**4. Antiangiogenesis in multiple myeloma**

The combination of biological drugs in the actual therapeutic strategies of MM have improved the outcome of MM patients because of their activity on microenvironment [17, 151–153].

#### **4.1. Proteasome inhibitors**

IL-27 exert strong antitumor activities against MM cells from patients by binding with its specific IL-27 receptor [116, 117] inhibiting the angiogenic potential of MM plasma cells. In animals injected with the U266 MM cell line, the expression of the genes encoding the chemokines CCL-2, CXCL-3, CXCL-5, and CXCL-6 is significantly downregulated by IL-27 treat-

Another important paracrine loop between MM endothelial cells and plasma cells involves CXC-chemokines and their cognate receptors have been evidenced in the BM of MM patients [118, 119]. In fact, BM endothelial cells express and secrete high amounts of the CXCchemokines CXCL8/IL-8, CXCL11/interferon-inducible T-cell alpha chemoattractant (I-TAC), CXCL12/stromal cell-derived factor (SDF)-1α, and CCL2/monocyte chemotactic protein (MPC)-1 [118] that mediate the interactions between plasma cells and stromal cells interact-

HIF-1α has been demonstrated to be stabilized in MM plasma cells, in hypoxic as in normoxic conditions [82, 83, 119, 121–123]. The constitutive stabilization of HIF-1α in myeloma cells is associated with the oncogenic c-Myc activity, suggesting that a common signaling pathway is active in MM plasma cells [122]. Among target genes controlled by HIF-1α, the genes coding for the pro-angiogenic cytokines VEGF, IL-8, and OPN have been evidenced, and HIF-1α silencing significantly suppresses the pro-angiogenic properties of MM cells reducing their secretion [87]. Moreover, MM endothelial cells from relapsed/refractory MM patients, but not those of newly diagnosed or non-active MM patients, showed a stabilization and activation of the HIF-1α protein in normoxic conditions [124]. This stabilization is induced by ROS and correlated with the expression of HIF-1α pro-angiogenic targets [124]. The inhibition of HIF-1α in MM plasma cells [123] as well as in endothelial cells [124] impaired the MM plasma cells/stromal cells communication, the angiogenesis-related functions, and revert bortezomib- and lenalidomide-resistance [123, 124]. It may also have prognostic significance because patients with MM endothelial cells

The mammalian target of rapamycin (mTOR) is an intracellular serine/threonine kinase that mediates intracellular metabolism, cell survival, and actin rearrangement. mTOR is made of two independent complexes, mTORC1, involved in protein synthesis and autophagy inhibition, and mTORC2, involved in progression promotion, survival, actin reorganization, and drug resistance [125–127]. In MM endothelial, a significantly higher activation of mTORC2 have been demonstrated. Its inhibition induces a reduction of the angiogenic abilities of MM endothelial cells, suggesting a major role of mTORC2 in the "angiogenic switch" and indi-

In MM endothelial, cell-to-cell contact-dependent homotypic activation of Notch pathway has been shown [128, 129]. MM plasma cells cocultured with MM endothelial cells trigger Jagged1/2-mediated Notch activation enhancing endothelial angiogenic activity. Moreover, halting Notch axis reduces angiogenesis *in vitro* and *in vivo* suggesting Notch pathway as a

The ephrins (Efn) and their receptors (Eph), a large family of receptor tyrosine kinases, are involved in several biological processes including cancer growth, progression, and angiogenesis [130–133]. Caivano et al. [134] recently demonstrated that EphA3 is highly overexpressed in MM endothelial cells and its expression correlates with disease progression. They have also

ing with the respective chemokine receptors (CXCR and CCR) [118, 120].

expressing the stabilized HIF-1α protein had shorter overall survival [124].

cates that mTORC2 might be a new antiangiogenic target in MM [127].

novel therapeutic target in MM [129].

ment [116, 117].

