signalling blockage by either GSI-XII or combination of Notch receptor-blocking antibodies in presence of chemotherapeutic agents significant lowered the supportive role of BM-MSCs towards AML blasts; conversely SAHM1 has a slight effect. The specific blockade of Notch-1, -2, -3 or Jagged-1, -2 reduced partially the chemoresistance, while blockade of Notch-4 or DLL-3 rescued totally the chemosensitivity of primary AML cells in co-culture with BM-MSCs. These results suggest that the inhi- bition of Notch signalling may represent a potential therapeutic strategy to improve AML treatment by overcoming bone marrow stromal-me- diated anti-apoptotic and chemoresistance effect