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M. Colombo et al.
Figure 5. Multiple myeloma (MM) cell-derived Jagged ligands promote resistance to apoptosis through the modulation of the CXCR4/SDF1α axis in the bone mar- row (BM) niche. We evaluated the effect of J1/2KD in human multiple myeloma cell lines (HMCL) on the CXCR4/SDF1α axis in the BM and the consequence on the pharmacological resistance. (A) qRT-PCR for SDF1α and HES5 gene expression in NIH3T3 cells co-cultured with J1/2KD or Scr OPM2 cells (left panel) or J1/2KD or Scr U266 cells (right panel) compared to NIH3T3 cultured alone (=1), calculated by the 2−ΔΔCt formula. HES5 was used as a control for Notch pathway activity. Mean±standard deviation of four experiments are shown. Statistical analysis was performed using two-tailed t-test (*P<0.05; ***P<0.001). (B) Intracellular SDF1α level in HS5 cells co-cultured with J1/2KD HMCL. Histograms show the levels of intracellular SDF1α (black lines) analyzed by flow cytometry in GFP+ HS5 cells cul- tured alone or co-cultured with J1/2KD or Scr OPM2 cells (left panel) and J1/2KD or Scr U266 cells (right panel), and the isotype-matched control (dotted line). Histograms are representative of at least three independent experiments. Due to a high percentage of SDF1α expressing HS5 cells cultured with OPM2, we also show ΔGeoMFI. The apparent discrepancy between the two different basal levels of SDF1α produced by HS5 cells used as control in the co-culture systems with OPM2 or U266 cells is due to the effect of the different HS5 cell concentrations (see Online Supplementary Methods). (C) SDF1α levels in conditioned media of Scr or J1/2KD HMCL, HS5 cells or co-culture systems have been assessed by ELISA. Statistical analysis was performed using one-way ANOVA and Tukey post-test (*P<0.05; **P<0.01). (D) Effect of stimulation with Jagged1 and Jagged2 peptides on the secretion of SDF1α by HS5 cells. Statistical analysis was performed using one-way ANOVA and Tukey post-test (*P<0.05; **P<0.01; ***P<0.001). (E) Contribution of the Notch pathway to the ability of stromal cells to produce SDF1α. SDF1α levels were measured in Scr or N1KD HS5 cells. Flow cytometry histograms (left) and graphs (right) display the levels of intracellular SDF1α (ΔGeoMFI) ana- lyzed in HS5 Scr (green) or HS5 N1KD cells (blue) and an isotype-matched control (gray); the graph shows mean±standard error of mean (SEM) of SDF1α expression levels. Statistical analysis was performed by t-test (*P<0.05). (F) Status of CXCR4 expression in Scr or J1/2KD HMCL used in co-culture experiments with HS5 cells. Values in the graph represent mean±SEM of CXCR4 expression levels (ΔGeoMFI) measured by flow cytometry. Statistical analysis was performed by t-test (*P<0.05; **P<0.01). (G) To evaluate if SDF1α contributes to BCL2, Survivin and ABCC1 expression, U266 cells were cultured in the presence of 500 ng/mL recombinant SDF1α for 48 h and analyzed by qRT-PCR. Graphs show the relative expression levels of the indicated genes compared with the corresponding values in BSA-treated cells (=1), calculated by the 2−ΔΔCt formula. Mean values +/- standard deviations of three independent experiments are shown. Statistical analysis was performed using two-tailed t-test (*P<0.05). (H) Results were further confirmed by western blot analysis. Images were acquired using the UV-tech Alliance system and are rep- resentative of three independent experiments. (I) To assess if the SDF1α/CXCR4 axis affects MM cell drug resistance, U266 cells cultured alone or with GFP+ HS5 cells were treated with 6 nM bortezomib (Bor), 30 μM melphalan (Melph), 15 μM lenalidomide (Len) or DMSO in the presence or absence of 50 μM AMD3100. Apoptotic MM cells were measured by flow cytometry as Annexin-V+/GFP– cells. Graph shows mean±SEM of at least three independent experiments. Statistical analy- sis was performed using one-way ANOVA and Tukey’s post-test: *P<0.05; **P<0.01; ****P<0.0001.
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Figure 6. Translational potential of Jagged1/2 inhibition: outcome on ex vivo cultures of multiple myeloma (MM) patients’ cells and treatment with small molecule affecting Notch-Jagged interaction. (A) Outcome of J1/2KD on primary CD138+ MM cells response to standard-of-care drugs in a primary co-culture system with bone marrow stromal cells (BMSC). Levels of apoptosis were analyzed by flow cytometry on primary MM cells transduced with the lentiviral vector pLL3.7 codifying for the Jagged1 and 2 shRNAs (J1/2) or the corresponding control (Ctrl), and then co-cultured with BMSC from MM patients. Co-cultures were maintained for 72 hours (h) and treated for the last 24 h with 6 nM bortezomib (Bor) (left panel; 8 patients) or 30 μM melphalan (Melph) (central panel; 10 patients) and for 48 h with 15 μM lenalidomide (Len) (right panel; 9 patients) or DMSO. The percentage of infected MM cells that underwent apoptosis (GFP+/AnnexinV+) was detected by flow cytom- etry. Statistical analysis was performed using one-way ANOVA and Tukey post-test (*P<0.05; **P<0.01; ***P<0.001). (B and C) Effect of the inhibitory small mole- cule, IGOR1, on MM drug resistance. OPM2 cells treated with 30 μM IGOR1 were cultured on a monolayer of HS5 GFP+ cells in the presence or the absence of dif- ferent drugs as described in the Methods. (B) Quantitative polymerase chain reaction assay shows that IGOR1 inhibits Notch pathway in OPM2 cells, as demonstrated by the downregulation of Notch target genes, HES1 and HES6. Relative gene expression variation was normalized to GAPDH and calculated by the 2-ΔΔCt formula. Mean values +/- standard deviations of three experiments are shown. Statistical analysis was performed using two-tailed t-test (*P<0.05). (C) The levels of apoptosis of OPM2 cells treated with IGOR1 and the indicated drugs were measured by staining with Annexin-V-APC (C). Graph shows mean values +/- standard deviations of at least three independent experiments. Statistical analysis was performed using one-way ANOVA and Tukey’s post test (*P<0.05; **P<0.01; ****P<0.0001).
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haematologica | 2020; 105(7)