Notch signalling drives bone marrow stromal cell-mediated chemoresistance in acute myeloid leukemia

Both preclinical and clinical investigations suggest that Notch signalling 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. Thus, 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*). As compared to hBM-MSCs, hBM-MSCs* showed higher level of Notch1, Jagged1 as well as the main Notch target gene HES1. Notably, hBM-MSCs* induced expression and activation of Notch signalling in AML cells, supporting AML proliferation and being more efficientin inducing AML chemoresistance than hBM-MSCs*. Pharmacological inhibition of Notch using combinations of Notch receptor-blocking antibodies or gamma-secretase inhibitors (GSIs), in presence of chemotherapeutic agents, significant lowered the supportive effect of hBM-MSCs and hBM-MSCs* towards AML cells, by activating apoptotic cascade and reducing protein level of STAT3, AKT and NF-κB. These results suggest that Notch signalling inhibition, by overcoming the stromal-mediated promotion of chemoresistance,may represent a potential therapeutic targetnot only for lymphoid neoplasms, but also for AML.

zolium bromide (MTT, Sigma-Aldrich) metabolic activity assay. AML lines (10 4 cells per wells) or AML primary cells (10 5 cells per wells) were seeded in 96 wells plates, with increasing concentrations of different drugs. Cell viability was then assessed by adding 10 µL of MTT into each well and keeping in incubator for 3 hours. Metabolically active, viable cells converted MTT into a colored formazan, which was solubilized with a volume of acid isopropanol equal to the volume of cell suspension. The product was then measured at 570nm in a spectrophotometric microplate reader (PerkinElmer VICTORX4). The viability was expressed as the percentage of optical density of treated cells compared to optical density of cells treated with the specific vehicle. The effective concentration to induce 50% reduction of AML cells viability (EC50) derived from the equations that best fit the linear range of the dose-response curve. Each experimental condition was done in hexaplicate and repeated at least twice.

Apoptosis, cell proliferation and TOPRO-3 viability assay
Apoptotic rate of AML cells was assessed using FITC-conjugated AnnexinV (AnnexinV-FITC) and Propidium Iodide (PI) staining. AML cells were washed twice with PBS and then stained with APC-conjugated anti-CD45 for 10 minutes in the dark at room temperature. Cells were resuspended in binding buffer (MiltenyiBiotec), and AnnexinV-FITC (MiltenyiBiotec) was added at 1 µg/mL final concentration. The mixture was incubated at room temperature for 15 minutes in dark. Membrane integrity was assessed by PI staining immediately before flow cytometry analysis on a FACS Canto II. In order to discriminate live cell population, samples were stained with TO-PRO-3 (1µM) and analyzed by FACS. Relative cell proliferation were expressed as the percentage of CFSE median fluorescence of treated cells compared to that of cells treated with the specific vehicle.

MayGrunwald-Giemsa staining
We applied this method to study whether the treatments with GSIs and Idarubicin affected hBM-MSCs* morphology. Briefly, adherent hBM-MSCs* were washed with PBS to remove AML cells, then cells were stained with three decreasing concentrations of MayGrunwald-Giemsa solution, i.e. 3 minutes with pure 100% MayGrunwald, 2 minutes with 1/3 diluted MayGrunwald solution, and finally 15 minutes with 1/10 diluted Giemsa solution.
Cells were then washed with distillated water and dried at room temperature. Stained cells were acquired with Axiovert Z1 Observer Microscope (Zeiss).      AML cells were cultured alone or co-cultured with hBM-MSCs* in presence of Idarubicin (0.5µM).
Viability of AML cells in culture or co-culture with hBM-MSCs in presence of either Ara-C (10µM), Etoposide (10µM) or Idarubicin (0.5µM) and increasing concentrations of GSI-XII.
After 48 hours, cells were collected and analyzed by AnnexinV/PI assay. Data are expressed as mean ± SEM of 3 independent experiments. Statistical analysis was performed with oneway Anova comparing all the culture conditions with AML + hBM-MSCs in presence of DMSO. *p<0.05, **p<0.01, ***p<0.001.