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M. Colombo et al.
ed by the interaction with five ligands (Jagged1-2 and Dll1-3-4) on adjacent cells.4-6 Notch receptors and ligands have been found to be aberrantly expressed in MM cells.7- 10 We recently demonstrated that Jagged1 and the Notch transcriptional target HES5 are increasingly expressed in MM and in primary plasma cell leukemia.11 Moreover, Jagged1 and Notch1 are over-expressed during progres- sion from the benign monoclonal gammopathy of uncer- tain significance (MGUS) to MM,12 while Jagged2 overex- pression is already detected at the MGUS stage13 and can be ascribed to aberrant acetylation of its promoter14 or to altered post-translational processing due to aberrant expression of the ubiquitin ligase Skeletrophin.15 Finally, Notch2 hyperexpression is associated with the high-risk translocations t(14;16)(q32;q23) and t(14;20)(q32;q11).16
Recently, we and other groups pointed out the impor- tance of Jagged ligands in providing MM cells with the ability to shape the surrounding microenvironment, inter- acting with osteoclast progenitors,17 and promoting a release of BM stromal cell (BMSC) key factors, including IL6, IGF1 and VEGF.11,13
Aberrant levels of Notch signaling are associated with pharmacological resistance in different tumor settings6 and correlate with the expression of anti-apoptotic genes, such as BCL218 and Survivin/BIRC5,19 or regulates the expression of ABCC1,46 which contributes to multidrug resistance in MM.20
Given this, we hypothesized that the aberrant expres- sion of Notch receptors and ligands in MM cells may foresee the development of drug resistance by inducing autonomous activation of Notch in MM cells, and by trig- gering Notch signaling in the surrounding BMSC and boosting their ability to support MM cell drug resist- ance.21,22
Previous studies investigated how BMSC support the development of drug resistance in MM cells by activating Notch signaling.23-25 Vice versa, here we show that also the overexpression of MM cell-derived Jagged ligands triggers Notch signaling dysregulation in the BM niche and pro- motes MM cell intrinsic pharmacological resistance as well as BMSC-dependent drug resistance.
Methods
Cell lines and primary cells
The human MM cell lines (HMCL), OPM2 (ACC-50) and U266 (ATCC® TIB-196) were purchased from the DSMZ and ATCC, respectively. Primary cells were isolated from patient BM aspirates and MM cells were purified using the Human Whole Blood CD138+ Selection Kit EasySep (StemCell Technologies). Detailed information is available in Online Supplementary Table S1. Primary BMSC were isolated as previously reported.11 The Ethical Committee of the Università degli Studi di Milano, Italy, approved this study (approval n. 8/15).
Details of all cell treatments are available in the Online Supplementary Appendix.
Luciferase reporter assay
HS5 cells were transiently transfected with a Notch reporter plasmid pNL2.1 carrying a 6xCSL Notch responsive element26 and with the vector constitutively expressing the firefly luciferase upon the thymidine kinase promoter (pGL4.54[luc2/TK]). After 24 hours (h), HS5 cells were cultured alone or placed in co-culture with scrambled (Scr) or Jagged1 and
Jagged2 knockdown (J1/2KD) HMCL and incubated for 24 h. Luciferase activity was measured using Nano-Glo® Dual- Luciferase® Reporter assay kit (Promega) on the Glowmax instrument (Promega).
In vivo experiments on xenografted zebrafish embryos Zebrafish AB strains obtained from the Wilson lab, University College London, UK, were maintained according to the national guidelines (Italian Ministerial Decree of 4/03/2014 2014, n. 26). All experiments were conducted within five days post fertiliza-
tion.
Dechorionated zebrafish embryos were injected with Scr or
J1/2KD U266 cells stained with the CM-Dil dye into the yolk (200 cells in 10 nl, 5-20 nl injection volume/embryo) with a man- ual microinjector (Eppendorf, Germany) using glass microinjec- tion needles.
Xenograft-positive embryos divided randomly into the follow- ing groups: Scr-injected embryos treated with DMSO, Scr-inject- ed embryos treated with 10 nM bortezomib, J1/2KD-injected embryos treated with DMSO, and J1/2KD-injected embryos treated with 10 nM bortezomib. Tumor growth was evaluated 48 h post injection (hpi) by fluorescence microscopy. Further details are available in the Online Supplementary Appendix.
Further details and information concerning cell cultures, RNA isolation and quantitative real-time polymerase chain reaction (qRT-PCR), RNAi assay, apoptosis assays, flow cytometry, ELISA, western blot and statistical analysis can be found in the Online Supplementary Appendix: experimental procedures.
Results
Jagged1/2 inhibition improves multiple myeloma cell response to standard-of-care drugs by increasing the anti-apoptotic background
To assess if Jagged1 and Jagged2 contribute to MM intrinsic drug resistance, we took advantage of an estab- lished knockdown (KD) approach using specific siRNAs for Jagged ligands11,17 and analyzed MM cell response to three standard-of-care drugs: bortezomib (Bor), melphalan (Melph), and lenalidomide (Len). Two HMCL, OPM2 and U266 cells, were transfected with Jagged1 and Jagged2 (J1/2KD) or the scrambled control (Scr) siRNAs and then were treated with 6 nM Bor or 30 μM Melph or with 15 or 30 μM Len (respectively for U266 and OPM2 cells) (Figure 1A). The efficacy of J1/2KD was assessed by eval- uating the expression of Jagged ligands and the active forms of the two Notch receptors expressed in MM cells, Notch intracellular domains 1 and 2 (NICD1 and NICD2), by western blot (Figure 1B).
The apoptosis rate of J1/2KD HMCL treated or not with Bor, Melph and Len was analyzed by flow cytometry. Figure 1C shows the effect of Bor, Melph and Len on HMCL normalized on DMSO-treated cells compared to J1/2KD HMCL treated with the drugs and normalized on untreated J1/2KD HMCL. J1/2KD induced an appreciable increase in HMCL sensitivity to standard-of-care drugs, with statistical significance reached in all cases, with the exception of U266 cells treated with Bor and Melph (P=0.06), that in any case confirmed the trend (Figure 1C). The basal apoptotic effect of J1/2KD is shown in Online Supplementary Figure S1. Concerning Len treatment, it is worth mentioning that, although Scr HMCL are resistant to this drug, J1/2KD cells acquire drug sensitivity. The selective inhibition of Jagged1 or Jagged2 is clearly less
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haematologica | 2020; 105(7)