106 Update on Multiple Myeloma

Bortezomib, a potent, highly selective, and reversible proteasome inhibitor targeting the 26S proteasome complex [154, 155] act on key cellular processes, such as cell cycle progression, inflammation, immune surveillance, growth arrest, and apoptosis [154]. Bortezomib acts by mean the modulation of NF-κB transcription factor, which mediates the expression and secretion of cytokines, chemokines, cell adhesion molecules involved also in anti-apoptosis and cellular growth control [154–156]. After phosphorylation by IκB kinase, IκB is polyubiquitinated and degraded by the 26S proteasome, which allows p50/p65 NF-κB nuclear translocation and binding to consensus motifs in the promoter region of target genes [155, 156]. NF-κB regulated also the expression of adhesion molecules, such as ICAM-1 and VCAM-1, on both MM cells and BM stromal cells [156], so, its inhibition downregulates these adhesion molecules favoring the susceptibility of MM plasma cells to therapeutic agents [156]. Moreover, NF-κB activation controls the production of IL-6 by BM stromal cells that increase production and secretion of VEGF-2 and FGF-2 from MM plasma cells [91]. By blocking NF-kB, bortezomib inhibits MM cell adherence to the BM stromal cells reducing MM cell growth and VEGF-2 and FGF-2 secretion [17, 91, 154, 155].

inhibit VEGF and FGF-2 secretion from both myeloma and BM stromal cells and block endothelial cell migration and proliferation in vivo and in vitro [169]. Lenalidomide, a first derivative of thalidomide, is less toxic and more potent than the parent drug, and in patients with relapsed or refractory MM, lenalidomide can overcome resistance not only to conventional chemotherapy but also to thalidomide [169]. De Luisi et al. [170] demonstrated that lenalidomide inhibits angiogenesis and migration of MM endothelial cells and that lenalidomidetreated MM endothelial cells show changes in VEGF/VEGFR-2 signaling pathway, and in several proteins controlling EC motility, cytoskeleton remodeling, and energy metabolism pathways. Both thalidomide and lenalidomide downregulate VEGF. Pomalidomide is a third generation IMiD with increased activity in vitro compared with thalidomide and lenalidomide [171, 172], which exerts anti-MM effects through multiple mechanisms, including induction of apoptosis via caspase-8, reduction of proliferation, inhibition of NF-κ B activation, reduction of stromal cell stimulatory cytokine secretion, and angiogenesis inhibition [172].

Angiogenesis and Antiangiogenesis in Multiple Myeloma

http://dx.doi.org/10.5772/intechopen.76931

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The bisphosphonates are other compounds that, although originally used to reduce bone loss in MM due to an anti-osteoclast activity, have also been shown to have antiangiogenic activity [173–175]. In fact, zoledronic acid has a direct cytotoxic activity on tumor cells and suppresses angiogenesis, inhibits FGF-2- and VEGF-dependent proliferation of endothelial cells and inhibits VEGFR-2 in an autocrine loop [173]. It has also been demonstrated that the addition of zoledronic acid to antimyeloma therapy, bortezomib-, lenalidomide-, or thalidomide-based, is associated with a benefit in term of skeletal-related event rate as well as in term of the progression-free survival rate of myeloma patients [174]. Neridronate exerts its antiangiogenic activity through both a direct effect on endothelial cell proliferative activity and inhibitory effect on the responsivity of

the endothelial cells to the proliferative stimuli mediated by angiogenic cytokines [175].

The most successful therapeutic approach to target VEGF in cancer is the use of a humanized monoclonal antibody against VEGF, bevacizumab [176]. Several clinical trials in MM tested the effects of bevacizumab used in conjunction with other agents including lenalidomide,

In addition to bevacizumab, other VEGFRs targeting compounds (including aflibercept-VEGFtrap), tyrosine kinase inhibitors (cabozantinib, dasatinib, pazopanib, sorafenib, sunitinib, and semaxanib), PI3K/Akt-MEK/ERK pathway inhibitors, FAK inhibitors, interleukin inhibitors (atiprimod), farnesyltransferase inhibitors, other monoclonal antibodies (anti-CD40), and marine cartilage extract (neovastat) have shown antiangiogenic activity but no significant results or only preliminary preclinical data have been reported with the use of this drugs in MM [177–181].

Despite the good results obtained in the last decades, MM remains an incurable malignancy, indicating that our knowledge on the mechanisms responsible for disease progression and

**4.3. Bisphosphonates**

**5. Conclusions**

**4.4. Monoclonal antibodies and other drugs**

dexamethasone, or bortezomib with discouraging results [177].

