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M. Colombo et al.
and contributing to the fatal outcome of this disease. The close interaction of MM cells with healthy BM cells repre- sents an important source of factors able to promote malignant cell growth and survival.
The Notch pathway is capable of mediating the cell-cell communication. Current evidence provided by different groups, including ours, highlighted the importance of Jagged ligands in the pathological communication between tumor and healthy cells within the myeloma BM. MM-derived Jagged ligands activate Notch receptors in the nearby BM cells inducing osteoclastogenesis, oste- olysis,17 angiogenesis,36 and BMSC-mediated release of key cytokines including IL6, IGF1 and VEGF.11,13 Moreover, the activation of Notch signaling in MM cells, induced by tumor cell-derived37,38 or BMSC-derived Jagged1,25 stimu- lates MM cell proliferation,38 resistance to apoptosis,37 and a decrease in drug sensitivity.25
This work is specifically focused on the pathological communication of MM cells and BMSC mediated by Notch signaling and on its outcome on MM drug resist- ance. Notably, the Notch pathway is known to be a key player in BM-induced drug resistance in other hematolog- ic malignancies. Indeed, Krampera’s group provided evi- dence of how the BM-driven activation of Notch3 and Notch4 in B-ALL39-41 and Notch1, Notch2 and Notch4 in chronic lymphocytic leukemia,42 results in chemoresis- tance, while Notch1-Jagged1 crosstalk supports BM- induced drug resistance in AML.43
As far as MM is concerned, in spite of the recent advances in the field, we still do not have a complete pic- ture of the bidirectional crosstalk between BMSC and MM cells, which is indicated by the expression of Notch receptors and ligands on both cell types.11,12,16,23,25,44 This work aims to fill some of those gaps by providing novel information about the effects of the aberrant expression of MM-derived Jagged ligands on the intrinsic tumor cell drug resistance and by investigating a key aspect that has never been previously explored, i.e. the outcome of MM- derived Jagged ligands on BMSC-induced drug resistance.
To address these issues, we interfered with the mRNA expression of MM-derived Jagged ligands and investigated J1/2KD outcomes in tumor cells and in surrounding BMSC. We observed in vitro that MM cell-derived Jagged ligands could trigger Notch signaling in the nearby MM cells by homotypic interaction. Notch activation resulted in the increased expression of anti-apoptotic effectors including BCL2, Survivin, and the multidrug resistance transporter ABCC1, along with the increase in MM cell survival to standard-of-care drugs, such as Bor, Len, and Melph. Notably, besides observing homotypic activation of Notch signaling among MM cells, we found that HMCL can trigger Notch signaling in the neighboring BMSC and, in turn, Notch activation boosts the ability of BMSC to increase the pharmacological resistance of MM cells. This effect was clearly dependent on MM-derived Jagged ligands, since J1/2KD completely abrogated BMSC support. At least in part, the pro-tumor effect of Notch- “educated” BMSC was due to their ability to increase SDF1α levels in the BM microenvironment. Indeed, solu- ble or MM cell-derived Jagged ligands may induce a Notch-dependent increase in SDF1α secretion by BMSC; on the contrary, J1/2KD HMCL lose this ability and N1KD interferes with BMSC to release SDF1α.
observed secretion induced by Notch activation in MM cells,29 since BMSC are the most effective producers of this cytokine in the BM.
The Notch-dependent activation of SDF1α secretion by BMSC is potentially more important than the previously
Although the downstream molecular mechanisms of Notch-associated drug resistance in MM still need to be fully elucidated, we showed that the secreted SDF1α can stimulate general mechanisms, including tumor cell anti- apoptotic background, by up-regulating BCL2 and Survivin, or drug extrusion mediated by ABCC1. These antiapoptotic proteins are particularly relevant to MM. Indeed, BCL2 and Survivin are over-expressed in MM cells, where they play an important role in cell survival, and significantly correlate with disease stage;20,27,45 on the other hand, xenobiotic transporters, such as ABCC1, are well known mediators of MM multidrug resistance,20 modulated by Notch in different cancer settings.46
The general validity of these novel findings stems from the observed improvement in drug-response promoted by J1/2KD in ex vivo co-culture systems of CD138+ MM cells and BMSC from BM aspirates of newly-diagnosed MM patients.
Additionally, in vivo validation of these findings in a zebrafish xenograft MM model engrafted with U266 cells confirmed that J1/2KD promoted an increased sensitivity to Bor in vivo, showing a wider decrease in tumor burden compared to the control.
The present results provide novel and important infor- mation to help improve the current picture on the effect of Notch-mediated communication in myeloma BM. Indeed, since both BMSC and MM cells carry Notch receptors and ligands, their bidirectional crosstalk needs to be taken into consideration. We sought to fill the gap in the available information on the role of MM cells, such as Notch signal- ing sending cells in the BM. Here we discuss our findings according to the previous literature data in order to sum- marize the overall picture (Figure 8). Previous work report- ed the consequences of Notch activity in MM cells (main- ly using g-secretase inhibitors), identifying the following molecular mechanisms: i) upregulation of p21 induced MM cell growth inhibition and increased survival;23 ii) Notch/HES1 mediated downregulation of the pro-apop- totic protein Noxa;24 iii) Notch up-regulated expression of integrin αvβ5 resulting in increased adhesion to vit- ronectin and consequent protection from pro-apoptotic drugs;47iv) upregulation of the enzyme cytochrome P450,44 implicated in drug metabolism and in the onset of several malignancies.48 Concerning the contribution of Notch in BMSC-dependent drug resistance, previous investigations were focused on the autonomous contribution of BMSC- derived Notch ligands in MM cell behavior (Figure 8).23,25,44
In this work, we found that the alteration induced in the BM by the presence of MM cells aberrantly expressing Jagged ligands is a key step in “educating” the tumor microenvironment to a pro-tumor type of behavior.
To complete the picture of a Notch signaling effect on the SDF1α/CXCR4 axis in myeloma BM, we also demon- strated that MM cell-derived Jagged ligands may further enhance the anti-apoptotic signaling of SDF1α by stimu- lating the expression of its receptor CXCR4 on the MM cell surface.
The contribution of the SDF1α/CXCR4 axis to MM pharmacological resistance was confirmed by the ability of the antagonist molecule AMD3100 to abrogate U266 cell resistance to (Bor), (Melph) and (Len) induced by BMSC, consistent with the findings of Azab et al.32
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haematologica | 2020; 105(7)