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Jagged1/2 and drug resistance in multiple myeloma
effective in comparison with the simultaneous J1/2KD, that maximizes the biological outcome (Online Supplementary Figure S2).
These results indicate that the expression of Jagged1 and 2 stimulates autonomous Notch activity in MM cells that, consequently, may be inhibited by Jagged silencing. This evidence prompted us to verify whether the increased pharmacological sensitivity of MM cells induced by J1/2KD was associated to variations in the expression of recognized anti-apoptotic Notch targets, such as BCL218 and Survivin/BIRC5,19 or with the levels of ABCC1 reported to have a significant impact in MM.19,20,27 J1/2KD, validated by the decrease in Jagged1, 2 and HES1 and 6 gene expression, significantly inhibited the expres- sion of the studied anti-apoptotic genes analyzed by qRT- PCR (Figure 2A and B). The effect of J1/2KD on gene expression was seen not to be due to an increased apop- tosis rate in HMCL (approx. 15%) (Online Supplementary Figure S1). J1/2KD effect on anti-apoptotic effectors was assessed at protein levels by flow cytometry (Figure 2C and D and Online Supplementary Figure S3) and western blot (Online Supplementary Figure S4). By contrast, the selective inhibition of Jagged1 or Jagged2 was not suffi- cient to significantly down-regulate the expression of these genes (Online Supplementary Figure S5).
Jagged1 and Jagged2 silencing contributes to multiple myeloma cell ability to promote bone marrow stromal cell-mediated drug resistance
Multiple myeloma cells localize within the BM and interact with several cell types, hijacking their functions to promote tumor progression. BMSC are a crucial target in this process that sustains malignant cell proliferation and survival.22 Since Jagged-mediated activation of Notch pathway is involved in cell-cell communication,6 we hypothesized that MM cell-derived Jagged ligands could activate Notch in BMSC, possibly determining BMSC- mediated drug resistance.
To explore this hypothesis, we first verified that HMCL- derived Jagged1 and Jagged2 were able to trigger the acti- vation of Notch signaling in a BMSC line, HS5, using a Notch reporter assay. Scr HMCL are able to activate Notch signaling in co-cultured HS5 cells (Figure 3A), while this ability is lost by J1/2KD HMCL, indicating that MM- derived Jagged may activate Notch signaling in BMSC.
To verify if Jagged-mediated activation of Notch in BMSC affected the ability of these cells to promote drug resistance in MM cells, we used flow cytometry to analyze the apoptotic rate of Scr or J1/2KD HMCL cultured alone or co-cultured with HS5 cells after treatment with stan- dard-of-care drugs. As expected, HS5 cells show a clear
A
B
administration administration administration
Figure 1. J1/2 silencing increases drug response in multiple myeloma (MM) cells. (A) Timeline of the experiment to study J1/2KD effect drug response in MM cell lines
C
(HMCL). h: hour. Representative western blots showing the expression of Jagged1, Jagged2, NICD1 and NICD2 in OPM2 and U266 cells following single and com- bined Jagged1 and/or Jagged2 silencing. β-actin was used as loading control. (C) The effect of J1/2KD on OPM2 (left) and U266 (right) cell response to bortezomib (Bor), melphalan (Melph) and lenalidomide (Len) was evalu- ated by Annexin V staining. MM cells were transfected with two specific siRNAs tar- geting Jagged1 and Jagged2 (J1/2KD) or the corresponding scrambled control (Scr) and treated with Bor, Melph or Len. Values of apoptosis of Scr HMCL were normalized to the corresponding DMSO-treated controls and values of J1/2KD HMCL treated with drugs were
P=0.06 normalized to DMSO-treated J1/2KD HMCL. Results are shown as the mean±standard error of at least three inde- pendent experiments, and sta- tistical analysis was per- formed using Mann-Whitney
test (*P<0.05; **P<0.01).
(B)
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