Bortezomib is directly cytotoxic on MM plasma cells by blocking proteasome activity that causes the accumulation of misfolded polyubiquitinated proteins and causes ROS production [155, 156]. The accumulation of misfolded proteins in the endoplasmic reticulum triggers caspase-4 activation, and ROS accumulation causes disruption of membrane potential and the release of cytochrome c from mitochondria, and then the caspase-9 activation. These cytoplasmic alterations consequently, initiate the apoptotic cascades causing apoptosis of the cell [155, 156]. Finally, bortezomib downregulates VEGF, IL-6, IGF-I, Ang-1, and Ang-2 production and secretion by MM plasma cells and BM stromal cells, targeting aberrant blood vessel development through a potent inhibition of proliferation of activated endothelial cells [17, 154].

Ixazomib (MLN2238) is a second-generation proteasome inhibitor with a similar activity of bortezomib on the inhibition of NF-kB [157, 158]. It has been demonstrated that ixazomib affects BM stromal cells triggered MM cell growth and BM stromal cells-induced endothelial cell proliferation suggesting that ixazomib not only directly targets MM plasma cells but also overcomes the cytoprotective effects of the MM host BM microenvironment [158]. In fact, ixazomib is able to impact angiogenesis in vivo decreasing the expression of angiogenic markers in mice as well as in vitro reducing the capillary formation by HUVEC in the Matrigel™ system [159].

The antiangiogenic activity of another proteasome inhibitor, carfilzomib, has not been clearly demonstrated but it seems to have inhibitory activity on tumor-stromal interactions and angiogenesis [137, 160]. Moreover, VEGF pathway polymorphisms have been associated with clinical outcomes in MM patients [161], and have been reported that polymorphisms of VEGF pathway are associated with response to the combination of carfilzomib and lenalidomide [162].

#### **4.2. Immunomodulators (IMIDs)**

Thalidomide, a first generation immunomodulatory drug (IMiD), has a direct tumoricidal activity, an antiangiogenic effect and modulates TNF-α signaling through direct and/or indirect effects on the tumor microenvironment [15, 163–167], reduces FGF-2, VEGF, and IL-6 secretion in BM stromal cells and by MM cells [163]. It also interferes with NF-κB activity by blocking its ability to bind to DNA abrogating inflammatory/angiogenic cytokine production [165, 166], and disrupts the direct interactions between MM plasma cells and BM stromal cells by modulation of cell surface adhesion molecules [167].

Two new IMiDs, including lenalidomide and pomalidomide, demonstrating up to 50,000 times more potent inhibition of TNF-α than thalidomide, has been developed [168–170]. They inhibit VEGF and FGF-2 secretion from both myeloma and BM stromal cells and block endothelial cell migration and proliferation in vivo and in vitro [169]. Lenalidomide, a first derivative of thalidomide, is less toxic and more potent than the parent drug, and in patients with relapsed or refractory MM, lenalidomide can overcome resistance not only to conventional chemotherapy but also to thalidomide [169]. De Luisi et al. [170] demonstrated that lenalidomide inhibits angiogenesis and migration of MM endothelial cells and that lenalidomidetreated MM endothelial cells show changes in VEGF/VEGFR-2 signaling pathway, and in several proteins controlling EC motility, cytoskeleton remodeling, and energy metabolism pathways. Both thalidomide and lenalidomide downregulate VEGF. Pomalidomide is a third generation IMiD with increased activity in vitro compared with thalidomide and lenalidomide [171, 172], which exerts anti-MM effects through multiple mechanisms, including induction of apoptosis via caspase-8, reduction of proliferation, inhibition of NF-κ B activation, reduction of stromal cell stimulatory cytokine secretion, and angiogenesis inhibition [172].

#### **4.3. Bisphosphonates**

growth control [154–156]. After phosphorylation by IκB kinase, IκB is polyubiquitinated and degraded by the 26S proteasome, which allows p50/p65 NF-κB nuclear translocation and binding to consensus motifs in the promoter region of target genes [155, 156]. NF-κB regulated also the expression of adhesion molecules, such as ICAM-1 and VCAM-1, on both MM cells and BM stromal cells [156], so, its inhibition downregulates these adhesion molecules favoring the susceptibility of MM plasma cells to therapeutic agents [156]. Moreover, NF-κB activation controls the production of IL-6 by BM stromal cells that increase production and secretion of VEGF-2 and FGF-2 from MM plasma cells [91]. By blocking NF-kB, bortezomib inhibits MM cell adherence to the BM stromal cells reducing MM cell growth and VEGF-2 and

Bortezomib is directly cytotoxic on MM plasma cells by blocking proteasome activity that causes the accumulation of misfolded polyubiquitinated proteins and causes ROS production [155, 156]. The accumulation of misfolded proteins in the endoplasmic reticulum triggers caspase-4 activation, and ROS accumulation causes disruption of membrane potential and the release of cytochrome c from mitochondria, and then the caspase-9 activation. These cytoplasmic alterations consequently, initiate the apoptotic cascades causing apoptosis of the cell [155, 156]. Finally, bortezomib downregulates VEGF, IL-6, IGF-I, Ang-1, and Ang-2 production and secretion by MM plasma cells and BM stromal cells, targeting aberrant blood vessel development through a potent inhibition of proliferation of activated endothelial cells [17, 154].

