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, 2 (5), e73

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

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

A H Nwabo Kamdje et al. Blood Cancer J.

Abstract

Stromal cells are essential components of the bone marrow (BM) microenvironment that regulate and support the survival of different tumors, including chronic lymphocytic leukemia (CLL). In this study, we investigated the role of Notch signaling in the promotion of survival and chemoresistance of human CLL cells in coculture with human BM-mesenchymal stromal cells (hBM-MSCs) of both autologous and allogeneic origin. The presence of BM-MSCs rescued CLL cells from apoptosis both spontaneously and following induction with various drugs, including Fludarabine, Cyclophosphamide, Bendamustine, Prednisone and Hydrocortisone. The treatment with a combination of anti-Notch-1, Notch-2 and Notch-4 antibodies or γ-secretase inhibitor XII (GSI XII) reverted this protective effect by day 3, even in presence of the above-mentioned drugs. Overall, our findings show that stromal cell-mediated Notch-1, Notch-2 and Notch-4 signaling has a role in CLL survival and resistance to chemotherapy. Therefore, its blocking could be an additional tool to overcome drug resistance and improve the therapeutic strategies for CLL.

Keywords: CLL; Notch signaling; chronic lymphocytic leukemia; drug resistance; hBM-MSCs; human bone marrow mesenchymal stromal cells.

Figures

Figure 1
Figure 1
The specific relative cell viability (%) of CLL cells cultured alone with increasing concentrations of chemotherapeutic agents for 1 day. CLL cells were cultured alone with increasing concentrations of GSI XII (a), Fludarabine (b), Cyclophosphamide (c), Bendamustine (d), Prednisone (e) and Hydrocortisone (f) for 1 day. Viable CLL cells were quantified by flow cytometry as percentage of Annexin V/7-AAD (lower left quadrant) after electronic gating on CD5+CD19+CD45+ CLL cells. The percentage of specific viable CLL cells was calculated according to the following formula: 100-((test (inhibitor-induced apoptosis)–control (spontaneous apoptosis)) × 100/(100–control)). Statistical analysis was carried out by using one-way analysis of variance, Holm–Sidak test. *P<0.05 was considered statistically significant. Data were represented as mean±SD of twelve independent experiments.
Figure 2
Figure 2
Effects of GSI XII, Fludarabine, Cyclophosphamide, Bendamustine, Prednisone and Hydrocortisone on the active caspase-3 expression by hBM-MSCs. MFI of active caspase-3 expression by CD73+CD45-hBM-MSCs after culture for 3 days with increasing concentrations of GSI XII (a), Fludarabine (b), Cyclophosphamide (c), Bendamustine (d), Prednisone (e) and Hydrocortisone (f). Statistical analysis was carried out by using analysis of variance test, Holm–Sidak test. Data were represented as mean±SD of 12 independent experiments. *P<0.05, **P<0.01, ***P<0.001 versus IgG control were considered statistically significant. GSI XII and Fludarabine were resuspended in dimethyl sulfoxide (DMSO). Cyclophosphamide, Bendamustine, Prednisone and Hydrocortisone were resuspended in NaCl (0.9%). DMSO and NaCl (0.9%) were used as controls for vehicle effects.
Figure 3
Figure 3
Percentage of viable CLL cells and specific relative cell viability (%) of CLL cells cultured alone or cocultured with hBM-MSCs in presence of increasing concentrations of GSI XII. (a) Percentage of viable CLL cells cultured alone or cocultured with hBM-MSCs at 10/1 ratio for 3, 7 and 28 days. (b, c) Specific relative cell viability (%) of CLL cells was evaluated by culturing alone or coculturing CLL cells with hBM-MSCs at 10/1 and 1/1 ratios in presence of increasing concentrations of GSI XII for 3 days. Statistical analysis was carried out by using one-way analysis of variance, Holm–Sidak test. *P<0.05 versus control was considered statistically significant. Data were represented as the mean±SD of 12 independent experiments. GSI XII was resuspended in dimethyl sulfoxide (DMSO) and DMSO was used as control for vehicle effects.
Figure 4
Figure 4
Viable CLL cells cultured alone and cocultured with hBM-MSCs or hBM-MSCs* in absence or presence of either neutralizing antibodies anti-Notch receptors or GSI XII. B-CLL cells were cultured alone or cocultured with either hBM-MSCs (a) or hBM-MSCs* (b) (10/1 ratio), in absence or presence of anti-Notch-1, -2, -3, -4 antibodies, or GSI XII for 3 days. Percentage of viable CLL cells (Annexin V/7-AAD, lower left quadrant) was measured by flow cytometry after electronic gating on CD5+CD19+CD45+ CLL cells. Statistical analysis was carried out by using one-way analysis of variance, Holm–Sidak test. *P<0.05, **P<0.01, ***P<0.001 versus IgG-treated control were considered statistically significant. GSI XII was resuspended in dimethyl sulfoxide (DMSO) and DMSO was used as control for vehicle effects.
Figure 5
Figure 5
Expression of IL-7R, VEGFR2, active caspase-3, Bcl-2, CD23, c-IAP protein, NF-κB and Hes-1 in CLL cells cocultured with hBM-MSCs. The expression of IL-7 R (a), VEGFR2 (b), active caspase-3 (c), CD23 (d), Bcl-2 (e), p53 (f) and NF-κB (g) by CD5+CD19+CD45+ CLL cells was analyzed by flow cytometry following coculture with hBM-MSCs (10/1 ratio), in absence or presence of anti-Notch-1,-2 or -4 antibodies or GSI XII for 3 days. Western blot analysis was performed to assess the expression of c-IAP2, Hes-1, PARP, NF-κB and Bcl-2 by CLL cells cocultured with hBM-MSCs in the same conditions (h). Hes-1 expression was used as functional read-out for Notch activation. NF-κB and Bcl-2 expression was evaluated to confirm CLL cell activation following coculture with hBM-MSCs in the same conditions. β-actin was included as loading control. Statistical analysis was carried out by using one-way analysis of variance, Holm–Sidak test. *P<0.05, **P<0.01, ***P<0.001 versus IgG-treated control were considered statistically significant. GSI XII was resuspended in dimethyl sulfoxide (DMSO) and DMSO was used as control for vehicle effects.
Figure 6
Figure 6
Percentage of viable CLL cells cocultured with hBM-MSCs in absence or presence of chemotherapeutic agents and neutralizing Notch receptor antibodies and GSI XII.Percentage of viable CLL cells (Annexin V/7-AAD, lower left quadrant) cultured alone in absence or presence of Fludarabine (5.0 μℳ) (a), Cyclophosphamide (100 μℳ) (b), Bendamustine (200.0 μℳ) (c), Prednisone (100.0 μℳ) (d) and Hydrocortisone (100.0 μℳ) (e), and cocultured with hBM-MSCs (10/1 ratio) in presence or absence of neutralizing Notch receptor antibodies or GSI XII for 3 days. CLL cells were analyzed by flow cytometry after electronic gating on CD5+CD19+CD45+ CLL cells. GSI XII was resuspended in dimethyl sulfoxide (DMSO) and DMSO was used as control for vehicle effects. One-way analysis of variance, Holm–Sidak test was used to compare the different groups. *P<0.05, **P<0.01, ***P<0.001 were considered statistically significant.

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