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Page 8 of 15 Sabol et al. J Cancer Metastasis Treat 2021;7:20 https://dx.doi.org/10.20517/2394-4722.2021.35

vitro work has shown that Jagged-mediated signals from MM cells can increase angiogenesis by activating
[77]
Notch and stimulating the release of Vegf in both endothelial cells and marrow stromal cells . In vitro,
genetic knockdown of Notch receptor 1/2 or blockade of Notch signaling with GSI decreased angiogenesis
induced by MM cells. Further, GSI treatment reduced the secretion of pro-angiogenic cytokines in
conditioned media and decreased angiogenesis in animal models of MM [48,78,79] . Until recently, MM cells or
stromal cells have been considered the main source of angiogenic factors in the MM niche. In a recent study
from our group, we examined if osteocytes contributed to the increased marrow vascular density in MM
patients. We found that the number of Vegf-A positive osteocytes is significantly increased in bones bearing
MM tumors and positively correlates with tumor vessel area . Hypoxia and MM cells increased Vegf
[80]
[80]
expression in osteocytes and increased the pro-angiogenic capacity of osteocytes . Vegf-A knockdown in
[80]
osteocytes completely blocked the increased endothelial activity induced by MM cells or hypoxia . These
results demonstrate that osteocytes are a source of Vegf-A, and potentially other pro-angiogenic factors, in
bones infiltrated with MM cells. However, whether Vegf-A production by osteocytes is dependent on Notch
remains to be determined. Despite the positive in vitro and in vivo results observed with pharmacological
blockade of Vegf in MM models, inhibition of Vegf in the clinical setting has not been successful, likely due
to the contribution of other pro-angiogenic factors to this phenomena . Further studies are needed to
[75]
clarify if Notch inhibition suppresses the production of other angiogenic factors besides Vegf, and if it could
be an efficacious strategy to contain angiogenesis in MM patients.

Notch and multiple myeloma-induced osteolytic bone disease
Bone is a very dynamic tissue, constantly being renewed by a lifelong process known as bone remodeling,
[81]
where mature bone is removed from the skeleton and new bone tissue is formed . This process is
orchestrated by the osteocytes, which coordinate the coupled and balanced activity of osteoclasts, bone
resorbing cells, and osteoblasts, bone forming cells [81,82] . Notch signals contribute to physiological bone
remodeling . However, the role of Notch in bone is complex and cell-dependent. The incomplete
[83]
understanding of the role of Notch in adult bone biology stems from the use of genetic manipulations in
mice, which result in alterations in skeletal development that inevitably affects the adult skeleton. The effects
of Notch on osteoclasts are controversial, with findings reporting both inhibition and stimulation of
osteoclast differentiation after Notch activation [84-86] . The effects of Notch in cells of the osteoblastic lineage
are dependent on the differentiation stage [87-91] . In osteocytes, Notch receptor 1 genetic activation from birth
[92]
results in inhibition of bone resorption due to Opg upregulation . In contrast, conditional activation of
[93]
Notch signaling in osteocytes in mature bones triggers bone formation .
The growth of MM cells in the marrow markedly alters bone remodeling, uncoupling the activity of
osteoclasts and osteoblasts, tilting the balance towards bone resorption [9,94,95] . The ability of MM cells to
shape the marrow into a pro-resorptive environment is mediated by several signaling pathways, including
[96]
Notch . Notch signaling can regulate osteoclastogenesis by 2 different mechanisms: (1) regulating the
expression of pro-osteoclastogenic cytokines in MM cells, and (2) mediating the communication between
MM cells and microenvironmental cells that leads to pro-resorptive effects. MM cells are a source of
cytokines that regulate osteoclast differentiation, including Rankl and M-Csf . Notch signaling regulates
[97]
Rankl expression in MM cells . Supporting this notion, we recently found that genetic deletion of Notch
[98]
receptors 2 and 3 in MM cells or treatment with GSI significantly decreases Rankl expression and impairs
their ability to stimulate osteoclastogenesis , Rankl expression in MM cells is also stimulated by stromal
[58]
cells, an effect depending on Notch activation . Additionally, MM cells can promote osteoclastogenesis by
[98]
direct activating Notch in osteoclasts, via Jagged 1 and 2 ligands [25,98] . MM cells also activate Notch signaling
in osteocytes, which are the major source of Rankl in adult bone [99,100] . Upon activation of Notch signaling by
MM cells, osteocytes undergo apoptosis , which in turn increases Rankl expression, decreases Opg
[43]
production, and enhances the ability of osteocytes to recruit osteoclast precursors .
[43]