Ixazomib (MLN2238) is a second-generation proteasome inhibitor with a similar activity of bortezomib on the inhibition of NF-kB [157, 158]. It has been demonstrated that ixazomib affects BM stromal cells triggered MM cell growth and BM stromal cells-induced endothelial cell proliferation suggesting that ixazomib not only directly targets MM plasma cells but also overcomes the cytoprotective effects of the MM host BM microenvironment [158]. In fact, ixazomib is able to impact angiogenesis in vivo decreasing the expression of angiogenic markers in mice as well as in vitro reducing the capillary formation by HUVEC in the Matrigel™ system [159].

The antiangiogenic activity of another proteasome inhibitor, carfilzomib, has not been clearly demonstrated but it seems to have inhibitory activity on tumor-stromal interactions and angiogenesis [137, 160]. Moreover, VEGF pathway polymorphisms have been associated with clinical outcomes in MM patients [161], and have been reported that polymorphisms of VEGF pathway

Thalidomide, a first generation immunomodulatory drug (IMiD), has a direct tumoricidal activity, an antiangiogenic effect and modulates TNF-α signaling through direct and/or indirect effects on the tumor microenvironment [15, 163–167], reduces FGF-2, VEGF, and IL-6 secretion in BM stromal cells and by MM cells [163]. It also interferes with NF-κB activity by blocking its ability to bind to DNA abrogating inflammatory/angiogenic cytokine production [165, 166], and disrupts the direct interactions between MM plasma cells and BM stromal cells

Two new IMiDs, including lenalidomide and pomalidomide, demonstrating up to 50,000 times more potent inhibition of TNF-α than thalidomide, has been developed [168–170]. They

are associated with response to the combination of carfilzomib and lenalidomide [162].

FGF-2 secretion [17, 91, 154, 155].

108 Update on Multiple Myeloma

**4.2. Immunomodulators (IMIDs)**

by modulation of cell surface adhesion molecules [167].

The bisphosphonates are other compounds that, although originally used to reduce bone loss in MM due to an anti-osteoclast activity, have also been shown to have antiangiogenic activity [173–175]. In fact, zoledronic acid has a direct cytotoxic activity on tumor cells and suppresses angiogenesis, inhibits FGF-2- and VEGF-dependent proliferation of endothelial cells and inhibits VEGFR-2 in an autocrine loop [173]. It has also been demonstrated that the addition of zoledronic acid to antimyeloma therapy, bortezomib-, lenalidomide-, or thalidomide-based, is associated with a benefit in term of skeletal-related event rate as well as in term of the progression-free survival rate of myeloma patients [174]. Neridronate exerts its antiangiogenic activity through both a direct effect on endothelial cell proliferative activity and inhibitory effect on the responsivity of the endothelial cells to the proliferative stimuli mediated by angiogenic cytokines [175].

#### **4.4. Monoclonal antibodies and other drugs**

The most successful therapeutic approach to target VEGF in cancer is the use of a humanized monoclonal antibody against VEGF, bevacizumab [176]. Several clinical trials in MM tested the effects of bevacizumab used in conjunction with other agents including lenalidomide, dexamethasone, or bortezomib with discouraging results [177].

In addition to bevacizumab, other VEGFRs targeting compounds (including aflibercept-VEGFtrap), tyrosine kinase inhibitors (cabozantinib, dasatinib, pazopanib, sorafenib, sunitinib, and semaxanib), PI3K/Akt-MEK/ERK pathway inhibitors, FAK inhibitors, interleukin inhibitors (atiprimod), farnesyltransferase inhibitors, other monoclonal antibodies (anti-CD40), and marine cartilage extract (neovastat) have shown antiangiogenic activity but no significant results or only preliminary preclinical data have been reported with the use of this drugs in MM [177–181].
