Role of Stromal Cell-Mediated Notch Signaling in AML Resistance to Chemotherapy

Introduction: Our group has recently shown that bone marrow-mesenchymal stromal cell (BM-MSCs)-mediated Notch signaling may control survival and chemoresistance of B-acute lymphoblastic leukemia (B-ALL) and chronic lymphocytic leukemia (CLL) cells. Conversely, the role of Notch signaling in acute myeloid leukemia (AML) remains controversial, as its contribution to the crosstalk between BM-MSCs and AML cells is still unknown. Thus, we evaluated the role of the Notch pathway in the proliferation, survival and chemoresistance of AML primary blast cells in co-culture with BM-MSCs.

Methods: AML blast cells were obtained after informed consent from bone marrow samples (30) and peripheral blood (20) of AML patients, according to the Institutional guidelines. BM-MSCs were expanded from bone marrow of 12 healthy donors (BM-MSCs) and of 12 AML-patients (BM-MSCs*). PCR, FACS analysis and western immunoblotting were used to study the expression of Notch receptors and ligands, as well as Notch activation status, in AML cells and BM-MSCs. AML cells were co-cultured with BM-MSCs or BM-MSCs* at 10:1 (AML:BM-MSCs) ratio for 2 to 3 days in presence of Cytarabine, Etoposide, Idarubicin, as well as in presence or absence of anti-Notch-1, -2, -3, -4, anti-Jagged1, -2 and anti-DLL3 blocking antibodies or gamma secretase inhibitor-XII (GSI-XII). Cell viability was evaluated by Annexin-V/Propidium Iodide (PI) and MTT; proliferation and cell cycle were assessed through CFSE dilution and PI methods, respectively.

Results: AML cells expressed Notch receptors and ligands, showing Notch-1, -2, Jagged-1, -2 and DLL-3 signature, while BM-MSCs/ BM-MSCs* showed expression of Notch-1, -2, -3, -4 and Jagged-1, -2 and DLL-1. We then analyzed Notch activation in different cell types by evaluating the expression of NICD-1, -2, -3 as well as Hes-1. We found that at least 50% of AML samples showed basal Notch activation while BM-MSCs/ BM-MSCs* showed slight Notch activation. The expression and activation pattern were modulated after 3 days of co-culture with either BM-MSCs or BM-MSCs*. The pan blockade of Notch signaling by GSI-XII were capable to inhibit AML cell proliferation as well as induce AML cell apoptosis in culture or in co-culture with BM-MSCs/ BM-MSCs*. The addition of chemotherapeutic agents decreased AML cell viability in culture, while a significant rescue from apoptosis was observed when cocultured with BM-MSCs or BM-MSCs*. Pan Notch signaling blockade by either GSI-XII or combination of Notch receptor-blocking antibodies in presence of chemotherapeutic agents significantly lowered the supportive role of BM-MSCs towards AML cell lines. The specific blockade of Notch-1, -2, -3 or Jagged-1, -2 rescued partially the chemosensibility, while blockade of Notch-4 or DLL-3 rescued totally the chemosensitivity of primary AML cells in co-culture with BM-MSCs.

Conclusions: These results suggest that Notch signaling may represent a potential therapeutic strategy to overcome bone marrow stromal-mediated survival and chemoresistance of AML. Moreover Notch blocking antibodies were able to impair the survival benefit imparted by bone marrow stromal cells. Therefore blocking Notch antibodies could be a useful strategy to improve the efficiency of AML chemotherapy.