Page 194 - Haematologica Vol. 107 - September 2022

ARTICLE - NOTCH2 in myeloma-derived extracellular vesicles D. Giannandrea et al.
For instance they may help preparing the premetastatic niche through the formation of new permeable vessels for the extravasation of tumor cells, and the destruction of the bone matrix to make space for metastatic cells. Taken as a whole, the in vitro and in vivo NOTCH reporter assays confirmed that NOTCH activity in target cells was due to NOTCH2 delivered by the injected MM-EV.
This evidence and the acknowledged effect of NOTCH sig- naling on OCL and EC, prompted us to verify if NOTCH2 delivery by MM-EV could affect the biology of these cells. Through the same specific RNA interference approach on two different HMCL, we provided an unequivocal demon- stration that vesicular NOTCH2 participates in MM-in- duced OCL differentiation and angiogenesis, assessed by a tube formation assay. The dependency of these pro- cesses on NOTCH signaling was clearly demonstrated by the fact that MM-EVN2KD impact was significantly lower. The presence of other NOTCH receptors in MM-EV cargo, even if at a lower level, (i.e. NOTCH1) suggests that they may also provide a contribution.
In order to strengthen the translational potential of our results we used a dual approach: i) the outcome of an anti-NOTCH therapeutic approach already tested in clinics was assessed in vitro and ex vivo; ii) ex vivo experiments were carried out with EV released in the BM of MM pa- tients, taking into account that a systemic treatment is expected to affect the communication of MM-EV, as well as that of EV from all the BM cell populations. In the first case, we showed that γ-secretase inhibitors (GSI), already used in clinics,15 greatly affected MM-EV ability to inhibit angiogenesis and osteoclastogenesis in vitro. Concerning the second point, EV collected from the BM of MM pa- tients, but not MGUS patients, displayed a clear pro-an- giogenic effect that could be hampered by GSI.
In conclusion, the RNA interfering approach specific for NOTCH2 on HMCL, complemented by a pan-NOTCH chemical inhibition on HMCL- and MM patients’ BM-de- rived EV, provides a new important evidence of the effect of NOTCH signaling pathway on EV-mediated pathological communication in myelomatous BM. The important in- hibitory effect of GSI suggests that the form of the NOTCH2 oncogene which mostly contributes to MM-EV mediated education is NOTCH2-TM and not NOTCH2-IC whose activity is GSI resistant. Although, the presence of
References
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NOTCH2-IC in MM-EV cargo is much intriguing since it may deliver an active oncogenic signal, we believe that vesicular NOTCH-IC might be more relevant in tumor that expresses the constitutively active form of NOTCH, such as T-cell acute lymphoblastic leukemia.
In conclusion, our results strengthen the rationale for therapeutic approaches directed to inhibit NOTCH activa- tion mediated by MM-EV, suggesting that they have the potential of interfering with the pathological communica- tion of the MM cells mediated by EV in the short and po- tentially in the long range and, thereby, they may influence the cross-talk with the surrounding microenvironment and the dissemination of the disease at distant skeletal sites.
Disclosures
No conflicts of interest to disclose.
Contributions
DG, NP and MC designed and performed experiments, ac- quire data and wrote the manuscript; MM, VC, RA, FM, SA, EL performed experiments and acquired data; VD and IG performed TEM morphologic analysis; MC, MM, AP per- formed the in vivo zebrafish experiment; DG, LCan and VB analyzed data; MT and EC collected patients' samples and clinical information, performed patients' sample first pro- cessing; DG, MT, AB and LCas performed statistical analy- sis; DG, MT, AB, AP, EL, VB and LCas revised the manuscript; LCas aquired data; RC, DG, AB and MT interpreted data; RC set up the experiment design and supervised the re- search, interpretated data and statistical analysis, drafted, wrote and critical revised the manuscript.
Funding
This study was supported by grants from Associazione Ita- liana Ricerca sul Cancro, Investigator Grant to RC (20614), My First Grant to AP (18714); Fondazione Italiana per la Ricerca sul Cancro to MC (post-doctoral fellowship 18013); Università degli Studi di Milano to RC (Linea 2B-2017 - Dept. Health Sciences), to NP (postdoctoral fellowship type A) and DG (PhD fellowship in experimental medicine).
Data-sharing statement
For any question, please contact the corresponding author.
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